( E - F ) Immunofluorescence was conducted to detect the expression levels of cGAS , NF - κB , and pyroptosis associated molecules ( GSDMD , caspase - 1 , and IL - 1β ) in BV2 cells . n = 3 in each group . Scale bars , 50 μm .

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(E-F) Immunofluorescence was conducted to detect the expression levels of cGAS, NF-κB, and pyroptosis associated molecules (GSDMD, caspase-1, and IL-1β) in BV2 cells. n = 3 in each group. Scale bars, 50 μm.


( A ) Gating strategy of brain - infiltrating immune cells including CD4 + T cells ( CD3 + CD4 + ) , CD8 + T cells ( CD3 + CD8 + ) , B cells ( CD3 - CD19 + ) , NK cells ( CD3 - NK1 . 1 + ) , monocyte / macrophages ( CD11b + CD45highF4 / 80 + ) , neutrophils ( CD11b + CD45highLy6G + ) , and microglia ( CD11b + CD45int ) and their expression of IL ( interleukin ) - 6 , TNF - α ( tumor necrosis factor - α ) , TGF - β ( transforming growth factor - β ) , and IL - 4 . FMO , fluorescence minus one . ( B ) Quantitative analysis of the counts of brain - infiltrating leukocytes in the brains of MCAO mice receiving indicated treatment . ( C ) Bar graph shows counts of leukocytes subtype in the spleens of MCAO mice receiving indicated treatment . n = 6 mice per group . ( D ) Quantitative analysis of the microglia in the brains of MCAO mice receiving indicated treatment . n = 6 mice per group . *

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(A) Gating strategy of brain-infiltrating immune cells including CD4+ T cells (CD3+ CD4+), CD8+ T cells (CD3+CD8+), B cells (CD3-CD19+), NK cells (CD3-NK1.1+), monocyte/macrophages (CD11b+CD45highF4/80+), neutrophils (CD11b+CD45highLy6G+), and microglia (CD11b+CD45int) and their expression of IL (interleukin)-6, TNF-α (tumor necrosis factor-α), TGF-β (transforming growth factor-β), and IL-4. FMO, fluorescence minus one. (B) Quantitative analysis of the counts of brain-infiltrating leukocytes in the brains of MCAO mice receiving indicated treatment. (C) Bar graph shows counts of leukocytes subtype in the spleens of MCAO mice receiving indicated treatment. n = 6 mice per group. (D) Quantitative analysis of the microglia in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group. *


( E ) Bar graph shows percentages of microglia expressing IL - 6 , TNF - α , TGF - β , and IL - 10 in the brains of MCAO mice receiving indicated treatment . n = 6 mice per group .

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(E) Bar graph shows percentages of microglia expressing IL-6, TNF-α, TGF-β, and IL-10 in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group.


( F ) Representative images of immunofluorescent staining for ly6G in peri - infarct areas of MCAO mice receiving indicated treatment . ( G ) Bar graph shows the number of ly6G + neutrophils . n = 6 mice per group .

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(F) Representative images of immunofluorescent staining for ly6G in peri-infarct areas of MCAO mice receiving indicated treatment. (G) Bar graph shows the number of ly6G+ neutrophils. n = 6 mice per group.


( H ) Representative images of immunofluorescent staining for Iba - 1 in peri - infarct areas of MCAO mice receiving indicated treatment . ( I ) Bar graph shows the number of Iba - 1 + microglia . n = 6 mice per group .

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(H) Representative images of immunofluorescent staining for Iba-1 in peri-infarct areas of MCAO mice receiving indicated treatment. (I) Bar graph shows the number of Iba-1+ microglia. n = 6 mice per group.


( B ) Neurological tests were performed to evaluate the motor , sensory , and balance functions in mice receiving A151 , or vehicle at days 1 , 3 , 7 , and 14 after MCAO . n = 10 mice per group .

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(B) Neurological tests were performed to evaluate the motor, sensory, and balance functions in mice receiving A151, or vehicle at days 1, 3, 7, and 14 after MCAO. n = 10 mice per group.


( C ) Representative TTC staining of brain sections from MCAO mice on day 3 after MCAO , the infarct area is shown in white .

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(C) Representative TTC staining of brain sections from MCAO mice on day 3 after MCAO, the infarct area is shown in white.


( D ) Bar graph shows percentages of infarct volume of indicated groups . n = 10 mice per group . *

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(D) Bar graph shows percentages of infarct volume of indicated groups. n = 10 mice per group. *


( E ) Flow cytometry plots and ( F ) summarized results show percentages of Annexin V + PI + cells in the brains of MCAO mice receiving A151 or vehicle . n = 6 mice per group .

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(E) Flow cytometry plots and (F) summarized results show percentages of Annexin V+ PI+ cells in the brains of MCAO mice receiving A151 or vehicle. n = 6 mice per group.


( G ) Representative histology images for TUNEL staining . ( H ) Bar gragh shows percentages of TUNEL - positive cells . n = 6 mice per group .

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(G) Representative histology images for TUNEL staining. (H) Bar gragh shows percentages of TUNEL-positive cells. n = 6 mice per group.


( A ) Representative double immunofluorescent stainings for Iba - 1 and cGAS , and for GFAP and cGAS . Scale bars , 10 μm .

(A) Representative double immunofluorescent stainings for Iba-1 and cGAS, and for GFAP and cGAS. Scale bars, 10 μm.


( D ) Representative genotyping analysis for WT , cGAS - KD , and cGAS - KO mice .

(D) Representative genotyping analysis for WT, cGAS-KD, and cGAS-KO mice.


( F ) Representative images of TTC staining of brain sections from vehicle - treated WT MCAO mice , vehicle - treated cGAS - KO mice , and A151 - treated cGAS - KO mice on day 3 after tMCAO . Three representative rostrocaudal levels of TTC staining are shown . ( G ) Bar graph shows percentages of infarct volume of indicated group . n = 6 mice per group . ( H ) Bar graph shows modified Neurological Severity Score ( mNSS ) in vehicle - treated WT MCAO mice , vehicle - treated cGAS - KO mice , and A151 - treated cGAS - KO mice n = 10 mice per group .

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(F) Representative images of TTC staining of brain sections from vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice on day 3 after tMCAO. Three representative rostrocaudal levels of TTC staining are shown. (G) Bar graph shows percentages of infarct volume of indicated group. n = 6 mice per group. (H) Bar graph shows modified Neurological Severity Score (mNSS) in vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice n = 10 mice per group.


( A ) Kinetics of cell numbers in Lineage - EPCR + CD150 + CD48 - ( L - ESLAM ) HSCs , Lineage - EPCR + CD150 + CD48 + ( CD150 + CD48 + ) progenitor cells , and Lineage - EPCR + CD150 - CD48 + ( CD150 - CD48 + ) progenitor cells after the administration of 5 - FU ( 250 mg / kg , i . v . ) . The small graph is an enlargement of 0 to 8 days after the administration of 5 - FU . ( * or * * ) in the small graph represents the statistical analysis of HSC between 3 and 4 or 7 days , respectively .

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(A) Kinetics of cell numbers in Lineage-EPCR+CD150+CD48- (L-ESLAM) HSCs, Lineage-EPCR+CD150+CD48+ (CD150+CD48+) progenitor cells, and Lineage-EPCR+CD150-CD48+ (CD150-CD48+) progenitor cells after the administration of 5-FU (250 mg/kg, i.v.). The small graph is an enlargement of 0 to 8 days after the administration of 5-FU. (* or **) in the small graph represents the statistical analysis of HSC between 3 and 4 or 7 days, respectively.


( A ) A principal component analysis ( PCA ) using the ATAC - seq data of HSCs in the steady state ( untreated ) , early phase ( 5 - FU 6d ) , or late phase ( 5 - FU 10d ) .

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(A) A principal component analysis (PCA) using the ATAC-seq data of HSCs in the steady state (untreated), early phase (5-FU 6d), or late phase (5-FU 10d).


mRNA ( C ) in Acly in HSCs after the administration of 5 - FU .

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mRNA (C) in Acly in HSCs after the administration of 5-FU.


protein ( D ) , and phosphorylation levels of Ser455 ( E ) in Acly in HSCs after the administration of 5 - FU .

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protein (D), and phosphorylation levels of Ser455 (E) in Acly in HSCs after the administration of 5-FU.


The levels of intracellular Glu ( G ) in HSCs obtained from untreated or 5 - FU - treated mice .

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The levels of intracellular Glu (G) in HSCs obtained from untreated or 5-FU-treated mice.


total acetylated lysine ( I ) in HSCs obtained from untreated or 5 - FU - treated mice .

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total acetylated lysine (I) in HSCs obtained from untreated or 5-FU-treated mice.


( B - C ) The effects of CTPI - 2 ( B ) or BMS303141 ( BMS ) ( C ) on H3K27ac in HSCs in the early phase after the administration of 5 - FU .

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(B-C) The effects of CTPI-2 (B) or BMS303141 (BMS) (C) on H3K27ac in HSCs in the early phase after the administration of 5-FU.


( D - E ) The effects of CTPI - 2 ( D ) , BMS303141 ( BMS ) ( E ) , A - 485 ( D ) , and TV - 3664 ( E ) on HSC expansion in the early phase after the administration of 5 - FU . Numbers in the dot plots represent the frequencies of the indicated fractions within total BM cells .

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(D-E) The effects of CTPI-2 (D), BMS303141 (BMS) (E), A-485 (D), and TV-3664 (E) on HSC expansion in the early phase after the administration of 5-FU. Numbers in the dot plots represent the frequencies of the indicated fractions within total BM cells.


Global H3K27ac ( A ) , Acly levels ( B ) in CD48 - HSCs and CD48 + progenitor cells ( HPCs ) in the late phase after the administration of 5 - FU .

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Global H3K27ac (A), Acly levels (B) in CD48- HSCs and CD48+ progenitor cells (HPCs)in the late phase after the administration of 5-FU.


acetylated lysine ( C ) , and ΔΨm ( D ) in CD48 - HSCs and CD48 + progenitor cells ( HPCs ) in the late phase after the administration of 5 - FU .

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acetylated lysine (C), and ΔΨm (D) in CD48- HSCs and CD48+ progenitor cells (HPCs)in the late phase after the administration of 5-FU.


( C ) The frequencies of the CD150 + CD48 - and CD48 + fractions within the lineage - EPCR + fraction after a 4 - day culture of HSCs in the late phase with or without BMS303141 ( BMS ; 100 μM ) , SB204990 ( SB ; 100 μM ) , an alternative Acly inhibitor , or CTPI - 2 ( 50 μM ) . ( D and E ) Total cell number ( D ) or CD48 + cell number ( E ) after the 4 - day culture of HSCs in the late phase with or without BMS303141 ( BMS ) , SB204990 ( SB ) , or CTPI - 2 . Numbers within the graph represent the inhibitory effects of each treatment ( fold decrease by an inhibitor treatment ) .

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(C) The frequencies of the CD150+CD48- and CD48+ fractions within the lineage-EPCR+ fraction after a 4-day culture of HSCs in the late phase with or without BMS303141 (BMS; 100 μM), SB204990 (SB; 100 μM), an alternative Acly inhibitor, or CTPI-2 (50 μM). (D and E) Total cell number (D) or CD48+ cell number (E) after the 4-day culture of HSCs in the late phase with or without BMS303141 (BMS), SB204990 (SB), or CTPI-2. Numbers within the graph represent the inhibitory effects of each treatment (fold decrease by an inhibitor treatment).


( a ) Mitochondrial tubules in HeLa cells ( top , TMRE stained ) exceeded the dimensions of that for autophagosomes ( labelled by EGFP - LC3B ) under either normal ( middle , same field as TMRE image ) or starvation conditions ( right ) .

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(a) Mitochondrial tubules in HeLa cells (top, TMRE stained) exceeded the dimensions of that for autophagosomes (labelled by EGFP-LC3B) under either normal (middle, same field as TMRE image) or starvation conditions (right).


( c ) Illuminating a selective region ( white dotted circle , top ) within a KR - dMito / EBFP2 - parkin - expressing HeLa cell led to the immediate loss of KR fluorescence ( top two panels ) . Third to fifth row : magnified view of the photo - impaired region , EBFP2 - parkin translocated onto impaired mitochondrial tubules synchronously ( 38 - 60 min after 559 illumination , white arrows ) . Scale bars ( all panels ) , 10 μm .

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(c) Illuminating a selective region (white dotted circle, top) within a KR-dMito/EBFP2-parkin-expressing HeLa cell led to the immediate loss of KR fluorescence (top two panels). Third to fifth row: magnified view of the photo-impaired region, EBFP2-parkin translocated onto impaired mitochondrial tubules synchronously (38-60 min after 559 illumination, white arrows). Scale bars (all panels), 10 μm.


( a ) A HeLa cell expressing KR - dMito , EGFP - LC3B and EBFP2 - parkin ( cyan ) were 559 nm illuminated ( white dotted circle region ; immediate loss of KR fluorescence ) . ( b - d ) Magnified view of the photo - inactivated region in a 34 - 93 min after 559 nm illumination . ( b ) Induction of parkin - mediated mitophagy on a single , long mitochondria tubule ( white arrows ) . ( c ) LC3B - coated structures were recruited onto distinct spots on the photo - impaired mitochondrial tubule ( white arrows : the location of the original impaired single mitochondrial tubule ) . ( d ) White dotted line region outlined in c , LC3 structures segregated from the main parkin - labelled cluster , carrying away bits of the parkin - labelled mitochondria with them . Two individual events are labelled in the image sequence with white and yellow arrows , respectively . Scale bars ( all panels ) , 5 μm .

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(a) A HeLa cell expressing KR-dMito, EGFP-LC3B and EBFP2-parkin (cyan) were 559 nm illuminated (white dotted circle region; immediate loss of KR fluorescence). (b-d) Magnified view of the photo-inactivated region in a 34-93 min after 559 nm illumination. (b) Induction of parkin-mediated mitophagy on a single, long mitochondria tubule (white arrows). (c) LC3B-coated structures were recruited onto distinct spots on the photo-impaired mitochondrial tubule (white arrows: the location of the original impaired single mitochondrial tubule). (d) White dotted line region outlined in c, LC3 structures segregated from the main parkin-labelled cluster, carrying away bits of the parkin-labelled mitochondria with them. Two individual events are labelled in the image sequence with white and yellow arrows, respectively. Scale bars (all panels), 5 μm.


( a ) A HeLa cell expressing KR - dMito ( red ) , EGFP - DFCP1 ( green ) and EBFP2 - parkin ( cyan ) were illuminated with 559 nm at the white dotted circle region , leading to the generation of a long parkin - labelled mitochondria tubule ( right , magnified view of the light - activated region ) for autophagic turnover ( white arrow ) . Scale bars , 10 μm .

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(a) A HeLa cell expressing KR-dMito (red), EGFP-DFCP1 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted circle region, leading to the generation of a long parkin-labelled mitochondria tubule (right, magnified view of the light-activated region) for autophagic turnover (white arrow). Scale bars, 10 μm.


( b ) After the generation of parkin - labelled mitochondrial tubules following treatments in a , discrete DFCP1 foci ( green , middle panel , ER - associated omegasomes ) formed along the parkin - labelled mitochondria ( white arrows indicate DFCP1 spots that colocalized with parkin ) . Bottom panel : merged image of parkin and DFCP1 . Scale bar , 10 μm .

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(b) After the generation of parkin-labelled mitochondrial tubules following treatments in a, discrete DFCP1 foci (green, middle panel, ER-associated omegasomes) formed along the parkin-labelled mitochondria (white arrows indicate DFCP1 spots that colocalized with parkin). Bottom panel: merged image of parkin and DFCP1. Scale bar, 10 μm.


( c ) Time - lapse imaging showing DCFP1 puncta formation on parkin - labelled mitochondria . A HeLa cell expressing KR - dMito , EGFP - DFCP1 ( green ) and EBFP2 - parkin ( pseudo - coloured red ) were 559 nm illuminated , leading to the generation of parkin - labelled mitochondria tubules . Time - lapse imaging ( 1 frame per min ) revealed that DFCP1 puncta ( green ) formed transiently at discrete sites on parkin - labelled mitochondrial tubules ( red ) . White arrows mark two DFCP1 puncta ( ER - associated omegasomes ) formation / disassembly events directly on parkin - labelled tubules . Scale bar , 5 μm .

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(c) Time-lapse imaging showing DCFP1 puncta formation on parkin-labelled mitochondria. A HeLa cell expressing KR-dMito, EGFP-DFCP1 (green) and EBFP2-parkin (pseudo-coloured red) were 559 nm illuminated, leading to the generation of parkin-labelled mitochondria tubules. Time-lapse imaging (1 frame per min) revealed that DFCP1 puncta (green) formed transiently at discrete sites on parkin-labelled mitochondrial tubules (red). White arrows mark two DFCP1 puncta (ER-associated omegasomes) formation/disassembly events directly on parkin-labelled tubules. Scale bar, 5 μm.


( d ) A COS - 7 cell expressing EBFP2 - parkin ( cyan ) , AcGFP - sec61β ( green ) and TagRFP - DFCP1 ( red ) were treated with 10 μM CCCP for 4 h and imaged . Omegasomes ( DFCP1 ) appeared where ER ( sec61β ) and impaired mitochondria ( parkin ) overlap . Scale bar , 10 μm .

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(d) A COS-7 cell expressing EBFP2-parkin (cyan), AcGFP-sec61β (green) and TagRFP-DFCP1 (red) were treated with 10 μM CCCP for 4 h and imaged. Omegasomes (DFCP1) appeared where ER (sec61β) and impaired mitochondria (parkin) overlap. Scale bar, 10 μm.


( e ) Histogram of the lifetime of individual DFCP1 foci on parkin - labelled mitochondria in CCCP - treated HeLa cells expressing TagRFP - DFCP1 , EGFP - LC3B and EBFP2 - parkin .

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(e) Histogram of the lifetime of individual DFCP1 foci on parkin-labelled mitochondria in CCCP-treated HeLa cells expressing TagRFP-DFCP1, EGFP-LC3B and EBFP2-parkin.


( f ) Wortmannin treatments delayed the initial LC3 recruitment times onto parkin - labelled mitochondria in HeLa cells expressing KR - dMito , EGFP - LC3B and EBFP2 - parkin . Each ' + ' marks the times measured in individual HeLa cells ( the red ' □ ' ( square ) denotes the average from five cells ) . P = 0 . 0172 ( Student ' s t - test ) .

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(f) Wortmannin treatments delayed the initial LC3 recruitment times onto parkin-labelled mitochondria in HeLa cells expressing KR-dMito, EGFP-LC3B and EBFP2-parkin. Each '+' marks the times measured in individual HeLa cells (the red '□' (square) denotes the average from five cells). P=0.0172 (Student's t-test).


( a ) A COS - 7 cell expressing EBFP2 - Ub ( red ) , AcGFP - sec61β ( green ) and mcherry - parkin ( cyan ) was treated with 10 μM CCCP for 1 h and imaged . Impaired mitochondria ( parkin ) displayed preferentially ubiquitinated foci ( white arrows ) at selected regions that overlap with the ER ( sec61β ) .

(a) A COS-7 cell expressing EBFP2-Ub (red), AcGFP-sec61β (green) and mcherry-parkin (cyan) was treated with 10 μM CCCP for 1 h and imaged. Impaired mitochondria (parkin) displayed preferentially ubiquitinated foci (white arrows) at selected regions that overlap with the ER (sec61β).


( b ) HeLa cells expressing KR - dMito ( red ) , EGFP - LC3 ( green ) and EBFP2 - parkin ( cyan ) were illuminated with 559 nm at the white dotted square regions , generating parkin - labelled mitochondria ( bottom right , magnified view of the light - activated regions ) . ( c ) Magnified view of the photo - inactivated region in b 80 min after illumination ( post - stained with anti - ubiquitin antibody ) . LC3 and ubiquitin colocalized at discrete sites along parkin - labelled mitochondria ( white arrows ) .

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(b) HeLa cells expressing KR-dMito (red), EGFP-LC3 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted square regions, generating parkin-labelled mitochondria (bottom right, magnified view of the light-activated regions). (c) Magnified view of the photo-inactivated region in b 80 min after illumination (post-stained with anti-ubiquitin antibody). LC3 and ubiquitin colocalized at discrete sites along parkin-labelled mitochondria (white arrows).


( d ) A MG - 132 treated HeLa cell expressing KR - dMito , EBFP2 - parkin ( green ) and EGFP - LC3B ( red ) was 559 nm illuminated , leading to the appearance of parkin - labelled mitochondria ( white arrows one to three indicated structures , top panels ) . The parkin - labelled structures did not fragment nor became engulfed by LC3 structures ( bottom panels ) in the presence MG - 132 . Scale bars ( all panels ) , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) A MG-132 treated HeLa cell expressing KR-dMito, EBFP2-parkin (green) and EGFP-LC3B (red) was 559 nm illuminated, leading to the appearance of parkin-labelled mitochondria (white arrows one to three indicated structures, top panels). The parkin-labelled structures did not fragment nor became engulfed by LC3 structures (bottom panels) in the presence MG-132. Scale bars (all panels), 5 μm.


B - Western blot verifying the expression of the CTD variants RPB1 - 52R and RPB1 - 25R in U2OS cells in whole cell extracts . U1 snRNP was used as a loading control .

B- Western blot verifying the expression of the CTD variants RPB1-52R and RPB1-25R in U2OS cells in whole cell extracts. U1 snRNP was used as a loading control.


C - Changes in RNA synthesis upon CTD shortening . MA plot showing RNA synthesis changes in TT - seq datasets upon CTD shortening in U2OS cells in steady state conditions . RPB1 - 52R cells are used as a control and the data was normalized using spike - in counts . 16214 expressed genes annotated in RefSeq were analyzed . Differentially expressed genes are in red . 473 genes were significantly upregulated ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) and 704 genes were significantly downregulated ( adjusted p value < 0 . 05 , log2 fold change ≤ 1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C- Changes in RNA synthesis upon CTD shortening. MA plot showing RNA synthesis changes in TT-seq datasets upon CTD shortening in U2OS cells in steady state conditions. RPB1-52R cells are used as a control and the data was normalized using spike-in counts. 16214 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red. 473 genes were significantly upregulated (adjusted p value < 0.05, log2 fold change >=1) and 704 genes were significantly downregulated (adjusted p value < 0.05, log2 fold change ≤1).


D - Box plot of estimated RNA synthesis rates of expressed RefSeq transcripts ( 12014 transcripts ) based on TT - seq and RNA - seq datasets in RPB1 - 52R and RPB1 - 25R cells . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D- Box plot of estimated RNA synthesis rates of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


E - Box plot of estimated RNA degradation rates of expressed RefSeq transcripts ( 12014 transcripts ) based on TT - seq and RNA - seq datasets in RPB1 - 52R and RPB1 - 25R cells . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range .

E- Box plot of estimated RNA degradation rates of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


F - Box plot of estimated RNA half - lives of expressed RefSeq transcripts ( 12014 transcripts ) based on TT - seq and RNA - seq datasets in RPB1 - 52R and RPB1 - 25R cells . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F- Box plot of estimated RNA half-lives of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


A - Box plots showing ratios of spliced TT - seq reads over total unspliced and spliced TT - seq reads for all constitutive 5 ' SSs and 3 ' SSs detected in major RNA isoforms in RPB1 - 52R and RPB1 - 25R cells . p value was calculated using Mann - Whitney U test . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed . B - D - Box plots showing ratios of spliced TT - seq reads over total unspliced and spliced TT - seq reads in RPB1 - 52R and RPB1 - 25R cells for all constitutive 5 ' SSs and 3 ' SSs detected in major RNA isoforms based on intron position in the transcript : ( B ) first intron junction , ( C ) intermediate intron junctions and ( D ) last intron junctions . p values were calculated using Mann - Whitney U test . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A- Box plots showing ratios of spliced TT-seq reads over total unspliced and spliced TT-seq reads for all constitutive 5'SSs and 3'SSs detected in major RNA isoforms in RPB1-52R and RPB1-25R cells. p value was calculated using Mann-Whitney U test. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. B-D- Box plots showing ratios of spliced TT-seq reads over total unspliced and spliced TT-seq reads in RPB1-52R and RPB1-25R cells for all constitutive 5'SSs and 3'SSs detected in major RNA isoforms based on intron position in the transcript: (B) first intron junction, (C) intermediate intron junctions and (D) last intron junctions. p values were calculated using Mann-Whitney U test. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


E - Differential mRNA isoform usage upon CTD shortening . Scatter plot showing read counts for mRNA isoforms detected in the chromatin fraction of RPB1 - 52R and RPB1 - 25R cells using long read sequencing . 47416 isoforms were detected , RPB1 - 52R condition was used as a control . 335 isoforms show differential expression ( p value < 0 . 05 , Fischer ' s exact test ) .

E- Differential mRNA isoform usage upon CTD shortening. Scatter plot showing read counts for mRNA isoforms detected in the chromatin fraction of RPB1-52R and RPB1-25R cells using long read sequencing. 47416 isoforms were detected, RPB1-52R condition was used as a control. 335 isoforms show differential expression (p value < 0.05, Fischer's exact test).


A - CTD shortening leads to a decreased Pol II occupancy in promoter - proximal region . Median coverage plot of PRO - seq signal at genes encoding major isoforms . Solid lines represent median signal and shaded area refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A- CTD shortening leads to a decreased Pol II occupancy in promoter-proximal region. Median coverage plot of PRO-seq signal at genes encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.


B , C - Pol II pause position remains unchanged upon CTD shortening . Histogram of estimated positions of paused Pol II in RPB1 - 52R and RPB1 - 25R cells at genes encoding major isoforms with constitutive first exon longer than 100 bp .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B,C- Pol II pause position remains unchanged upon CTD shortening. Histogram of estimated positions of paused Pol II in RPB1-52R and RPB1-25R cells at genes encoding major isoforms with constitutive first exon longer than 100 bp.


D - Median coverage plot of TT - seq signal at genes encoding major isoforms in RPB1 - 52R and RPB1 - 25R cells . Solid lines represent median signal and shaded area refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D- Median coverage plot of TT-seq signal at genes encoding major isoforms in RPB1-52R and RPB1-25R cells. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.


E - Box plot showing productive transcription initiation rate in RPB1 - 52R and RPB1 - 25R cells for genes encoding major isoforms . p value = 0 . 13 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed .

E- Box plot showing productive transcription initiation rate in RPB1-52R and RPB1-25R cells for genes encoding major isoforms. p value=0.13 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


F - Box plot showing Pol II pausing duration in RPB1 - 52R and RPB1 - 25R cells for genes encoding major isoforms with constitutive first exon longer than 100 bp . p value < 2 . 2e - 16 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F- Box plot showing Pol II pausing duration in RPB1-52R and RPB1-25R cells for genes encoding major isoforms with constitutive first exon longer than 100 bp. p value<2.2e-16 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


G - Median scaled coverage plot of TT - seq signal at genes encoding major isoforms . Solid lines represent median signal and shaded area refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G- Median scaled coverage plot of TT-seq signal at genes encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.


H - Pol II velocity remains unchanged upon CTD shortening . Metagene profile of transcription elongation velocity at 4480 genes longer than 10 kb encoding major isoforms . Solid lines represent median signal and shaded area refer to 95 % bootstrap confidence intervals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H- Pol II velocity remains unchanged upon CTD shortening. Metagene profile of transcription elongation velocity at 4480 genes longer than 10 kb encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals.


I - Median coverage plot of TT - seq signal centered at PAS at genes encoding major isoforms in RPB1 - 52R and RPB1 - 25R cells . Solid lines represent median signal and shaded area refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I- Median coverage plot of TT-seq signal centered at PAS at genes encoding major isoforms in RPB1-52R and RPB1-25R cells. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.


J - Box plot showing length of the poly ( A ) tail in RPB1 - 52R and RPB1 - 25R cells measured using Nanopore sequening data . p value < 2 . 2e - 16 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J - Box plot showing length of the poly(A) tail in RPB1-52R and RPB1-25R cells measured using Nanopore sequening data. p value<2.2e-16 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


K - Box plot showing distance from the polyadenylation site ( PAS ) to the transcription termination site ( TTS ) in RPB1 - 52R and RPB1 - 25R cells for 1958 genes encoding major isoforms . p value = 0 . 8 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K- Box plot showing distance from the polyadenylation site (PAS) to the transcription termination site (TTS) in RPB1-52R and RPB1-25R cells for 1958 genes encoding major isoforms. p value= 0.8 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


B - Box plots showing RNA synthesis levels ( RPKs ) of putative eRNAs annotated using TT - seq data in RPB1 - 52R and RPB1 - 25R cells . p value = 9 . 27e - 103 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B- Box plots showing RNA synthesis levels (RPKs) of putative eRNAs annotated using TT-seq data in RPB1-52R and RPB1-25R cells. p value =9.27e-103 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


C - Box plots showing lengths of putative eRNAs annotated using TT - seq data in RPB1 - 52R and RPB1 - 25R cells . p value = 0 . 000519 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C- Box plots showing lengths of putative eRNAs annotated using TT-seq data in RPB1-52R and RPB1-25R cells. p value =0.000519 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


D - Histogram showing a distribution of number of putative eRNAs paired to genes in RPB1 - 52R and RPB1 - 25R cells .

O O O O O O O O O O O O O O O O O O O O O O O O

D - Histogram showing a distribution of number of putative eRNAs paired to genes in RPB1-52R and RPB1-25R cells.


A - Changes in RNA synthesis in response to 15 min of TPA treatment . MA plot showing RNA synthesis changes in TT - seq datasets upon 15 min treatment with TPA ( 200 nM ) in RPB1 - 52R and RPB1 - 25R cells . DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization . 14636 expressed genes annotated in RefSeq were analyzed . Differentially expressed genes are in red ( adjusted p value < 0 . 05 , log2 fold change > = 1 for upregulated genes and adjusted p value < 0 . 05 , log2 fold change ≤ 1 for downregulated genes ) . B - Box plots showing log2 fold change of genes significantly upregulated upon 15 min treatment with 200 nM TPA in RPB1 - 52R and RPB1 - 25R cells ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) . P value = 0 . 02257 ( Mann - Whitney test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed . C - Changes in RNA synthesis in response to 30 min of TPA treatment . MA plot showing RNA synthesis changes in TT - seq datasets upon 30 min treatment with TPA ( 200 nM ) in RPB1 - 52R and RPB1 - 25R cells . DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization . 14636 expressed genes annotated in RefSeq were analyzed . Differentially expressed genes are in red ( adjusted p value < 0 . 05 , log2 fold change > = 1 for upregulated genes and adjusted p value < 0 . 05 , log2 fold change ≤ 1 for downregulated genes ) D - Box plots showing log2 fold change in RNA synthesis of genes significantly upregulated upon 30 min treatment with 200 nM TPA in RPB1 - 52R and RPB1 - 25R cells ( adjusted p value < 0 . 05 , log2 fold chage > = 1 ) . P value = 0 . 001749 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A- Changes in RNA synthesis in response to 15 min of TPA treatment. MA plot showing RNA synthesis changes in TT-seq datasets upon 15 min treatment with TPA (200 nM) in RPB1-52R and RPB1-25R cells. DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization. 14636 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red (adjusted p value < 0.05, log2 fold change >=1 for upregulated genes and adjusted p value < 0.05, log2 fold change ≤1 for downregulated genes ). B- Box plots showing log2 fold change of genes significantly upregulated upon 15 min treatment with 200 nM TPA in RPB1-52R and RPB1-25R cells (adjusted p value < 0.05, log2 fold change >=1). P value =0.02257 (Mann-Whitney test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. C- Changes in RNA synthesis in response to 30 min of TPA treatment. MA plot showing RNA synthesis changes in TT-seq datasets upon 30 min treatment with TPA (200 nM) in RPB1-52R and RPB1-25R cells. DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization. 14636 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red (adjusted p value < 0.05, log2 fold change >=1 for upregulated genes and adjusted p value < 0.05, log2 fold change ≤1 for downregulated genes) D- Box plots showing log2 fold change in RNA synthesis of genes significantly upregulated upon 30 min treatment with 200 nM TPA in RPB1-52R and RPB1-25R cells (adjusted p value < 0.05, log2 fold chage >=1). P value =0.001749 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


A - Histogram showing the distribution of a number of putative eRNAs paired to genes in RPB1 - 52R and RPB1 - 25R cells upon 15 and 30 min TPA treatment ( 200 nM ) as well as in the DMSO controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

A- Histogram showing the distribution of a number of putative eRNAs paired to genes in RPB1-52R and RPB1-25R cells upon 15 and 30 min TPA treatment (200 nM) as well as in the DMSO controls.


B - Box plots showing RNA synthesis levels ( RPK ) of putative eRNAs paired with genes upregulated ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) in RPB1 - 52R and RPB1 - 25R cells upon 15 min of TPA treatment ( 200 nM ) . p value = 0 . 000732 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed . C - Box plots showing RNA synthesis levels ( RPK ) of putative enhancer eRNAs paired with genes upregulated ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) in RPB1 - 52R and RPB1 - 25R cells upon 30 min of TPA treatment ( 200 nM ) . p value = 2 . 77e - 5 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed .

B- Box plots showing RNA synthesis levels (RPK) of putative eRNAs paired with genes upregulated (adjusted p value < 0.05, log2 fold change >=1) in RPB1-52R and RPB1-25R cells upon 15 min of TPA treatment (200 nM). p value = 0.000732 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. C- Box plots showing RNA synthesis levels (RPK) of putative enhancer eRNAs paired with genes upregulated (adjusted p value < 0.05, log2 fold change >=1) in RPB1-52R and RPB1-25R cells upon 30 min of TPA treatment (200 nM). p value = 2.77e-5 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


The Dyf phenotype of the ts - che - 3 mutant was confirmed , with animals grown at the permissive temperature ( 15ºC ) showing the ability to uptake dye , while those raised at the restrictive temperature ( 25ºC ) are Dyf . Scale bar , 20

The Dyf phenotype of the ts-che-3 mutant was confirmed, with animals grown at the permissive temperature (15ºC) showing the ability to uptake dye, while those raised at the restrictive temperature (25ºC) are Dyf. Scale bar, 20 µm


Using the homodimeric kinesin OSM - 3 as a marker for IFT , proper localisation is seen in the ts - che - 3 mutant at the permissive temperature , with the strongest signal at the basal body ( bb ) . At the restrictive temperature , most of the OSM - 3 signal is found within the axoneme of the short , bulbous cilia . Scale bar is 4

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Using the homodimeric kinesin OSM-3 as a marker for IFT, proper localisation is seen in the ts-che-3 mutant at the permissive temperature, with the strongest signal at the basal body (bb). At the restrictive temperature, most of the OSM-3 signal is found within the axoneme of the short, bulbous cilia. Scale bar is 4 µm


The ts - che - 3 mutant shows normal osmotic avoidance at permissive temperature but not at the restrictive temperature , indicating defective chemosensation . The osm - 9 mutant is included as a positive control . n = 50 animals ; error bars are standard

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The ts-che-3 mutant shows normal osmotic avoidance at permissive temperature but not at the restrictive temperature, indicating defective chemosensation. The osm-9 mutant is included as a positive control. n = 50 animals; error bars are standard error


Three TZ markers ( NPHP - 4 , CEP - 290 and MKS - 6 ) are shown at both the permissive ( 15ºC ) and restrictive ( 25ºC ) temperatures in the ts - che - 3 mutant . At the permissive temperature , all three markers localise to the TZ , while at the restrictive temperature exhibit ectopic localisation ( leakage ) distally into the ciliary axoneme . While NPHP - 4 and MKS - 6 show significant accumulations , CEP - 290 is grossly normal . At the permissive temperature , both IFT co - markers ( CHE - 13 and XBX - 1 ) display strong localisation to the basal body ( bb ) and along the axoneme . At the restrictive temperature , the IFT markers show accumulation in the axoneme . Accumulations emphasized using

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Three TZ markers (NPHP-4, CEP-290 and MKS-6) are shown at both the permissive (15ºC) and restrictive (25ºC) temperatures in the ts-che-3 mutant. At the permissive temperature, all three markers localise to the TZ, while at the restrictive temperature exhibit ectopic localisation (leakage) distally into the ciliary axoneme. While NPHP-4 and MKS-6 show significant accumulations, CEP-290 is grossly normal. At the permissive temperature, both IFT co-markers (CHE-13 and XBX-1) display strong localisation to the basal body (bb) and along the axoneme. At the restrictive temperature, the IFT markers show accumulation in the axoneme. Accumulations emphasized using asterisks


To determine if ciliary compartmentalisation could be restored , young ts - che - 3 mutant adults were transferred to the permissive temperature after being raised at the restrictive temperature . The TZ proteins reassembled at the TZ , with loss of the ectopic accumulations , and IFT protein localisation was also restored - strongest at the basal body , similar to animals grown at the permissive temperature . ( c ) After 24 hours at the restrictive temperature , TZ proteins appear to begin to accumulate distally within the axoneme in ts - che - 3 mutants . This is seen , in small amounts , for NPHP - 4 and CEP - 290 but with stronger ectopic localisation for MKS - 6 . The IFT proteins CHE - 13 and XBX - 1 also show accumulations in the axoneme after 24 hours at the restrictive temperature . Data information : For ( A ) , ( B ) , and ( C ) , fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right . Each point in the plot represents one pixel along the centre of the basal body ( BB ) , transition zone ( TZ ) and middle segment ( MS ) regions . Dotted areas ( three pixels ) in the MS were used to quantify ectopic localisation ( TZ markers ) or accumulation ( IFT markers ) for statistical analyses . n = 10 cilia ( 4 - 7 animals ) , Kruskal - Wallis test , * P < 0 . 05 , * * P < 0 . 01 . tz , transition zone ; bb , basal body ; scale bars are 4

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

To determine if ciliary compartmentalisation could be restored, young ts-che-3 mutant adults were transferred to the permissive temperature after being raised at the restrictive temperature. The TZ proteins reassembled at the TZ, with loss of the ectopic accumulations, and IFT protein localisation was also restored-strongest at the basal body, similar to animals grown at the permissive temperature. (c) After 24 hours at the restrictive temperature, TZ proteins appear to begin to accumulate distally within the axoneme in ts-che-3 mutants. This is seen, in small amounts, for NPHP-4 and CEP-290 but with stronger ectopic localisation for MKS-6. The IFT proteins CHE-13 and XBX-1 also show accumulations in the axoneme after 24 hours at the restrictive temperature. Data information: For (A), (B), and (C), fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (TZ markers) or accumulation (IFT markers) for statistical analyses. n = 10 cilia (4-7 animals), Kruskal-Wallis test, *P<0.05, **P<0.01. tz, transition zone; bb, basal body; scale bars are 4 µm


In the ts - che - 3 mutant grown at the permissive temperature ( 15ºC ) , both RPI - 2 / RP2 and TRAM - 1 / TRAM1 are localised to the PCMC , just proximal to the ciliary axoneme . At the restrictive temperature , TRAM - 1 shows leakage into the axoneme with its strongest localisation at the PCMC . Intriguingly , RPI - 2 now displays strong localisation to the axoneme , indicating that in the absence of IFT , RPI - 2 may be targeted into the cilium . When ts - che - 3 mutant animals grown at the restrictive temperature ( 25ºC ) are shifted at young adult to the permissive ( 15ºC ) for 24 hours , both TRAM - 1 and RPI - 2 exhibit restoration of their PCMC localisation . Consistent with other TZ markers , both MKS - 2 and MKSR - 1 show ectopic localisation in the axoneme when grown at the restrictive temperature compared to the permissive temperature . Also consistent with what was observed for other markers , after 24 hours at the permissive temperature , animals grown at the restrictive temperature show loss of the ectopic localisation . Fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right . Each point in the plot represents one pixel along the centre of the basal body ( BB ) , transition zone ( TZ ) and middle segment ( MS ) regions . Dotted areas ( three pixels ) in the MS were used to quantify ectopic localisation ( MKS - 2 and MKSR - 1 ) or accumulation ( RPI - 2 and TRAM - 1 ) for statistical analyses . n = 10 cilia ( 4 - 6 animals ) , Kruskal - Wallis test , * P < 0 . 05 , * * P < 0 . 01 ; scale bars are 4

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In the ts-che-3 mutant grown at the permissive temperature (15ºC), both RPI-2/RP2 and TRAM-1/TRAM1 are localised to the PCMC, just proximal to the ciliary axoneme. At the restrictive temperature, TRAM-1 shows leakage into the axoneme with its strongest localisation at the PCMC. Intriguingly, RPI-2 now displays strong localisation to the axoneme, indicating that in the absence of IFT, RPI-2 may be targeted into the cilium. When ts-che-3 mutant animals grown at the restrictive temperature (25ºC) are shifted at young adult to the permissive (15ºC) for 24 hours, both TRAM-1 and RPI-2 exhibit restoration of their PCMC localisation. Consistent with other TZ markers, both MKS-2 and MKSR-1 show ectopic localisation in the axoneme when grown at the restrictive temperature compared to the permissive temperature. Also consistent with what was observed for other markers, after 24 hours at the permissive temperature, animals grown at the restrictive temperature show loss of the ectopic localisation. Fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (MKS-2 and MKSR-1) or accumulation (RPI-2 and TRAM-1) for statistical analyses. n = 10 cilia (4-6 animals), Kruskal-Wallis test, *P<0.05, **P<0.01; scale bars are 4 µm


Loss of CHE - 3 function results in short , bulbous cilia containing electron dense accumulations and abnormal membrane - microtubule connections . Shown are transmission electron micrograph ( TEM ) cross sections of an amphid channel cilium in wild - type and che - 3 ( e1124 ) null mutant animals . Representative images of wild - type cilia show intact middle segment ( containing doublet microtubules ) and transition zone ( with Y - shaped links ) ( left top and bottom panels ) regions . Representative images of che - 3 ( e1124 ) cilia reveal apparently intact transition zones , with visible Y - link structures , but enlarged ciliary ends filled with electron - dense accumulations , which often appear vesicular . The bulbous structures reveal doublet microtubules associated with the membrane , and occasionally ectopic microtubule - to - membrane connections , which sometimes appear similar to transition zone Y - links in the region just distal to the seemingly " normal " TZ structure . Schematics show longitudinal ( left images ) and cross section ( right images ) representations of wild - type and che - 3 ( e1124 ) cilia . tz , transition zone ; pcmc , periciliary membrane compartment ; scale bars are as indicated in ( nm )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Loss of CHE-3 function results in short, bulbous cilia containing electron dense accumulations and abnormal membrane-microtubule connections. Shown are transmission electron micrograph (TEM) cross sections of an amphid channel cilium in wild-type and che-3(e1124) null mutant animals. Representative images of wild-type cilia show intact middle segment (containing doublet microtubules) and transition zone (with Y-shaped links) (left top and bottom panels) regions. Representative images of che-3(e1124) cilia reveal apparently intact transition zones, with visible Y-link structures, but enlarged ciliary ends filled with electron-dense accumulations, which often appear vesicular. The bulbous structures reveal doublet microtubules associated with the membrane, and occasionally ectopic microtubule-to-membrane connections, which sometimes appear similar to transition zone Y-links in the region just distal to the seemingly "normal" TZ structure. Schematics show longitudinal (left images) and cross section (right images) representations of wild-type and che-3(e1124) cilia. tz, transition zone; pcmc, periciliary membrane compartment; scale bars are as indicated in (nm) for


Using the IFT marker BBS - 7 , cilia lengths were measured for wild - type and the ts - che - 3 mutant animals raised at the permissive ( 15ºC ) and shifted to the restrictive temperature ( 25ºC ) for three time points . After 12 hours some accumulations ( indicated by asterisks in the fluorescence images ) are observed within the cilia but there is not a significant reduction in cilium length . After 18 and 24 hours , significant reduction in ciliary length is observed and large accumulations of BBS - 7 is seen within short bulbous cilia , similar to what is observed in ts - che - 3 animals raised at the restrictive temperature . n = 30 - 34 cilia , one per animal ; Kruskal - Wallis test , * * P < 0 . 01 ; error bars are mean absolute deviation ; scale bars are 4

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Using the IFT marker BBS-7, cilia lengths were measured for wild-type and the ts-che-3 mutant animals raised at the permissive (15ºC) and shifted to the restrictive temperature (25ºC) for three time points. After 12 hours some accumulations (indicated by asterisks in the fluorescence images) are observed within the cilia but there is not a significant reduction in cilium length. After 18 and 24 hours, significant reduction in ciliary length is observed and large accumulations of BBS-7 is seen within short bulbous cilia, similar to what is observed in ts-che-3 animals raised at the restrictive temperature. n = 30-34 cilia, one per animal; Kruskal-Wallis test, **P<0.01; error bars are mean absolute deviation; scale bars are 4 µm


IFT velocities were measured for wild - type or ts - che - 3 mutant animals ( grown at permissive temperature ) and imaged at the permissive temperature , restrictive temperature or after 3 hours at the restrictive temperature . Compared to wild - type , anterograde velocities in the ts - che - 3 dynein mutant are similar , while IFT retrograde rates are highly reduced for both the middle and distal ciliary segments . Interestingly , the ts - che - 3 IFT dynein mutant appears to not only have slow retrograde IFT trains , but the trains also appear much larger and less frequent . Initially upon the temperature shift ( < 15min ) IFT rates are much faster for both wild - type and ts - che - 3 dynein mutant , although retrograde remains much slower in the mutant . After 3 hours at the restrictive temperature , IFT velocities are intermediate between the rates seen at 15ºC and those observed at the initial shift to

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IFT velocities were measured for wild-type or ts-che-3 mutant animals (grown at permissive temperature) and imaged at the permissive temperature, restrictive temperature or after 3 hours at the restrictive temperature. Compared to wild-type, anterograde velocities in the ts-che-3 dynein mutant are similar, while IFT retrograde rates are highly reduced for both the middle and distal ciliary segments. Interestingly, the ts-che-3 IFT dynein mutant appears to not only have slow retrograde IFT trains, but the trains also appear much larger and less frequent. Initially upon the temperature shift (<15min) IFT rates are much faster for both wild-type and ts-che-3 dynein mutant, although retrograde remains much slower in the mutant. After 3 hours at the restrictive temperature, IFT velocities are intermediate between the rates seen at 15ºC and those observed at the initial shift to 25ºC


To assess the effects of IFT restoration on TZ assembly , ts - che - 3 mutant animals were shifted from the restrictive to the permissive temperature at different time points during their development ( days 1 , 4 and 8 of adulthood ) . The TZ markers MKS - 6 ( a ) and NPHP - 4 ( b ) show strong ectopic ciliary accumulations when raised at the restrictive temperature until day 1 of adulthood . After a shift to the permissive temperature for 24 hours , day 2 adults show " repaired " TZ protein localisation resembling that of wild - type animals . For both markers , Day 4 adults ( maintained at the restrictive temperature ) show less ectopic TZ protein localisation than Day 1 adults . The correction of ectopic localisation following shift to the permissive temperature for 24 hours was reduced in day 4 ( to day 5 ) of adulthood compared to that observed in day 1 ( to day 2 ) adults . For MKS - 6 , but not NPHP - 4 , day 8 adults ( maintained at the restrictive temperature ) also display less ectopic accumulation than day 1 adults . The correction of ectopic TZ protein localisation in animals shifted to the permissive temperature after 8 days of adulthood is also much smaller in magnitude for both markers , indicating that this repair is most effective in day 1 adults . In summary , as the adults age , ectopic TZ protein localisation is reduced , while the ability to correct this phenotype by restoring IFT function

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

To assess the effects of IFT restoration on TZ assembly, ts-che-3 mutant animals were shifted from the restrictive to the permissive temperature at different time points during their development (days 1, 4 and 8 of adulthood). The TZ markers MKS-6 (a) and NPHP-4 (b) show strong ectopic ciliary accumulations when raised at the restrictive temperature until day 1 of adulthood. After a shift to the permissive temperature for 24 hours, day 2 adults show "repaired" TZ protein localisation resembling that of wild-type animals. For both markers, Day 4 adults (maintained at the restrictive temperature) show less ectopic TZ protein localisation than Day 1 adults. The correction of ectopic localisation following shift to the permissive temperature for 24 hours was reduced in day 4 (to day 5) of adulthood compared to that observed in day 1 (to day 2) adults. For MKS-6, but not NPHP-4, day 8 adults (maintained at the restrictive temperature) also display less ectopic accumulation than day 1 adults. The correction of ectopic TZ protein localisation in animals shifted to the permissive temperature after 8 days of adulthood is also much smaller in magnitude for both markers, indicating that this repair is most effective in day 1 adults. In summary, as the adults age, ectopic TZ protein localisation is reduced, while the ability to correct this phenotype by restoring IFT function al


B ) Electrophoretic mobility shift assay ( EMSA ) to examine binding affinities of the GST - KNL2518 - 560 fragment for the CENP - A or H3 nucleosome . The molar ratio of KNL2518 - 560 against nucleosomes are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Electrophoretic mobility shift assay (EMSA) to examine binding affinities of the GST-KNL2518-560 fragment for the CENP-A or H3 nucleosome. The molar ratio of KNL2518-560 against nucleosomes are indicated.


D ) - F ) EMSA to examine binding affinities of the GST - KNL2 fragment for the CENP - A nucleosome : D , GST - KNL2347 - 560 ; E , GST - KNL2456 - 560 ; F , GST - KNL2456 - 516 ( CC - upstream ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D)-F) EMSA to examine binding affinities of the GST-KNL2 fragment for the CENP-A nucleosome: D, GST-KNL2347-560; E, GST-KNL2456-560; F, GST-KNL2456-516 (CC-upstream).


B ) EMSA to examine contribution of the ggKNL2 R527 residue to the CENP - A nucleosome binding .

B) EMSA to examine contribution of the ggKNL2 R527 residue to the CENP-A nucleosome binding.


D ) Expression of the WT or R527A mutant GFP - KNL2 in KNL2 conditional knock out chicken DT40 cells ( cKO - KNL2 ) . Expression of full - length KNL2 WT was conditionally turned off by tetracycline ( tet ) addition in cKO - KNL2 cells ( None ) . GFP - fused KNL2 WT or R527A mutant was stably expressed in the cKO - KNL2 cells . These cells were cultured in the presence or absence of tet ( + tet : KNL2 OFF or − tet : KNL2 ON ) for 48 h . α - Tubulin ( Tub ) was probed as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Expression of the WT or R527A mutant GFP-KNL2 in KNL2 conditional knock out chicken DT40 cells (cKO-KNL2). Expression of full-length KNL2 WT was conditionally turned off by tetracycline (tet) addition in cKO-KNL2 cells (None). GFP-fused KNL2 WT or R527A mutant was stably expressed in the cKO-KNL2 cells. These cells were cultured in the presence or absence of tet (+tet: KNL2 OFF or −tet: KNL2 ON) for 48 h. α-Tubulin (Tub) was probed as a loading control.


E ) Localization analyses of GFP - fused KNL2 WT or R527A mutant ( green ) . CENP - T was stained as a kinetochore marker ( red ) , and DNA was stained using DAPI ( blue ) . Magnified views are shown in insets . Scale bar indicates 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Localization analyses of GFP-fused KNL2 WT or R527A mutant (green). CENP-T was stained as a kinetochore marker (red), and DNA was stained using DAPI (blue). Magnified views are shown in insets. Scale bar indicates 10 μm.


F ) Growth of the cKO - KNL2 cells expressing GFP - fused KNL2 WT or R527A mutant . The upper panel shows examined cell numbers at the indicated time after tet addition ( + tet : KNL2 OFF ) . The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number . The lower panel shows examined untreated cells ( - tet : KNL2 ON ) . Parental cKO - KNL2 chicken DT40 cells ( None ) were also examined .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or R527A mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.


B ) EMSA to examine contribution of the F535 and W536 residues in ggKNL2 to the CENP - A nucleosome binding .

O O O O O O O O O O O O O O O O O O O O O O

B) EMSA to examine contribution of the F535 and W536 residues in ggKNL2 to the CENP-A nucleosome binding.


D ) Expression of GFP - KNL2 WT or CENP - A C - terminal tail binding mutants in cKO - KNL2 cells . Expression of full - length KNL2 WT was conditionally turned off by tetracycline ( tet ) addition to the cKO - KNL2 cells ( None ) . GFP - fused KNL2 WT , F535A , W536A or F535A / W536A was stably expressed in the cKO - KNL2 cells . These cells were cultured in the presence or absence of tet ( + tet : KNL2 OFF or − tet : KNL2 ON ) for 48 h . α - Tubulin ( Tub ) was probed as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Expression of GFP-KNL2 WT or CENP-A C-terminal tail binding mutants in cKO-KNL2 cells. Expression of full-length KNL2 WT was conditionally turned off by tetracycline (tet) addition to the cKO-KNL2 cells (None). GFP-fused KNL2 WT, F535A, W536A or F535A/W536A was stably expressed in the cKO-KNL2 cells. These cells were cultured in the presence or absence of tet (+tet: KNL2 OFF or −tet: KNL2 ON) for 48 h. α-Tubulin (Tub) was probed as a loading control.


E ) Localization analyses of GFP - fused KNL2 WT and F535A , W536A or F535A / W536A mutant ( green ) . CENP - T was stained as a kinetochore marker ( red ) , and DNA was stained using DAPI ( blue ) . Magnified views are shown in insets . Scale bar indicates 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Localization analyses of GFP-fused KNL2 WT and F535A, W536A or F535A/W536A mutant (green). CENP-T was stained as a kinetochore marker (red), and DNA was stained using DAPI (blue). Magnified views are shown in insets. Scale bar indicates 10 μm.


F ) Growth of the cKO - KNL2 cells expressing GFP - fused KNL2 WT or each CENP - A C - terminal tail binding mutant . The upper panel shows examined cell numbers at the indicated time after tet addition ( + tet : KNL2 OFF ) . The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number . The lower panel shows examined untreated cells ( - tet : KNL2 ON ) . Parental cKO - KNL2 chicken DT40 cells ( None ) were also examined .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or each CENP-A C-terminal tail binding mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.


A ) Immunostaining with anti - Mis18α antibody in AID - based KNL2 knockout cells expressing GFP fused KNL2 wild type or each KNL2 mutant in the presence of IAA for 15 h . Clear colocalization of KNL2 signals with Mis18α was observed ( Insets ) . DNA was stained with DAPI . Scale bar indicates 10 µm .

A) Immunostaining with anti-Mis18α antibody in AID-based KNL2 knockout cells expressing GFP fused KNL2 wild type or each KNL2 mutant in the presence of IAA for 15 h. Clear colocalization of KNL2 signals with Mis18α was observed (Insets). DNA was stained with DAPI. Scale bar indicates 10 µm.


B ) AID - based KNL2 knockout cells expressing GFP fused each KNL2 mutant and SNAP - tagged CENP - A were used . After addition of IAA , SNAP - CENP - A signals were visualized by TMR - Star staining . CENP - T was stained as a kinetochore marker . Representative images were presented . Mitotic centromere localization of each KNL2 mutant was shown as a merged image ( Insets ) : SNAP - CENP - A ( red ) and each KNL2 mutant ( green ) . DNA was stained with DAPI . Scale bar indicates 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) AID-based KNL2 knockout cells expressing GFP fused each KNL2 mutant and SNAP-tagged CENP-A were used. After addition of IAA, SNAP-CENP-A signals were visualized by TMR-Star staining. CENP-T was stained as a kinetochore marker. Representative images were presented. Mitotic centromere localization of each KNL2 mutant was shown as a merged image (Insets): SNAP-CENP-A (red) and each KNL2 mutant (green). DNA was stained with DAPI. Scale bar indicates 10 µm.


A ) KNL2 ( red ) localization in CENP - C conditional knockout chicken DT40 cells ( cKO - CENP - C ) with or without CENP - C expression . DNA was stained with DAPI ( blue ) . CENP - T was stained as a kinetochore marker ( light blue ) . Scale bar indicates 10 μm .

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A) KNL2 (red) localization in CENP-C conditional knockout chicken DT40 cells (cKO-CENP-C) with or without CENP-C expression. DNA was stained with DAPI (blue). CENP-T was stained as a kinetochore marker (light blue). Scale bar indicates 10 μm.


B ) KNL2 intensities were evaluated in six cells before and after CENP - C knock out . KNL2 signal intensities on the mitotic centromeres ( 14 - 90 centromeres ) in each cell were plotted with the average and the standard deviation . The ratio of KNL2 intensities between cells before and after CENP - C knock out was estimated . KNL2 intensities were reduced by ~ 55 % , when CENP - C was knocked out .

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B) KNL2 intensities were evaluated in six cells before and after CENP-C knock out. KNL2 signal intensities on the mitotic centromeres (14-90 centromeres) in each cell were plotted with the average and the standard deviation. The ratio of KNL2 intensities between cells before and after CENP-C knock out was estimated. KNL2 intensities were reduced by ~55%, when CENP-C was knocked out.


C ) Competitive pull - down assays for the CENP - A nucleosome binding of ggKNL2 and ggCENP - C . A complex of GST - KNL2456 - 560 with the CENP - A nucleosome ( CA - nuc ) was incubated in the absence or presence of MBP - CENP - C619 - 690 ( right panel ) or phosphorylated MBP - CENP - C619 - 690 ( middle panel ) and pulled down by GST affinity . The concentration of CENP - C619 - 690 was increased from 0 μM to 1 μM ( lanes 3 - 7 ) . The ratio of band intensities of pull - downed histones ( * ) to GST - KNL2456 - 560 in each lane ( lanes 3 - 7 ) was estimated and presented in the bar graph ( left panel ) . Data are mean with standard deviations calculated from the technical replicates , n = 3 .

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C) Competitive pull-down assays for the CENP-A nucleosome binding of ggKNL2 and ggCENP-C. A complex of GST-KNL2456-560 with the CENP-A nucleosome (CA-nuc) was incubated in the absence or presence of MBP-CENP-C619-690 (right panel) or phosphorylated MBP-CENP-C619-690 (middle panel) and pulled down by GST affinity. The concentration of CENP-C619-690 was increased from 0 μM to 1 μM (lanes 3-7). The ratio of band intensities of pull-downed histones (*) to GST-KNL2456-560 in each lane (lanes 3-7) was estimated and presented in the bar graph (left panel). Data are mean with standard deviations calculated from the technical replicates, n=3.


A , B Immunoblot analyses of the protein abundance of the METTL3 / METTL14 / WTAP heterotrimer in PBMCs ( A ) and BMDMs ( B ) upon challenge with VSV - eGFP , poly ( I : C ) , HSV - 1 or HSV60 at the indicated time points .

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A, B Immunoblot analyses of the protein abundance of the METTL3/METTL14/WTAP heterotrimer in PBMCs (A) and BMDMs (B) upon challenge with VSV-eGFP, poly(I:C), HSV-1 or HSV60 at the indicated time points.


E A549 cells were stimulated with poly ( I : C ) for 16 hrs and then treated with PR - 171 ( 10 μM ) or PS - 341 ( 10 μM ) for additional 4 hrs and 8 hrs . The cell lysates were analyzed by immunoblotting . Representative images from 3 independent biological experiments are shown ( left ) , and the relative levels of WTAP are presented as the mean ± SD ( right ) . F A549 cells were stimulated with poly ( I : C ) for 16 hrs and then treated with CQ ( 50 mM ) or Baf A1 ( 0 . 2 mM ) for additional 4 hrs and 8 hrs . The cell lysates were analyzed by immunoblotting . LC3B served as a good marker of the autophagy process . Representative images from 3 independent biological experiments are shown ( left ) , and the relative levels of WTAP are presented as the mean ± SD ( right ) .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with PR-171 (10 μM) or PS-341 (10 μM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right). F A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with CQ (50 mM) or Baf A1 (0.2 mM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. LC3B served as a good marker of the autophagy process. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right).


H Coimmunoprecipitation and immunoblot analyses of extracts of 293T cells transfected with various combinations of plasmids encoding FLAG - tagged WTAP , and HA - tagged K48 - linked , K63 - linked or wild - type ubiquitin and treated with DMSO or PR171 ( 10 μM ) .

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H Coimmunoprecipitation and immunoblot analyses of extracts of 293T cells transfected with various combinations of plasmids encoding FLAG-tagged WTAP, and HA-tagged K48-linked, K63-linked or wild-type ubiquitin and treated with DMSO or PR171 (10 μM).


C qRT - PCR analyses to detect the mRNA abundances of IFNB1 , RANTES , DDX58 , and ISG15 in WT and WTAP - / - 293T cells followed by treatments with intracellular poly ( I : C ) , poly ( dA : dT ) , or VSV - eGFP .

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C qRT-PCR analyses to detect the mRNA abundances of IFNB1, RANTES, DDX58, and ISG15 in WT and WTAP-/- 293T cells followed by treatments with intracellular poly(I:C), poly(dA:dT), or VSV-eGFP.


I , J Immunoblot analyses to detect the protein expression of ISGs in shCtrl and shWTAP A549 cells that were stimulated with poly ( I : C ) - LMW ( I ) or poly ( dA : dT ) ( J ) at different time points with the indicated antibodies .

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I, J Immunoblot analyses to detect the protein expression of ISGs in shCtrl and shWTAP A549 cells that were stimulated with poly(I:C)-LMW (I) or poly(dA:dT) (J) at different time points with the indicated antibodies.


A , B Immunoblot analyses of total and phosphorylated ( p - ) TBK1 and IRF3 in A549 cells transfected with Ctrl or WTAP - specific siRNA , followed by stimulation with poly ( I : C ) ( A ) or poly ( dA : dT ) ( B ) at different time points .

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A, B Immunoblot analyses of total and phosphorylated (p-) TBK1 and IRF3 in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by stimulation with poly (I:C) (A) or poly (dA:dT) (B) at different time points.


E , F Immunoblot analyses of total and phosphorylated ( p - ) STAT1 in shCtrl and shWTAP A549 cells , followed by stimulation with IFN - α ( E ) or - β ( F ) at different time points .

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E, F Immunoblot analyses of total and phosphorylated (p-) STAT1 in shCtrl and shWTAP A549 cells, followed by stimulation with IFN-α (E) or -β (F) at different time points.


A , B Immunoblot analyses to detect the protein expression of critical adaptors in the IFN - I and Jak - STAT signaling pathway in A549 cells after transfection with Ctrl or WTAP - specific siRNAs for 48 hrs .

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A, B Immunoblot analyses to detect the protein expression of critical adaptors in the IFN-I and Jak-STAT signaling pathway in A549 cells after transfection with Ctrl or WTAP-specific siRNAs for 48 hrs.


F , G Abundance of IRF3 ( F ) and IFNAR1 ( G ) transcripts ( detection site - 1 and - 2 ) among mRNA immunoprecipitated with anti - m6A antibody from shCtrl and shWTAP A549 cells . n = 3 independent biological replicates , and error bars represent standard deviations .

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F, G Abundance of IRF3 (F) and IFNAR1 (G) transcripts (detection site-1 and -2) among mRNA immunoprecipitated with anti-m6A antibody from shCtrl and shWTAP A549 cells. n = 3 independent biological replicates, and error bars represent standard deviations.


A Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 cells stimulated with poly ( I : C ) or poly ( dA : dT ) at the indicated time points . B Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 and L929 cells infected with VSV - eGFP at the indicated time points .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 cells stimulated with poly (I:C) or poly (dA:dT) at the indicated time points. B Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 and L929 cells infected with VSV-eGFP at the indicated time points.


H , Immunoblot analysis of IRF3 and IFNAR1 in A549 cells transfected with poly ( I : C ) - LMW or infected with VSV - GFP for 16 hrs and then treated with PR - 171 or DMSO for additional 4 and 8 hrs . Representative images from 3 independent biological experiments are shown ( H ) ,

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H, Immunoblot analysis of IRF3 and IFNAR1 in A549 cells transfected with poly (I:C)-LMW or infected with VSV-GFP for 16 hrs and then treated with PR-171 or DMSO for additional 4 and 8 hrs. Representative images from 3 independent biological experiments are shown (H),


protein abundance of IRF3 and IFNAR1 in A549 cells transfected with poly ( I : C ) - LMW or infected with VSV - GFP for 16 hrs and then treated with PR - 171 or DMSO for additional 4 and 8 hrs . the relative levels of IFNAR1 ( left ) and IRF3 ( right ) are presented as the mean ± SD ( I ) .

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protein abundance of IRF3 and IFNAR1 in A549 cells transfected with poly (I:C)-LMW or infected with VSV-GFP for 16 hrs and then treated with PR-171 or DMSO for additional 4 and 8 hrs. the relative levels of IFNAR1 (left) and IRF3 (right) are presented as the mean ± SD (I).


B . Significant increase of survival in Ppt1 - / - animals transplanted with HSCPs or BM isolated from Ppt1 + / + mice ; Log - rank : HSPCs / BM + / + tx vs BM - / - tx p < 0 . 001 ; BM - / - tx vs Ppt1 - / - UT p < 0 . 001 .

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B. Significant increase of survival in Ppt1-/- animals transplanted with HSCPs or BM isolated from Ppt1+/+ mice; Log-rank: HSPCs/BM+/+ tx vs BM-/- tx p<0.001; BM-/- tx vs Ppt1-/- UT p < 0.001.


D . Representative fluorescence microscope photomicrographs showing the distribution of donor - derived cells ( transduced with D . NGFR reporter gene , red ) and autofluorescent storage material ( AF , green ) in the brain of a Ppt1 - / - mouse euthanized at 240 days of age , after transplantation with HSPCs isolated from Ppt1 + / + mice . Insets in D show high magnification representative confocal scanning photomicrographs of DNGFR + AF + cells in different regions of the CNS . Arrows show DNGFR + cells engulfed with autofluorescent ( AF ) storage material . Scale bar ( main figure ) = 1mm ; scale bar ( insets ) = 25μm .

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D. Representative fluorescence microscope photomicrographs showing the distribution of donor-derived cells (transduced with D.NGFR reporter gene, red) and autofluorescent storage material (AF, green) in the brain of a Ppt1-/- mouse euthanized at 240 days of age, after transplantation with HSPCs isolated from Ppt1+/+ mice. Insets in D show high magnification representative confocal scanning photomicrographs of DNGFR+AF+ cells in different regions of the CNS. Arrows show DNGFR+ cells engulfed with autofluorescent (AF) storage material. Scale bar (main figure) = 1mm; scale bar (insets) = 25μm.


G . Representative brightfield photomicrographs of Nissl - stained brain sections from wild type Ppt1 + / + mice and untreated or transplanted Ppt1 - / - mice . Black dashed box highlights a portion of the cortex , shown in the high magnification insets . Yellow lines represent the segments applied to measure cortical thickness ( see Materials and Methods for details on the quantification ) . Scale bar ( main figure ) = 1mm ; scale bar ( insets ) = 25μm . H . Histograms showing the quantification of cortical thickness . SM = somatomotor ; SS = somatosensory ; V = visual cortex . * * = p < 0 . 01 ; Kruskal Wallis followed by Dunn ' s post - hoc test ( for SM ) or ANOVA followed by Tukey ' s post - hoc test ( for SS and V ) .

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G. Representative brightfield photomicrographs of Nissl-stained brain sections from wild type Ppt1+/+ mice and untreated or transplanted Ppt1-/- mice. Black dashed box highlights a portion of the cortex, shown in the high magnification insets. Yellow lines represent the segments applied to measure cortical thickness (see Materials and Methods for details on the quantification). Scale bar (main figure) = 1mm; scale bar (insets) = 25μm. H. Histograms showing the quantification of cortical thickness. SM = somatomotor; SS = somatosensory; V = visual cortex. ** = p < 0.01; Kruskal Wallis followed by Dunn's post-hoc test (for SM) or ANOVA followed by Tukey's post-hoc test (for SS and V). D


C . Significant increase of the survival in Ppt1 - / - mice receiving the administration of hPPT1 - LV transduced HSPCs .

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C. Significant increase of the survival in Ppt1-/- mice receiving the administration of hPPT1-LV transduced HSPCs.


E , F . Significant prevention of pathological symptoms ( DSS higher than 4 and limbs stiffness ) in Ppt1 - / - mice transplanted with hPPT1 - LV transduced HSPCs .

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E, F. Significant prevention of pathological symptoms (DSS higher than 4 and limbs stiffness) in Ppt1-/- mice transplanted with hPPT1-LV transduced HSPCs.


E . Representative fluorescence microscope photomicrographs showing autofluorescent storage material ( AF , green ) in different brain regions of Ppt1 - / - mice transplanted with hPPT1 - LV transduced HSPCs at 400 - 420 days of age , when the study was terminated . Mock transplanted and untreated Ppt1 - / - mice at about 250 days , i . e . humane end point , are shown as reference ; 450 days - old untreated wild type Ppt1 + / + are shown as control . Neurotrace fluorescent Nissl staining ( NT , red ) is used to highlight neurons in different brain regions . Arrows highlight the surviving Purkinje cells in the cerebellum of IV and ICV + IV transplanted mice ; arrowheads highlight the high number of AF + cells in the Purkinje cell layer of ICV transplanted or Ppt1 - / - untreated ( UT ) mice . Scale bar = 100μm .

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E. Representative fluorescence microscope photomicrographs showing autofluorescent storage material (AF, green) in different brain regions of Ppt1-/- mice transplanted with hPPT1-LV transduced HSPCs at 400-420 days of age, when the study was terminated. Mock transplanted and untreated Ppt1 -/- mice at about 250 days, i.e. humane end point, are shown as reference; 450 days-old untreated wild type Ppt1+/+ are shown as control. Neurotrace fluorescent Nissl staining (NT, red) is used to highlight neurons in different brain regions. Arrows highlight the surviving Purkinje cells in the cerebellum of IV and ICV+IV transplanted mice; arrowheads highlight the high number of AF+ cells in the Purkinje cell layer of ICV transplanted or Ppt1-/- untreated (UT) mice. Scale bar = 100μm.