Supplementary Figure S1

A C Complete medium Complete medium

1.61,6 24 h 48 h 72 h Cells treated with siPFDN2 siControl 120 1.41,4 siPFDN2 siC siPFDN2 siC siPFDN2 siC siPFDN2 100 1.21,2 ** ** ** / siControl 11 PFDN2 15 kDa 80

0.80,8 / siControl levels 1 0.97 1 0.54 1 0.33 60

0.60,6 cells ± 0.35 ± 0,32 ± 0.06 40 0.40,4 GAPDH 37 kDa 0.20,2 % living 20

mRNA PFDN2 00 1 1 1 1 1 1 0 24 h 48 h 72 h 24 h 48 h 72 h Time after transfection Time after transfection

1.61,6 Cells treated with siPFDN5 siControl 24 h 48 h 72 h 140 1.41,4 ** siPFDN5 1.21,2 siC siPFDN5 siC siPFDN5 siC siPFDN5 120 ** * * / siControl 11 100 PFDN5

15 kDa / siControl 0.80,8 80 levels

1 0.51 1 0.77 1 0.45 cells 0.60,6 60 ± 0.17 ± 0.12 ± 0.10 0.40,4 40

0.20,2 GAPDH 37 kDa % living 20 mRNA PFDN5 00 24 h 48 h 72 h 1 1 1 1 1 1 0 Time after transfection 24 h 48 h 72 h Time after transfection

B 48 h of serum starvation D 48 h of serum starvation 1,6 1.6 72 h

1,4 1.4 siControl Cells treated with siPFDN2 siC siPFDN2 120 1,2 1.2 siPFDN2 PFDN2 15 kDa 11 100

0.8 1 0.377 0,8 80 ± 0.109 0,6 0.6

37 kDa 60 0,4 0.4 GAPDH

0,2 0.2 40 mRNA PFDN2 levels / siControl 1 1 00 % living cell / siControl 20 72 h Time after transfection 0 72 h Time after transfection 1.2 1,2 siControl Cells treated with siPFDN5 72h 120 1 1 siPFDN5

siC siPFDN5 100 0.8 0,8 PFDN5 15 kDa 80 0.6 0,6 siControl 1 0.25

/ ± 0.16 60 0.4 0,4 cell GAPDH 37 kDa 40 0.2 0,2

1 1 % living mRNA PFDN5 levels / siControl 20 0 0 72h 0 Time after transfection 72 h Time after transfection Supplementary Figure S2

A siPFDN2 siPFDN5

22 38 755 999 619 3056

Before serum After serum Before serum After serum stimulation stimulation stimulation stimulation

B C

GOs whose expression level changes in deficient cells in PFDN2 and PFDN5 after serum stimulation GO p siPFDN2 p siPFDN5 cell adhesion 2.78E-14 6.30E-09 nervous system development 7.14E-12 0 22 38 1580 animal organ morphogenesis 3.17E-10 1.09E-07 regulation of cell differentiation 1.26E-08 8.76E-07 vasculature development 2.33E-08 6.24E-07 negative regulation of developmental 4.56E-06 1.89E-05 process siPFDN2 siPFDN5 synapse organization 2.30E-05 2.87E-05 cytoskeleton organization 2.78E-05 2.32E-07

heart development 4.70E-05 1.94E-06 n = 10322 p = 7.24e-17

D

2

2 1.5

1,5 1

0.5 1 siPFDN2 0,5 0 siPFDN5

-0.5 0 FC 2 log -0,5 -1

-1.5 -1

-1,5 -2

-2

Genes Supplementary Figure S3

A Before serum induction After serum induction

FC) FC) 2 1.0 1.0

(Log * * 0.5 0.5 expression

0.0 0.0 siControl

-0.5 -0.5 Differential -1.0 -1.0 siPFDN2 / ] ] ] ] ] ] ] ] ] ] 1 4 .9 .5 0 1 4 .9 .5 0 -1 -2 5 3 0 -1 -2 5 3 0 6 1 4 9 9 6 1 4 9 9 0 1 - - -1 0 1 - - -1 2 ( 4 .9 5 2 ( 4 .9 5 . (2 5 . . (2 5 . [0 4 3 [0 4 3 ( (9 ( (9 Lenght (kb) B Before serum induction After serum induction 1.0

1.0 FC) FC) 2 * * * 0.5 0.5 (Log

expression 0.0 0.0

siControl -0.5 -0.5

Differential -1.0 -1.0 4 7 1 7 2 -4 -7 1 7 2 - - 1 1 6 siPFDN2 / 0 5 -1 -1 6 0 5 - - 8 2 -3 8 2 -3 1 8 1 8 1 1 Number of introns

C

1 FC) 2

siControl siPFDN2

0.5 Number of introns: Lower tercil Middle tercil Upper tercil

0 Inductionserumafter addition (Log

-1 0 1 2 3

Log10 (gene length, kb) Supplementary Figure S4

Expression level (CPM) Supplementary Figure S5

A PFDN5

E1 E2 E3 E4 E5 E6

B E2 E3 E4

WT PFDN5 GTACAGACCAAGTATGTGGAAGCCAAGGACTGTCTGAACGTGCTGAACAAGAGCAACGAGGGGAAAGAATTACTCGTCCCACTGACGAGTTCTATGTATGTCCCTGGGAAGCTGCATGAT V Q T K Y V E A K D C L N V L N K S N E G K E L L V P L T S S M Y V P G K L H D 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78

PFDN5 KO GTACAGACCGAGTTATGTGGAAGCCAAGGACTGTCTGAACGTGCTGAACAAGAGCAACGAGGGGAAAGAATTACTCGTCCCACTGACGAGTTCTATGTATGTCCCTGGGAAGCTGCATGA V Q T E L C G S Q G L S E R A E Q E Q R G E R I T R P T D E F Y V C P W E A A STOP 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78

C

PFDN5 15 kDa

GAPDH 37 kDa Supplementary Figure S6

A

14 SE MX A5 A3

12 RI AF AL

10

8

6

% affected events %affected 4

2

0 Events that occur less frequently in EventsNew events that appear in siPFDN5 cells that occur more frequently in PFDN5-deficient cells. PFDN5-deficient cells.

B FASN Ratio short isoform / long isoform 33

2.52,5

22

1.51,5

11

0.50,5

00 WT PFDN5 KO PFDN5 KO + PFDN5 Supplementary Figure S7

OPA1 A 104668 bp

Ex18 Ex19

Ex19 In19

11

0.8 0,8

0.60,6

siControl 0.40,4 siPFDN5

0.20,2 Relative levels of pre-mRNA Relativelevels pre-mRNA of

0 0 0 10 20 30 40 50 60 70 Time post-DRB wash (min)

B Intron 18 removed

Ex18 Ex19 In19

0.80,8

0.60,6

0.40,4 siControl siPFDN5

0.20,2 Relative levels of pre-mRNA Relativelevels pre-mRNA of

00 0 10 20 30 40 50 60 70 Time post-DRB wash (min) Supplementary Figure S8

A

Flag-PFDN5 20 kDa

PFDN5 15 kDa

GAPDH 37 kDa

B

ChIP after RNAse treatment

PFDN5 in CTNNBL1 PFDN5 in CD44 18 10 **** 16 **** 9 8 14 **** 7 12 **** **** 6 10 5 8 **** 4 6 3 4 Flag/negative control 2 Flag / negativeFlag / control 2 1 0 0 Ex1 Ex5 Ex16 Ex1 Ex6 Ex19

FLAG FLAG-PFDN5 Supplementary Figure S9

6.0 ) M P C ( (CPM) (CPM)

n o i s

s 5.5 e

r expression p x

e

n RR == 0.06780.0678 a e Pp == 0.50430.5043

M Mean 5.0 -1.0 -0.5 0.0 0.50.5 1.01.0 1.5 2.0 2.0 PFDN5 PFDN5 (ChIP-seq (ChIP-seq signal signal)) Supplementary Figure S10

A

Non-snRNP snRNP PRP19C prp46- syf1- lsm8- smx3- hsh49- cef1-13 clf1-1 prp2-1 lsm6∆ 5001 5001 5003 5001 5002 pfdn1∆ X X X pfdn2∆ X X X pfdn3∆ X pfdn4∆ X X X X X pfdn5∆ X pfdn6∆ X X X X

Table. Pearson correlation coefficient ≥ 0.13 Source: https://thecellmap.org/

B Supplementary Figure S11

A B C

PRPF19 PRLG1 U2AF65 50 kDa 50 kDa 50 kDa 1 0.80 1 1.34 1 1.05 ± 0.10 ± 0.28 ± 0.65 GAPDH GAPDH GAPDH 37 kDa 37 kDa 37 kDa 1 1 1 1 1 1 Supplementary Figure S12

A PRP19 in CTNNBL1 PRP19 in CD44 12 10 II 10 II * pol

pol 8 8 siControl 6 ** 6 siPFDN2 4 4

2 2 PRP19 / Total RNA RNA Total PRP19 / PRP19 / Total RNA RNA Total PRP19 / 0 0 Ex1 Ex5 Ex16 Ex1C1 V5Ex9 C19Ex19

B UA2F65 in CTNNBL1 UA2F65 in CD44 30 25 II II 25

** pol

pol 20 * 20 ** 15 siControl 15 10 siPFDN2 10 5 5 UA2F65/ Total RNA RNA Total UA2F65/ UA2F65 / Total RNA RNA Total UA2F65 / 0 0 Ex1 Ex5 Ex16 Ex1C1 Ex9V5 Ex19C19 Supplementary Figure S13

A

Total RNA pol II in CTNNBL1 Total RNA pol II in CD44 0,006 0,006 * * 0,004 0,004 siControl siControl

0,002 siPFDN2 0,002 siPFDN2 RNA pol II / Input RNA RNA pol II/ Input RNA

0,000 0 Ex1 Ex5 Ex16 Ex1 Ex9 Ex19

B Total RNA pol II in CTNNBL1 Total RNA pol II in CD44

0,006 0,006

0,004 WT 0,004 WT PFDN5 KO PFDN5 KO 0,002 0,002 RNA pol II / Input RNA RNA pol II / Input RNA

0 0 Ex1 Ex5 Ex16 Ex1 Ex9 Ex19 Supplementary Figure S14

Nascent pre-mRNA

P PRP19C P P P U2AF65 P CTD P P P P CDK9 Prefoldin RNA pol II

Transcritpion-dependent spliceosome pre-catalytic activation

P P P PRP19C P U2AF65 P CTD P P P P CDK9 Prefoldin RNA pol II

Co-transcriptional splicing

P P P PRP19C P U2AF65 P CTD P P P P CDK9 Prefoldin RNA pol II Supplementary Figure S15

A B PFDN5 in promoters 2.0 ) l

a *** n g

i 1.5 s

q

e 1.0 s - P I

h 0.5 C (

5

N 0.0 D F P -0.5

p p e e d d - n c -i y c M y M Supplementary figure S1. Efficiency of the siRNA-mediated depletion of PFDN2 and PFDN5. A) Left panels: PFDN2 and PFDN5 mRNA levels in HCT116 cells, 24, 48 and 72 hours after being transfected with siPFDN2 or siPFDN5, respectively, or with a siRNA control (siControl). Right panels: Protein levels analyzed by Western blotting using antibodies against PFDN2 or PFDN5 and GAPDH as a loading control, after transfecting the cells with siPFDN2, siPFDN5, respectively, or with siControl. The results were quantified with Image Lab software. B) Left panels: PFDN2 and PFDN5 mRNA levels in HCT116 cells treated with siPFDN2, siPFDN5, or siControl for 24 h and serum starved for 48 h more (72 hours total siRNA treatment). Right panels: Protein levels of the same samples analyzed by western blotting using antibodies against PFDN2 or PFDN5 and GAPDH as a loading control. C) Cell viability assay on the same cell line transfected with siPFDN2 or siPFDN5 and the siControl separately. The cells were counted every 24 hours for 3 days, and the percentage of cells relative to the control was represented. D) Cell viability assay in the same conditions as in B. In all panels, averaged values ± the standard deviation obtained from three different experiments are represented. * Student´s t test with p value < 0.05 and ** Student´s t test with p value < 0.005.

Supplementary figure S2. Differential expression of PFDN2- and PFDN5-deficient cells. A) Representation of the number of genes differentially expressed in the cells transfected withsiPFDN2 or with siPFDN5 at each time, before and after serum stimulation. B) The genes affected by siPFDN2 or siPFDN5 after serum stimulation were functionally grouped into Gene Ontology categories. Those categories that were commonly affected by both siRNAs are represented in the table. C) Representation of the number of genes differentially expressed in the cells transfected with siPFDN2 or with siPFDN5 before serum stimulation, and the genes that coincide. p value of the hypergeometric test is shown. n indicates total number of genes included in the analysis. D) Parallelism of the expression changes produced by siPFDN2 and siPFDN5. Changes in mRNA levels of genes affected by both siPFDN2 and siPFDN5 before serum stimulation are shown.

Supplementary figure S3. PFDN2 deficiency impairs the induction of long genes with a high number of introns. A) Expression differences between siPFDN2 and siControl cells (log2(FC)) with respect to the gene length, before and after serum stimulation. The genes were separated into quintiles. The * represents p < 0.005 in a Student´s t test comparing each quintile to quintile 1. B) The same analysis of A with respect to the number of introns that each gene contains, before and after serum stimulation. The genes were separated into quintiles. The * represents p< 0.005 in a Student´s t test comparing each quintile to quintile 1. C) Serum regulated genes (|log2(FC)| > 0 and FDR < 0.05, N = 1035) were divided into three groups according to the tercile of the number of introns. Serum-dependent fold change (Log2) is represented against the gene length (Log10), in cells transfected with the siControl (solid line) or siPFDN2 (dotted line).

Supplementary figure S4. Exonic ratio distribution before and after serum stimulation. The exonic ratio (vertical axis) is represented against the level of gene expression (horizontal axis) of the cells transfected with the siControl (red), siPFDN2 (green) and siFDN5 (blue) before and after serum stimulation. Supplementary figure S5. The sequence of the PFDN5 KO. A) A frameshift mutation of PFDN5 was originated by CRISPR-Cas9 technology in its exon 2. B) The sequence of the three exons affected by the translational consequences of the frameshift mutation is shown, comparing the WT and the PFDN5 KO cell line. The bases in red represent the two mutations caused by CRISPR-Cas9 (an A to G transition and a T insertion, marked with arrows). Predicted translation is shown under each DNA sequence. The numbers under the aminoacid residues correspond to the position in the PFDN5 WT protein from its N-terminus. The residues in red are those not found in the WT protein in each position. A premature stop codon in exon 4 is underlined. C) Western blot showing the absence of PFDN5 protein in the PFDN5 KO cell line.

Supplementary figure S6. PFDN5 deficiency has a mild general impact on alternative splicing. A) PFDN5 depletion causes general alteration in alternative splicing. SUPPA computational tool (19) was used for exploring the impact of siPFDN5 on alternative splicing. Bars histogram representing the percentage of annotated alternative splicing events that occur significantly less frequently in PFDN5-deficient cells is shown in the left panel. On the right, bars histogram representing the frequency of alternative splicing events that occur more frequently in PFDN5-deficient cells. SE: skipping exon; MX: mutually exclusive exons; A5: alternative 5’ splice-site; A3: alternative 3’ splice-site; RI: retained intron; AF: alternative first exon; AL: alternative last exon. B) Lack of PFDN5 causes accumulation of intron reads after serum stimulation in a FASN minigene assay. A schematic drawing of the alternative isoforms of the Fas minigen is shown on the left. On the right, bar histogram of short isoform / long isoform ratios of the Fas minigen, in control cells, PFDN5 KO cells, and PFDN5 KO cells transfected with GFP-PFDN5. The graph represents the average values and the standard deviation obtained from five different experiments. According to the Student´s t test, the difference between WT and KO cells and between KO and KO+GFP-PDFN5 cells did not generated p values lower than 0.05.

Supplementary figure S7. PFDN5 depletion impairs co-transcriptional splicing in the long OPA1 gene. A) Pre-mRNA levels from the OPA1 gene in cells transfected with control siRNA (red line) or PFDN5 siRNA (blue line). Cells were treated with 100 µM DRB for 3 hours. Samples were taken every 10 minutes after washing DRB out. Pre-mRNA data was normalized first to the mature mRNA, and next to the DRB untreated sample. A scheme of the OPA1 gene is also shown, and the amplicon used is depicted above the graph. B) Event of co-transcriptional splicing (measured as the presence of pre-mRNA without the indicated intron) in the OPA1 gene in cells treated as in A). The amplicon used is depicted above the graph. The average and standard error of at least three biological replicates with three technical replicates each are represented.

Supplementary figure S8. PFDN5 presence in the chromatin is not RNA dependent. A) HCT116 cell line expressing Flag-PFDN5. Expression of Flag-PFDN5 in HCT116 stable cell line. A western blot is shown, after using antibodies against PFDN5 and GAPDH as a loading control. B) The level of Flag-PFDN5 in the chromatin at different loci throughout the CTNNBL1 gene (left panel) and the CD44 gene (right panel), measured after treatment of the extracts with RNase. The graphs represent the average values ± the standard error obtained from three different experiments. **** Student´s t test with p value < 0.0001. A total number of 12 technical replicates were considered for Student´s t test.

Supplementary figure S9. Weak correlation between prefoldin ChIP-seq signal and total gene expression. The promoter (TSS +/- 500 bp) signal of PFDN5 was represented with respect to the level of gene expression. Expressed genes (>0.1 reads per kilobase per million, RPKM) were ordered according to the level of expression and divided into 100 bins. The mean signal of each group was the calculated and represented. The R and p values from Pearson´s correlation are shown.

Supplementary figure S10. Correlations between prefoldin and splicing mutants in the CellMap database of genetic interactions. A) Genetic correlations between prefoldin and splicing mutants were explored in the Cell Map database (thecellmap.org). Pairs of prefoldin/pre-mRNA splicing mutants showing Pearson correlations coefficients higher than 0.13 are shown in the table. B) The two factors that showed general correlations with prefoldin mutants, PRP19C and PRP2, participate in the activation step of pre-mRNA splicing. Spliceosome assembly and splicing reaction. snRNPs subunits U1, U2, U4, U5 and U6 are represented; as well as the splicing factors Prp2 and Prp19C.

Supplementary figure S11. Total levels of different splicing factors and RNA pol II remain constant in the absence of prefoldin. A-C) Protein levels determined by western blotting, using antibodies against PRPF19 (A), PRGL1 (B) and U2AF65 (C) splicing factors and GAPDH as a loading control. The results were quantified with Image Lab software. Averaged values ± the standard deviation obtained from three different experiments are represented.

Supplementary figure S12. Splicing factors recruitment is impaired in PFDN2 knockdown cells. A-B) The levels of PRP19 (A) and U2AF65 (B) were measured by ChIP in different regions of the CTNNBL1 gene (left panels) and the CD44 gene (right panels) in cells treated with siControl (red bars) and with siPFDN2 (green bars). In all the panels, values were normalized to total RNA pol II levels. The graphs represent the average values ± the standard deviation obtained from three different experiments. * p value < 0.05; ** < 0.005. A total number of 12 technical replicates were considered for Student´s t test.

Supplementary figure S13. Total levels of RNA pol II on genes did not decrease after prefoldin perturbation. A and B) The levels of RNA pol II were measured by ChIP in different regions of the CTNNBL1 gene (left panels) and the CD44 gene (right panels), using the 8WG16 antibody in A) cells treated with siControl (red bars) and with siPFDN2 (green bars); B) control (red bars) and PFDN5 KO cells (blue bars). The graphs represent the average values ± the standard deviation obtained from three different experiments. * p value < 0.05. A total number of 12 technical replicates were considered for Student´s t test.

Supplementary figure S14. Graphic model illustrating how prefoldin could contribute to co-transcriptional splicing. Prefoldin helps recruit CDK9 to transcribed chromatin. CDK9 phosphorylates the RNA pol II CTD at Ser2 residues. In turn, the phosphorylated CTD recruits U2AF65 and the PRP19 complex. These splicing factors, acting from the CTD, activate the spliceosome assembled on the nascent pre-mRNA, thereby favouring co-transcriptional pre-mRNA splicing.

Supplementary figure S15. PFDN5 binds both Myc-dependent and -independent genes. A) Box plot comparing the presence of PFDN5 in promoters of Myc-dependent or independent genes. Data from ChIP-seq analysis were used. B) Box plot comparing PFDN5/RNA pol II ChIP ratios in promoters of Myc-dependent or independent genes. *** Mann-Whitney U test p value < 0.0005.

Supplementary Table S1. Primers used in this work

Primers pair Sense 5’-3’ Antisense 5’-3’ PFDN5 CGCAGCTAGAAATGCTCAAGAA GCAATGGACGTGGACAAGAA PFDN2 GTTGGAGATGGAGTTGAATG CTCCTTGTTGTTCTCCAAAG CTNNBL1 AGTGCAGGGAAGTGGAGTATTTGC AGAGGAGGTGAGATGAAAGGGCT Ex1 CTNNBL1 TACCACCTTCTGGTGGAGCTGAAT GGAGAAAGAGTAACAGCACTTCCC Ex4 CTNNBL1 TGAAGAGGGAGCAGAAGTGCTCAT TATTCACTCCAGCCTCACCACACT Ex5 CTNNBL1 TTCTGGGAAAGGTATGAAGCGACG TAGAAGTTCTCCAGCAAGCCCA Ex16 CTNNBL1 TACCACCTTCTGGTGGAGCTGAAT TATTCACTCCAGCCTCACCACACT Ex4-In5 CTNNBL1 TGAAGAGGGAGCAGAAGTGCTCAT CAAAGCAACTCAGGCAATGGCA Ex5-In6 CTNNBL1 TGGGTAGCTGAGAGGGAGGTG ATTATTGGCCCCACTGTGCAG STOP + 4000 bp CD44 Ex1 TCCCTCCGTCTTAGGTCACTGTTT CCTCGGAAGTTGGCTGCAGTTT CD44 Ex9 GAAACTGGAACCCAGAAGCACA TGATGCTCATGGTGAATGAGGG CD44 Ex19 TGATCAACAGTGGCAATGGAGC TCTGACGACTCCTTGTTCACCA CD44 STOP TGGGTAGCTGAGAGGGAGGTG CTCACTCTTCACCCAGGCTG + 4000 bp NT Chr.V CTGTACCTGGGGTTCATTCATT CAGTAAGCCGTTCACTCTCAC FASN Ex2. CAGAGTCGGAGAACTTGCAGGAG GAGCTTCACACCCCAGGCAC In2 FASN Ex22. AGAACATGCCCAGCCTGAAG TGTCTGTGCTGTCTCCTGCA In22 SRRM2 CGGGAGTCTAGCAGTTCTCG ATTGGTCCTCTGGCTATCA Ex5.In5 SRRM2 AGATGCGCCTTTCAGTGAAC TTTGCAATCCAATGAACATCA Ex10.In10 CDK9 TATATACCTGGTGTTCGACTTCTGC GTGGATGTAGTAGAGGCCGTTAAG Ex4.In4 OPA1 Ex18- GGAACAGCTCTGAAAGCATTG TCGTATGGATGCCAAAGATTGCCAG In19 OPA1 Ex19- GGACAAGCATGCTAAAGGCACACC GGACAAGCATGCTAAAGGCACACC In19