1 Termination Control by an RNA

Termination Control by an RNA Polymerase II CTD R1810me2s-SMN Interaction

One-sentence summary: Symmetric dimethylation of the human RNA polymerase II C-terminal domain (CTD) residue R1810 by the Protein Arginine Methyltransferase 5 directly recruits the protein Survival of Motor Neuron (SMN) and indirectly recruits the helicase Senataxin to resolve R-loops in transcription termination regions and possibly contribute to preventing the neurodegenerative disorder spinal muscular atrophy (SMA).

Dorothy Yanling Zhao1,2,3, Gerald Gish2ǂ, Ulrich Braunschweig1ǂ, Yue Li4, Zuyao Ni1, Frank W. Schmitges1, Guoqing Zhong1, Ke Liu5, Weiguo Li5, Jason Moffat1,3, Masoud Vedadi5, Jinrong Min5, Tony J. Pawson2,3, Ben J. Blencowe1,3, Jack F. Greenblatt1,3 ,6

1. Donnelly Centre and Banting & Best Department of Medical Research, University of Toronto, Toronto, ON M5S 3E1, Canada 2. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada 3. Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada 4. Department of Computer Science, The Donnelly Centre, University of Toronto, Toronto, ON M5S 3G4, Canada 5. Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada 6. To whom correspondence should be addressed. E-mail: [email protected] ǂ Equal contribution

ABSTRACT

The C-terminal domain (CTD) of the RNA polymerase II subunit POLR2A is a platform for modifications specifying the recruitment of factors that regulate transcription, messenger RNA processing, and chromatin remodeling. We now find that a CTD arginine residue (R1810 in human) that is conserved across vertebrates is symmetrically dimethylated (me2s). This R1810me2s modification requires Protein Arginine Methyltransferase 5 and recruits the Tudor domain of the

Survival of Motor Neuron (SMN) protein, which is mutated in spinal muscular atrophy (SMA). SMN interacts with Senataxin, which is sometimes mutated in Ataxia Oculomotor Apraxia 2 (AOA2) and

Amyotrophic Lateral Sclerosis (ALS4). Because R1810me2s and SMN, like Senataxin, are required for resolving R-loops created by RNA polymerase II in transcription termination regions, we propose that R1810me2s, SMN, and Senataxin are components of a pathway for R-loop resolution in which defects can influence transcription termination and may contribute to neurodegenerative disorders like

SMA, AOA, and ALS.

INTRODUCTION

The CTD of the mammalian RNA polymerase II (RNAPII) subunit POLR2A contains 52 heptapeptide repeats, the N-terminal half containing mostly consensus heptads (Tyr1-Ser2-Pro3-Thr4-

Ser5-Pro6-Ser7) and the C-terminal half many more heterogeneous repeats 1. These repeats can be phosphorylated on Tyr1, Thr4, and all three Ser residues, and specific phosphorylation patterns are important for various aspects of transcription, as well as co-transcriptional RNA processing and histone modifications 2-6. Two non-consensus human CTD Arg residues, R1603 and R1810, are conserved in vertebrate species. It was recently found that asymmetric dimethylation (me2a) of R1810 by the

CARM1/PRMT4 methyltransferase in human inhibits expression of snRNA and snoRNA genes7. It was also shown that this R1810me2a mark can be bound by the Tudor domain of TDRD3 in vitro7. We now show that the CTD residue R1810 can also be symmetrically dimethylated (me2s), a modification that requires the Protein Arginine Methyltransferase 5 (PRMT5). This R1810me2s modification recruits SMN, which then interacts with Senataxin, a helicase needed for resolving R-loops in transcription regions.

RESULTS

Identification of symmetric dimethylation of R1810 on the RNAPII CTD

We performed immunoprecipitations using tagged TDRD3 and the RNAPII POLR2D subunit and observed that both tagged proteins could co-immunoprecipitate POLR2A with the me2a modification as detected by western blotting with the ASYMM24 antibody specific for Arg-me2a, whereas only precipitation of POLR2D, and not TDRD3, co-immunoprecipitated a form of POLR2A with an me2s modification that could be detected by the SYMM10 or Y12 antibodies specific for Arg- me2s (Extended Data Fig. 1a). We thus generated polyclonal antibodies against a CTD R1810me2s- containing 7mer peptide and found that immunoprecipitated POLR2A is recognized by the R1810me2s antibody (Fig.1a). To further determine whether the Arg-me2s modification indeed involved R1810,

Raji cells stably expressing α-amanitin-resistant, HA-tagged, wild type or R1810A mutant POLR2A were generated7. After treatment with α-amanitin to deplete endogenous α-amanitin-sensitive RNAPII, followed by immunoprecipitation of RNAPII with anti-HA antibody, western blotting with antibodies recognizing R1810me2a7 or R1810me2s, as well as Y12 antibody, revealed that the R1810A mutation causes loss of both modifications, R1810me2a and R1810me2s (Fig. 1b, Extended Data Fig. 1b). The precipitated RNAPII was dephosphorylated prior to western blotting to enable more sensitive detection

of R1810me2s (Extended Data Fig. 1c). The specificities of the Y12 and R1810me2s antibodies are illustrated in the slot blots of Extended Data Fig.1d: they recognize an Arg-me2s peptide bracketing

CTD R1810 much better than peptides with no Arg modification or R1810me2a. Therefore, R1810 is symmetrically dimethylated in cell extracts. The experiment of Extended Data Fig. 1a also indicates that TDRD3 recognizes R1810me2a in cell extracts, as well as in vitro7, although TDRD3 does not mediate inhibition of snRNA and snoRNA gene expression by R1810me2a8.

Symmetric dimethylation of CTD residue R1810 requires PRMT5

The PRMT5/WDR77 complex is known to associate with RNAPII through the CTD phosphatase FCP19, which is consistent with our observation that PRMT5 co-purifies with RNAPII

(Fig. 1a), so we tested whether PRMT5 might be needed to symmetrically dimethylate R1810.

HEK293 cell lines stably expressing shRNAs for CARM1, PRMT5, and GFP were generated, and endogenous RNAPII was precipitated. Western blotting revealed that CARM1 knock-down causes loss of the R1810me2a mark but not the R1810me2s mark on RNAPII, whereas PRMT5 knock-down causes loss of the R1810me2s mark but not the R1810me2a mark (Fig. 1c, Extended Data Fig. 1e-f).

Consistently, transient siRNA-mediated knock-down of PRMT5 also reduced R1810me2s, as detected by R1810me2s and Y12 antibodies, whereas over-expression of FLAG-tagged PRMT5 increased

R1810me2s (Fig. 1d, Extended Data Figs. 1g, 3a, 3c). These experiments indicated that PRMT5 is required in vivo for the R1810me2s modification on the RNAPII CTD.

PRMT5 is the catalytic subunit of the methylosome, which also contains WDR77 (MEP50)10,11.

To test whether PRMT5 can directly methylate CTD arginine residues, we incubated recombinant

PRMT5/WDR77 with tritiated S-adenosyl methionine (SAM [3H] ) and recombinant GST-N-CTD containing CTD repeats 1-29, which includes R1603, or GST-C-CTD containing CTD repeats 24-52, which includes R1810. Scintillation counting then monitored SAM [3H] labeling following

glutathione-agarose pull-down of the GST fusion proteins. This revealed that GST-C-CTD and GST-

N-CTD were both methylated above the background defined by GST alone (which contains 14 arginines) (Fig. 1e). When the PRMT5 methylation assays were repeated with biotinylated 13-mer peptides containing R1810 or R1603, methylation was again observed on both R1603 and R1810 (Fig.

1f). These experiments indicated that PRMT5/MEP50 may require an additional co-factor9 and/or appropriate CTD phosphorylation to specifically methylate R1810.

Recognition of R1810me2s by SMN

Modified CTD residues and dimethylated Arg residues usually mediate their biological effects via interacting proteins, in the latter case by Tudor domains12,13. Because the Tudor domains of SMN and SPF30, as well as TDRD1, 2, 9, and 11, specifically bind Arg-me2s12, we used them in fluorescence polarization assays to identify whether any could bind an FITC-tagged 13mer CTD peptide containing R1810me2s. Of these, only SMN’s exhibited binding, with much lower affinity for

R1810me2a and R1603me2s peptides and no detectable affinity for the unmodified peptides (Fig. 2a,

Extended Data Fig. 2a, 2c). In contrast, the TDRD3 Tudor domain showed weak affinity (not shown) only for R1810me2a > R1603me2a above background (no modification or Arg-me2s), in line with published data7,14. Compared to a CTD peptide with R1810me2s alone, the presence of additional phospho-Tyr1 or –Ser2 modifications, or both, on the peptide only slightly enhanced its binding to

SMN in fluorescence polarization assays and had no significant effect in isothermal titration calorimetry assays (Fig 2b, Extended Data Fig 2b, 2d ). Other phosphorylations near R1810 also had no effect on SMN binding in vitro (data not shown), indicating SMN association with R1810me2s is not greatly influenced by CTD phosphorylation.

We also found, using co-immunoprecipitation, that SMN and POLR2A interact in cell extracts

(Fig. 1a). Consistent with specific recognition of the R1810me2s modification by SMN,

immunoprecipitation of SMN from HEK293 cell extracts co-precipitated endogenous RNAPII with the

R1810me2s modification but not R1810me2a (Fig. 1a, Extended Data Fig. 3b). To test whether R1810 is important for the association of RNAPII with SMN in vivo, HA-tagged wild type or mutant

(R1810A) RNAPII were immunoprecipitated with anti-HA antibody. Western blotting showed that the

R1810A mutation causes loss of the association of RNAPII with both SMN and TDRD3 (Fig. 2c). As expected, immunoprecipitation of endogenous RNAPII from HEK293 cells expressing shRNAs for

GFP, CARM1, or PRMT5 revealed that only knock-down of PRMT5 reduced co-precipitation of SMN

(Fig. 2d). Similarly, transient siRNA-mediated knock-down of PRMT5 reduced RNAPII-SMN interaction, whereas over-expression of PRMT5 enhanced RNAPII-SMN interaction (Extended Data

Fig. 3a, 3c). Consistent with R1810me2s recognition by SMN, immunostaining of SMN and RNAPII revealed substantial co-localization (Extended Data Fig. 3d).

These experiments showed that R1810me2s controls the SMN-RNAPII interaction in cell extracts. To examine whether SMN also displays R1810me2s-dependent association with RNAPII during transcription, SMN ChIP was performed. As shown in Fig. 2e and Extended Data Fig. 4a, SMN associates with the β-Actin and Gapdh genes from their promoter regions to their termination regions.

Moreover, PRMT5 knock-down with shRNA (Fig. 2f, Extended Data Fig. 4b) or siRNA (not shown) strongly reduced the SMN ChIP signals along these genes, as did shRNA for SMN (Fig.2f), showing the specificity of the SMN antibody for ChIP. As well, the SMN ChIP signals on β-Actin and Gapdh decreased in the RNAPII R1810A mutant, compared to wild type, after α-amanitin treatment to eliminate endogenous POLR2A (Fig. 2g, Extended Data Fig. 4c). These experiments indicated that

R1810me2s recruits SMN to RNAPII elongation complexes. Consistent with our observation that

SMN is recruited from promoters to 3’-ends, we found that immunoprecipitation of SMN co- precipitated RNAPII with CTD phosphorylation on Ser2 and Ser5 (Extended Data Fig. 3b). SMN

ChIPseq was also performed, but probably because SMN doesn’t bind to DNA directly, enriched but weak SMN signals were only observed at promoter and termination regions (Extended Data Fig. 3e).

Interaction of SMN with the RNAPII termination factor Senataxin

SMN is known to interact with Senataxin15, a DNA:RNA helicase encoded by the SETX gene and important for termination by RNAPII15,16. By immunoprecipitating SMN, we confirmed that SMN indeed interacts with Senataxin (Fig. 1a, 3a), and immunoprecipitation of Senataxin also co- precipitated SMN (Fig.3b). The SMN-Senataxin interaction is apparently not mediated by RNAPII, as the interaction persists after 3 days of treatment with α-amanitin to eliminate the bulk of the RNAPII

(Fig. 3b). Moreover, the SMN-Senataxin interaction is reduced when PRMT5 is stably knocked down, indicating that the interaction is likely mediated by the Tudor domain in SMN and an Arg-me2s modification on Senataxin (Fig. 3c). Senataxin also co-precipitated with HA-tagged, α-amanitin- resistant, wild type RNAPII, and the interaction was reduced by the R1810A mutation (Fig.2c), indicating that the Senataxin-RNAPII interaction is likely stabilized by SMN. Consistent with this, in cells with stable shRNA-mediated or transient siRNA-mediated knock-down of PRMT5 or SMN, the association of Senataxin with RNAPII was reduced (Fig. 3d, 3e, Extended Data Figs 3c, 1h-j).

Therefore, we used ChIP to test whether SMN and R1810 are important for Senataxin recruitment to elongating transcription complexes. Senataxin ChIP in Raji cells depleted of endogenous POLR2A and expressing wild type or R1810A mutant RNAPII revealed that R1810 is important for Senataxin recruitment all along the β-Actin and Gapdh genes, including their termination regions (Fig. 3g,

Extended Data Fig. 4d, 4e). As well, SMN or PRMT5 knock-down revealed that both are important for

Senataxin recruitment (Fig. 3h, Extended Data Fig. 4f). Knock-down of Senataxin showed that the

Senataxin antibody used for ChIP was specific for Senataxin (Fig. 3h). Because Senataxin is a termination factor20, 21, these experiments showed that R1810, SMN, and PRMT5 should also be

important for termination by RNAPII. Consistent with this, XRN2, a 5’→3’ exonuclease involved in termination by RNAPII17, also co-precipitated with SMN and RNAPII (Fig. 1a).

SMN and R1810me2s are important for R-loop resolution and termination by RNAPII

To test this hypothesis, we carried out ChIP on α-amanitin-resistant wild type or R1810A

RNAPII after depleting endogenous α-amanitin-sensitive RNAPII. There was enrichment of the

R1810A mutant over wild type RNAPII downstream of the cleavage and polyadenylation sites where

RNAPII pauses and terminates transcription on β-actin (Fig. 4b, 4d, Extended Data Fig. 5) and Gapdh

(Extended Data Fig. 6c, 6d), as detected by various anti-POLR2A antibodies (4H8, H224, 8WG16).

The effect of R1810 on RNAPII accumulation in termination regions was confirmed by carrying out a variant of the global run-on procedure in which nuclei are incubated with BrUTP and short run-on

RNAs are isolated by binding to anti-BrU antibodies 18. This experiment also indicated that the

R1810A mutation leads to over-accumulation of active RNAPII in regions downstream of the poly(A) sites on β-actin and Gapdh (Fig.4c, Extended Data Fig. 6e). When Senataxin is stably knocked down,

RNAPII similarly over-accumulates in the termination region of β-actin 16 (Fig. 4a), suggesting

Senataxin and R1810 are both needed for termination and release of RNAPII from DNA at the pause sites. Consistent with the idea that it is the R1810me2s modification that leads to a failure of RNAPII release, stable shRNA-mediated, transient siRNA-mediated knock-down or CRISPR-mediated knock out of SMN or PRMT5 led to similar over-accumulation of RNAPII in termination regions (Fig. 4a, 4d,

Extended Data Fig. 6b, 6d, 7, 8a). We then performed ChIP-seq of RNAPII for shSMN versus shGFP, and R1810A versus WT RNAPII samples in Raji cells, generating ~10 million unique reads per sample. When normalizing reads to gene bodies and comparing to the control, SMN knock down and the R1810A mutation on CTD both led to RNAPII over-accumulation in termination regions in a

genome wide pattern (Fig. 4e, Extended Data Fig 9). Similar patterns were observed with antibodies against phospho (4H8), non-phospho (8WG16), or total (N20) RNAPII.

It was recently shown that formation of RNA/DNA hybrids (R-loops) by elongating RNAPII over pause sites downstream of poly(A) signals and their resolution by the Senataxin helicase are important for recruiting the 5’→3’ exonuclease XRN2 and termination by RNAPII 16. The monoclonal antibody S9.6 binds RNA/DNA hybrids (Fig. 5a), and, when we used this antibody for DNA immunoprecipitation (DIP), we found that, like depletion of Senataxin16 (Fig. 5b), mutation of R1810

(Fig. 5c) or knock-down of SMN or PRMT5 (Fig. 5b) led to over-accumulation of R-loops in the termination region on the β-actin gene. A second method for R loop detection using a GFP fusion construct that includes the RNase H1 R loop binding domain (GFP-HB) was also used19. Between control and SMN CRISPR knock out cells that stably express GFP-HB, increased R loop formation was detected in the termination regions in SMN knock out cells by GFP ChIP (Extended Data Fig 8b).

These experiments indicated that R1810me2s, PRMT5, and SMN lie upstream of Senataxin in a common pathway important for R-loop resolution and transcription termination.

To show that the phenotypes for RNAPII and R loop accumulation in the termination region are relevant to the SMA disease state, human cell lines (fibroblasts and B lymphocytes) were obtained from the Coriell Institute (Extended Data Fig. 8d) that include cells from 2 children with SMA and their normal parents. RNAPII ChIP and R loop DIP performed as before on the β-actin gene showed that, compared to the control (average of the 2 parents), RNAPII and R loops tend to accumulate in the termination region in the SMA cells (Extended Data Fig. 8e, 8f).

An important issue is why R-loop accumulation in termination regions might contribute to neurodegeneration. By using RNA-seq to examine the effects on gene expression of the R1810A mutation in Raji cells, we confirmed (data not shown) that there are few changes in gene expression aside from the previous noted up-regulation of the expression of various snoRNAs and snRNAs7.

Another possibility is that the R1810me2s-SMN-SETX pathway prevents SMA by affecting splicing, and deeper analysis of our RNA-seq data (not shown) has revealed that the R1810A mutation causes many alterations in splicing. However, it is not clear whether these splicing changes are caused by enhanced expression of snRNA genes, control by the R1810me2s-SMN-SETX pathway of RNAPII elongation kinetics20,21, or recruitment by this pathway of TDP-43 and FUS, which do affect splicing 22-

25. Still another possibility is that genome instability caused by less efficient removal of R-loops 26 may contribute to the neurodegeneration characteristic of SMA. To test whether R-loop accumulation in the β-actin gene termination region would lead to DNA damage, we used ChIP to assay the effects of the R1810A mutation and the depletion of SMN or PRMT5 on the accumulation of gammaH2AX27

(Fig. 5e, f Extended Data Fig. 10a, b). As expected, we observed an accumulation of gammaH2AX and increased gammaH2AX/H2AX ratio in the termination region where RNAPII and R-loops accumulate.

DISCUSSION

That R1810 in the RNAPII CTD is symmetrically dimethylated by PRMT5 is substantiated by many observations described here: first, immunoprecipitated RNAPII binds in a western blot two different antibodies, SYMM10 and Y12, specific for Arg-me2s, as well as antibodies raised against an

R1810me2s peptide and specific for the Arg-me2s modification; second, the SDS gel mobility of this band is the same as that of the largest subunit of RNAPII; third, recognition of the modification by the antibodies depends on R1810; fourth, the modification recognized by the antibodies depends on

PRMT5; and fifth, R1810 and PRMT5 are needed for the association with RNAPII and the recruitment to transcribed regions of SMN, whose Tudor domain is specific for Arg-me2s. Symmetric dimethylation of R1810 in the RNAPII CTD causes the direct recruitment of SMN and indirect

recruitment of the RNA/DNA helicase Senataxin, followed by R-loop resolution and, in termination regions, recruitment of the 5’→3’ exonuclease XRN2 and efficient termination by RNAPII (see model in Extended Data Fig. 10c). Mutation of R1810 or depletion of SMN or PRMT5 may also sometimes lead to RNAPII accumulation in promoter-proximal regions, perhaps because some RNAPII molecules that pause downstream of promoters 18 terminate prematurely in a process that depends on R1810me2s and SMN.

PRMT5, the only type II PRMT seemingly important for symmetrically dimethylating arginine

28, has many other known substrates, including the SPT5 subunit of RNAPII elongation factor DSIF 29.

R1810 is also asymmetrically dimethylated by the Type I PRMT CARM17, but it is not uncommon for

Type I and Type II PRMTs to compete for deposition of me2a and me2s on the same substrates 30.

Other examples of alternative modifications of the same Arg residue are PRMT5 antagonizing

PRMT1’s activating histone H4R3me2a mark by depositing repressive me2s marks on histones H4R3 and H3R8 13,31,32, and alternative modifications of SPT5 R698 by PRMT1 and PRMT5 to regulate its role in RNAPII elongation 29.

SMN, which recognizes R1810me2s, can self-aggregate through its N-terminal K-rich domain and C-terminal YG box, amplifying its potential for Arg-me2s-mediated protein-protein interactions33-

35. Complete loss of SMN is lethal in yeast and embryonic lethal in mammals33. While the human genome contains two SMN genes (SMN1, SMN2), mutating one copy of SMN1 decreases total SMN protein enough to cause the SMA disease phenotype, with disease severity reflecting insufficiency of the remaining SMN for its full range of functions 33. In particular, mutations in SMN1’s oligomerization and Tudor domains can cause SMA33,36.

The GEMIN-containing SMN complex serves as an assembly factor for various snRNPs that participate in spliceosome assembly33,37, although splicing deficiencies may be insufficient to account for SMA 38. Just as SMN facilitates snRNP assembly through binding to Arg-me2s modifications on

Sm proteins, SMN may act similarly to assemble an R-loop resolving complex on the RNAPII CTD through binding to Arg-me2s-modified proteins. The ability of SMN to oligomerize may enable it to recruit multiple molecules of Senataxin and other termination factors to facilitate R-loop disassembly and transcription termination. Indeed, many of the RNAPII elongation and termination factors are known to contain dimethylated Arg, including XRN2, 3 subunits of CPSF (1, 5, and 6), CSTF2, 3 of

PolyA binding proteins (PABP1, 2, and 4), RBBP6, WDR33, PCF11, SPT5, CTDP1, DHX9, FUS, and

EWSR139-41. Persistence of the R-loops may then lead to DNA damage and genome instability, as has been shown to be the case when Senataxin is depleted27. Because Senataxin is the locus for ALS4 and

AOA2 mutations 42,43, defects in this pathway may lead to various types of neurodegenerative disease, and, in view of the fact that TDP-43 and FUS, which have been implicated in ALS, interact with each other 44,45 and SMN 46, it has already been argued that ALS and SMA are related diseases46.

METHODS SUMMARY

Details regarding the sources of reagents, DNA constructs, and antibodies, the preparation of peptides and recombinant proteins, and the various published and modified procedures used for methyltransferase assays, fluorescence polarization assays, isothermal titration calorimetry assays, cell culture, RNA interference, immunostaining, immunoprecipitation, western blotting, chromatin immunoprecipitation (ChIP), ChIP-seq, and DNA immunoprecipitation (DIP) are to be found in

Methods. The number of times each of the experiments described in this work was performed with similar results is shown in Extended Table 1.

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Acknowledgements We thank: Dr. J. Manley for GST-CTD constructs; Dr. D. Reinberg for RNAPII me2a antibodies; Dr. D. Eick for the wild type and mutant RNAPII R1810A constructs; and Dr. Leppla for purified S9.6 antibodies. We also thank Dr. D. Torti and Danica Leung for Illumina library preparation and sequencing, T. Hajian for the purified PRMT5/WDR77 complex, J. Li for the purified

8WG16 antibodies, S. Kulkarni for microscope training, and Drs. B. Blencowe and D. Durocher for constructive criticism and advice during the course of this work. This project was supported by the

Ontario Research Fund from the Ontario Ministry of Research and Innovation (to J.F.G. and T.P).

D.Y.Z was supported by a National Science and Engineering Research Council of Canada Studentship and an Ontario Graduate Scholarship.

Author contributions J.F.G. supervised the project. D.Y.Z performed the experiments. J.F.G. and

D.Y.Z. wrote the manuscript. T.P., B.J.B, Z.N., and G.G commented on experiments and edited the manuscript. G.G. prepared the FITC peptides. F.W.S. performed ChIPseq experiments. U.B. and Y.L. performed computational data analysis for ChIP-seq. Vectors for shRNAs were provided by J.M., and

G.Z. generated stable shRNA-mediated knock-down cell lines. Z.N. generated CRISPR knock out cell lines. J.M. and K.L provided the purified Tudor domains. M.V. provided the purified

PRMT5/WDR77 complex.

Author information Reprints and permissions information is available at www.nature.com/reprints.

The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to J.F.G. ([email protected])

FIGURE LEGENDS

Figure 1 | Symmetric dimethylation of R1810 on the RNAPII CTD and a requirement for

PRMT5. a-d, Immunoprecipitations (IP) with the indicated antibodies from HEK293 (a,c,d) or Raji

(b) whole cell lysates (WCL) after stable (sh) or transient (si) knock-downs of the indicated proteins

(c,d) followed by western blotting with the indicated antibodies. Anti-HA was used to precipitate HA- tagged wild-type (WT) or R1810A mutant POLR2A in (b). Relative quantification of the western blots is shown under the blots in (b,c,d). e,f, Quantification of methylation with [3H]S-adenosyl-methionine

(SAM) of the indicated GST-CTD fusion proteins (e), as shown in the Coomassie-stained SDS gel of the insert, or 13-mer CTD peptides (f) with the indicated concentrations of recombinant

PRMT5/WDR77. Error bars denote s.e.m. (n=3).

Figure 2 | R1810me2s in the RNAPII CTD is recognized by SMN. a, Fluorescence polarization assays showing binding of SMN to the indicated FITC-labeled CTD peptides containing R1810. b,

Isothermal titration calorimetry assays showing binding of SMN to a CTD peptide containing

R1810me2s. c,d, Immunoprecipitations (IP) with the indicated antibodies from HEK293 (d) or Raji (c) whole cell lysates (WCL) after stable (sh) knock-downs of the indicated proteins (d) followed by western blotting with the indicated antibodies. Anti-HA was used to precipitate HA-tagged wild-type

(WT) or R1810A mutant POLR2A in (c). Relative quantification of the western blots is shown under the blots in (c,d). e,f,g, Quantification of chromatin immunoprecipitation data in HEK293 (e,f) or Raji

(g) cells, expressed as percent input or SMN/POLR2A ratio, using the indicated primer positions for qPCR along the β-actin gene. Also shown are the relative effects of knocking down (KD) PRMT5 or

SMN (f) or mutating R1810 to alanine (g). Error bars denote s.e.m. (n=3).

Figure 3 | R1810me2s and SMN recruit Senataxin to RNAPII. a-e, Immunoprecipitations (IP) with the indicated antibodies from HEK293 whole cell lysates (WCL) after stable (sh) knock-downs of the indicated proteins (c,d,e) followed by western blotting with the indicated antibodies. The cells were grown for 3 days in the presence of α-amanitin in the lower panel of (b) to eliminate endogenous

POLR2A as shown in the western blot of the upper panel. Relative quantification of the western blots is shown under the blots in (c,d,e). f,g,h, Quantification of chromatin immunoprecipitation data in

HEK293 (f,h) or Raji (g) cells, expressed as percent input or Senataxin/POLR2A ratio, using the indicated primer positions for qPCR along the β-actin gene (see Fig. 2). Also shown are the relative effects of knocking down (KD) PRMT5, SMN, or Senataxin (h) or mutating R1810 to alanine (g).

Error bars denote s.e.m. (n=3).

Figure 4 | SMN and R1810me2s regulate transcription termination by RNAPII. a-d,

Quantification of RNAPII chromatin immunoprecipitation using POLR2A antibody (4H8, 8WG16,

N20) (a,b,d) or nuclear run-on (c) in HEK293 (a) or Raji (b,c) cells, using the indicated primer positions for qPCR along the β-actin gene, after knocking down PRMT5, SMN, or Senataxin (a), with

GFP knock-down as a negative control, or mutating R1810 to alanine (b). Normalized ratios to control

POLR2A for a, b are displayed in d. Error bars denote s.e.m. (n=5 for a,b,d; n=3 for c). e. RNAPII

ChIP-seq for SMN (purple) versus GFP (grey) stable knock down (Raji), and of R1810A (red) versus

WT (blue) POLR2A (Raji) with signals normalized to the gene body. The 5% most highly expressed genes (~800) with reliable ChIPseq signals were analyzed.

Figure 5 | SMN and R1810me2s are important for resolving R-loops created by elongating

RNAPII and preventing DNA damage a-d, Quantification of DNA immunoprecipitation (DIP) with the S9.6 antibody in HEK293 cells (a,b) with or without knock-down of SMN, PRMT5, or Senataxin

(b) or in Raji cells after mutating R1810 to alanine (c). Error bars denote s.e.m. (n=3 to 5).

Normalized ratios to control POLR2A for b, c are displayed in d. The effect of treating the nucleic acid with RNase H prior to DIP is shown in a. e,f, Quantification of gammaH2AX chromatin immunoprecipitation in HEK293 (e) or Raji (f) cells, along the β-actin gene, after knocking down

PRMT5 or SMN (e), with GFP knock-down as a negative control, or mutating R1810 to alanine (f).

Error bars denote s.e.m. (n=4).

EXTENDED DATA FIGURE LEGENDS

Extended Data Figure 1 | The R1810me2s and R1810me2a modifications on POLR2A depend on

PRMT5 and CARM1, respectively. a, POLR2A carries Rme2a and Rme2s modifications. Whole cell lysates (WCL) from HEK293 cells stably expressing Flag-tagged TDRD3 or the POLR2D subunit of RNAPII were used for immunoprecipitation using beads conjugated with M2 anti-Flag antibody, and the precipitates were western blotted with the indicated antibodies. Cells expressing Flag-GFP were used as negative control. Precipitated TDRD3 and POLR2D contained POLR2A with the Arg-me2a modification (ASYMM24 antibody), whereas precipitated POLR2D, and not TDRD3, contained

POLR2A with the Arg-me2s modification (SYMM10 and Y12 antibodies). b, Whole cell lysate western blot controls for Figure 1b. c, Y12 and R1810me2s recognition of RNAPII CTD R1810me2s is blocked by surrounding phosphorylated residues. The detection of R1810me2s improves for both antibodies when the precipitated samples are treated with alkaline phosphatase. d, Slot blots illustrating that the Y12 and R1810me2s antibodies specifically recognize peptides containing RNAPII

R1810me2s. The indicated amounts of biotin-labelled 7mer CTD peptides bracketing R1810 with no modification, Arg-me2a, and Arg-me2s were blotted before incubating with the R1810me2s or Y12

antibodies. e, Western blot confirming efficient PRMT5 knock-down, and RT-qPCR assay confirming efficient CARM1 knock-down for experiment of Fig. 1c. f, g, Whole cell lysate western blot controls for Figures 1c and 1d, respectively. h, i, j, Whole cell lysate western blot controls for Figures 3c, 3d,

3e, respectively.

Extended Data Figure 2 | Recognition of R1810me2s by SMN. a-c, In vitro fluorescence polarization (FP) peptide binding assays. a, Recombinant SMN Tudor domain was incubated with

FITC-labeled 13mer CTD peptides bracketing either R1810 or R1603. SMN preferentially bound CTD peptides in the order R1810me2s > R1603me2s ~ R1810me2a > R1603me2a, and exhibited no detectable affinity for the unmodified peptides. b, Recombinant SMN Tudor domain was incubated with FITC-labelled CTD peptides bracketing R1810me2s also containing Y1P, S2P, or both upstream of R1810me2s, showing slightly preferential binding to the peptides when the phospho-modification(s) are present. c, FITC-CTD R1810me2s or FITC-CTD R1810me2a is not recognized by other recombinant Tudor domains from SMNDC1/SPF30, TDRD1, TDRD2, TDRD9, TDRD11/SND1. d,

Isothermal titration calorimetry assays showing that the recombinant SMN Tudor domain has no enhanced binding to R1810me2s containing peptide also carrying S2P or both Y1P and S2P.

Extended Data Figure 3 | Interaction of SMN and Senataxin with RNAPII depends on PRMT5. a, PRMT5 over-expression increases the R1810me2s modification and the SMN and Senataxin associations with RNAPII. Left: western blots with the indicated antibodies of HEK293 whole cell lysates (WCL) over-expressing Flag-tagged PRMT5 or GFP. Over-expressing PRMT5 does not increase the amount of SMN or Senataxin. Right: endogenous POLR2A was immunoprecipitated (N20 antibody) from HEK293 cell lysates with over-expressed Flag-tagged PRMT5 or GFP. b, SMN associates with many phosphor- isoforms of RNAPII. Immunoprecipitation with SMN antibody, but

not control IgG, co-precipitated RNAPII with unmodified CTD repeats (8WG16 antibody) and CTD repeats phosphorylated on Ser5 and Ser2 as detected by western blotting, but not RNAPII with the

R1810me2a modification. c, requirement of PRMT5 for association of SMN and Senataxin with

RNAPII. Left: western blots with the indicated antibodies of HEK293 whole cell lysates (WCL) expressing siRNAs for PRMT5 or SMN. Right: endogenous POLR2A was immunoprecipitated (N20 antibody) from cells with transient siRNA-mediated knock-down of PRMT5 or SMN. Western blots were performed with the indicated antibodies. PRMT5 knock-down causes loss of R1810me2s on

RNAPII, as well as reduced association of SMN and Senataxin with RNAPII. SMN knock-down causes reduced association of Senataxin with RNAPII. d, SMN co-localizes with active RNAPII in the nucleus. HEK293 cells were used for co-staining of SMN (green) and POLR2A with its CTD phosphorylated on Ser2 (red). Hoechst was used to stain the nucleus (blue). e, SMN ChIPseq (with

GFP antibody against inducible GFP-SMN, or with SMN specific antibody in Hek293 cells). Both methods observe enriched SMN signals at promoter and termination regions.

Extended Data Figure 4 | Association of SMN and Senataxin with the Gapdh gene depends on

R1810 and PRMT5. a, Chromatin immunoprecipitation (ChIP) was used to determine the distribution of SMN along the human Gapdh gene in HEK293 cells, expressed as percent input or as a ratio of

SMN to RNAPII. Error bars represent technical variation in a single experiment (mean ± s.e.m., n=3). b, SMN ChIP was performed in HEK293 cells stably expressing shRNAs for PRMT5 or GFP (as a control). With the control normalized to 1, knock-down of PRMT5 caused strong reductions of the

SMN ChIP signals all along Gapdh. Error bars represent biological replicates (mean ± s.e.m., n=3). c,

SMN ChIP signals on Gapdh decrease in Raji cells expressing HA-tagged R1810A mutant RNAPII after 3 days of treatment with α-amanitin to eliminate endogenous POLR2A. ChIP results with wild type HA-tagged RNAPII were normalized to 1. Error bars represent biological replicates (mean ±

s.e.m., n=3). d, Chromatin immunoprecipitation (ChIP) in HEK293 cells showing the distribution of

Senataxin along the human Gapdh gene, expressed as percent input or as a ratio to RNAPII. Error bars represent technical variation in a single experiment (mean ± s.e.m., n=3). e, Senataxin ChIP signals on

Gapdh decrease in the POLR2A R1810A mutant after 3 days of treatment of Raji cells with α-amanitin to eliminate endogenous POLR2A. Results are normalized to wild type POLR2A and expressed as the ratio of Senataxin to RNAPII. Error bars represent biological replicates (mean ± s.e.m., n=3). f,

Knock-down of PRMT5 or SMN causes reductions of the Senataxin ChIP signals all along Gapdh.

ChIP against Senataxin was performed in HEK293 cells with shRNA-mediated knock-down of PRMT5 or SMN. Results were normalized to a control knock-down of GFP and expressed as the ratio of

Senataxin to RNAPII. Error bars represent biological replicates (mean ± s.e.m., n=2).

Extended Data Figure 5 | The R1810 mutation causes RNAPII to accumulate in the termination region of the β-actin gene. Chromatin immunoprecipitation (ChIP) with three different POLR2A antibodies (8WG16, H224, 4H8), as indicated, was performed on the β-actin gene in Raji cells expressing HA-tagged wild type or mutant (R1810A) POLR2A 3-days after treatment with α-amanitin to eliminate endogenous POLR2A. Shown are single experiments with error bars representing technical variation (mean ± s.e.m., n=3).

Extended Data Figure 6 | R1810, PRMT5, and SMN regulate transcription termination on the

Gapdh gene. a, Chromatin immunoprecipitation (ChIP) with the N20, 4H8, and 8WG16 antibodies to show the distribution of wild type POLR2A along the human Gapdh gene. Error bars represent technical variation (mean ± s.e.m., n=3). b, POLR2A ChIP along the Gapdh gene was performed in

HEK293 cells after stable knock-down of PRMT5 or SMN, using stable knockdown of GFP as a negative control. RNAPII over-accumulates at the termination sites on Gapdh after knock-down of

PRMT5 or SMN. Error bars represent biological replicates (mean ± s.e.m., n=5).c,POLR2A ChIP on the Gapdh gene was performed in Raji cells that express HA-tagged wild type or mutant (R1810A)

POLR2A 3-days after α-amanitin treatment to eliminate endogenous POLR2A. R1810A mutant

RNAPII over-accumulates downstream of the cleavage and polyadenylation sites where RNAPII pauses and terminates transcription on Gapdh. Error bars represent biological replicates (mean ± s.e.m., n=4). d, Data from b, c are displayed as normalized ratios to the control (GFP knock-down, or

HA wild type POLR2A), with the ratio for the intron 5 qPCR primers at 2436 set as 1. e, Nuclear run- on experiment in which nuclei from Raji cells expressing wild type or mutant (R1810A) POLR2A 3- days after α-amanitin treatment to eliminate endogenous POLR2A were incubated with BrUTP for 30 minutes, and short run-on RNAs were isolated by binding to anti-BrU antibodies. The R1810A mutation led to over-accumulation of active RNAPII in the region downstream of the poly(A) site on

Gapdh. Error bars represent technical variation (mean ± s.e.m., n=3).

Extended Data Figure 7 | PRMT5 and SMN but not CARM1 regulate transcription termination by RNAPII on β-actin and Gapdh. a, Chromatin immunoprecipitation (ChIP) for POLR2A with 4H8 antibody was performed after stable shRNA-mediated PRMT5, CARM1 or SMN knock-down in

HEK293 cells to show that only PRMT5 and SMN knock-downs lead to the over-accumulation of

RNAPII in the termination regions of β-actin. The graph shows a single experiment with error bars representing technical variation (mean ± s.e.m., n=3). b, POLR2A ChIP with the 8WG16 antibody was performed after transient siRNA-mediated knock-down of PRMT5 or SMN in HEK293 cells to show that PRMT5 or SMN knock-down leads to the over-accumulation of RNAPII in the termination region of β-actin. The graph shows a single experiment with error bars representing technical variation (mean

± s.e.m., n=3). c, ChIP for POLR2A with 4H8 antibody was performed after stable shRNA-mediated knock-down of PRMT5, SMN, or GFP (as a control) in HEK293 cells to show that knock-down of

PRMT5 or SMN leads to the over-accumulation of RNAPII in the termination region of Gapdh. The graph shows a single experiment with error bars representing technical variation (mean ± s.e.m., n=3). d, POLR2A ChIP with 8WG16 antibody was performed after transient siRNA-mediated knock-down of PRMT5 or SMN in HEK293 cells to show that knock-down of PRMT5 or SMN leads to the over- accumulation of RNAPII in the termination region of Gapdh. The graph shows a single experiment with error bars representing technical variation (mean ± s.e.m., n=3).

Extended Data Figure 8| RNAPII Pausing Defect and R Loop Accumulation are observed in

SMN CRISPR knock-out cells and in SMA Disease Cells. a, Chromatin immunoprecipitation

(ChIP) for POLR2A with N20 antibody was performed after stable SMN knock-out (CRISPR) in

HEK293 cells shows that RNAPII accumulates in the termination regions of β-actin. Scrambled guide

RNA treatment was used as a negative control. Error bars represent biological variation (mean ± s.e.m., n=3). b, Accumulation of R-loops in the termination regions of the β-actin gene after SMN knock-out. A fusion protein of GFP-RNaseH DNA:RNA hybrid binding domain was stably expressed in HEK293 cells. ChIP with GFP antibody (abcam 290) was used for the detection of the R-loops

(DNA:RNA hybrids), using the indicated primer positions for qPCR along the β-actin gene. Scrambled guide RNA treatment was used as a negative control. Error bars represent biological variation (mean ± s.e.m., n=3). c, Western blot with anti-SMN antibody showing that SMN expression is knocked out by

CRISPR. d, Human cell lines (3 fibroblast, 3 B lymphocyte) were obtained from the Coriell Institute for Medical Research. These include cells from two children with SMA disease and their normal parents. e, ChIP for POLR2A (N20, 8WG16 antibodies) was performed on the β-actin gene using the averaged value of the parents as control. POLR2A in the SMA disease cells (from both fibroblast and

B cell lines) accumulates in the termination regions of the β-actin gene. Error bars represent biological variation (mean ± s.e.m., n=4). f, R loop DIP with the S9.6 antibody shows that R loops accumulate in

the termination regions of the β-actin gene in the SMA disease cells. The averaged value of the parents was used as a control. Error bars represent biological variation (mean ± s.e.m., n=5).

Extended Data Figure 9| SMN and POLR2A R1810 effects on transcription termination by

RNAPII occur on a genome wide level (RNAPII ChIPseq) a, Western blotting using the N20 antibody for POLR2A and the SMN antibody to verify shSMN knock-down of SMN for the ChIPseq experiment, shGFP was used as a control of Fig. 4e. b, Western blot using N20 and HA antibodies to verify gene expression of HA-tagged wild type and R1810A PORL2A and the effect of α-amanitin treatment on cells without an HA-tagged construct for the ChIPseq experiment of Fig 4e. c, RNAPII

ChIPseq results for several housekeeping genes are displayed in detail (B2M, CD40, ATF4- a short gene, JUND– an intronless gene, and TUBA1B) using Integrative genomics viewer. The promoter peaks are displayed to the left, and the regions with red underlines are RNAPII termination regions.

IgG ChIPseq was used as negative control. ~10 million unique RNAPII ChIPseq reads (4H8, 8WG16) were obtained from GFP or SMN stable knock down Raji cells. ~10-12 million unique RNAPII

ChIPseq reads (N20) were obtained from WT or R1810A RNAPII Raji cells upon 3 days of amanitin treatment (2ug/ml).

Extended Data Figure 10 | Model of the pathway that regulates R-Loop Accumulation to prevent

DNA damage a,b Quantification of gammaH2AX and H2AX ratio through chromatin immunoprecipitation in HEK293 (a) or Raji (b) cells, along the β-actin gene, after knocking down

PRMT5, or SMN, with GFP knock-down as a negative control (a), or mutating R1810 to alanine (b) .

Error bars denote s.e.m. (n=4). c, Pathway (boxed) and model showing influence of PRMT5,

R1810me2s, and SMN on R-loop resolution and transcription termination

Extended Data Table 1 |Numbers of times each type of experiment described in this work was performed with the reported results.

IP a c POLR2A (4H8) IP

Western blots Western blots POLR2A (N20) POLR2A (N20) POLR2A 1 1 1 R1810 me2s POLR2A SMN Rme2s (Y12) 1 0.9 0.5 PRMT5 POLR2A R1810me2a 1 0.5 1 Senataxin POLR2A (N20) IP XRN2 d HA IP b Western blots Western blots POLR2A (N20) POLR2A (N20) 1 1.1 1 1 1 POLR2A POLR2A (HA) (8WG16) 1 1 1 1 1.1 POLR2A POLR2A R1810me2a R1810me2s 1 0.3 1 0.5 0.5 POLR2A POLR2A Rme2s (Y12) 1 0.25 Rme2s (Y12) POLR2A 1 0.5 0.5 R1810me2s PRMT5 1 0.5 1 0.6 0.5 e f CTD R1810 2500 GST 6000 CTD R1603 GST-N-CTD 2000 5000 no substrate GST-C-CTD 4000 1500 3000 1000 2000 3H SAM (c.p.m.) SAM3H 500

3H SAM (c.p.m.) SAM3H 1000 0 0 0 0.3 0.5 1 1.5 0 0.1 0.5 1 2 [PRMT5/WDR77] (μM) [PRMT5/WDR77] (μM)

Fig 1| Symmetric dimethylation of R1810 on the RNAPII CTD Time (min) -10 0 10 20 30 40 50 60 70 80 a B b 120 YSPSSPR(me2s)YTPQSP 0.00 110 Kd = 132 ± 13 µM

100 Data: Data1_B,Data1_C,Data1_D

-20.00 Model: OneSiteBind 90 Chi^2/DoF = 23.94598 80 R^2 µWatts = 0.9769 -40.00

70 Bmax 8.34443 ±2.56899 k1 9.27857 ±14.04192 60 YSPSSPR(me2a)YTPQSP Bmax_2 95.76171 ±35.28288 k1_2 900.27001-60.000.00 ±559.09295 50 Kd = 325.7 ± 66 µM Bmax_3 115.22465 ±4.50274 k1_3 69.29586 ±9.54389 40 -4.00 Y Axis Title Y 30 -8.00 20 YSPSSPRYTPQSP 10 Kd = n.b. -12.00 Fluorescence polarization Fluorescence 0 Kd= 175 ± 13.6 µM -10 -16.00 SPSYSPSSPR(me2s)YTPQS KJoules/Mole of Injectant 0 200 400 600 800 1000 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Molar Ratio SMN concentrationX Axis Title (μM) c d POLR2A (4H8) IP WCL(10%) HA IP

Western blots Western blots POLR2A (N20) POLR2A (N20) 1 1 1 1 1 SMN PRMT5 1 1.1 0.5 1 0.1 SMN TDRD3 1 1.1 0.5 1 0.2 β-actin polyA Senataxin 1 0.6

pause sites

f e 12% 1 SMN PRMT5 KD: SMN/POLR2A 0.8 9% SMN KD: SMN/POLR2A 0.6 6% 0.4 3% 0.2 Percent Input Percent 0% 0 80% g 1 R1810A: SMN/POLR2A 60% SMN/WT 0.8 POLR2A 0.6 40%

Ratio 0.4 20% 0.2 0% 0 Normalized to SMN/POLR2A in Control (=1) Control in SMN/POLR2A to Normalized -72 332 -72 332 1671 3560 3752 4657 5590 1671 2911 3560 3752 4657 5590 Positions along the beta-actin gene

Fig 2| RNAPII CTD R1810 contains me2s that is recognized by SMN a IP c Senataxin IP Western blots Western blots SMN Senataxin Senataxin 1 1 1 SMN 1 0.6 0.3 b d POLR2A (N20) IP POLR2A 0.1 1 IP Western blots POLR2A (N20) Western blots 1 0.8 1 Senataxin Senataxin 1 1 0.4 SMN e POLR2A (N20) IP IP (Amanitin+) Western blots Western blots POLR2A (N20) Senataxin 1 0.8 1 Senataxin SMN 1 1 0.4 3% g 1 f Senataxin R1810A: Senataxin/POLR2A 2% 0.8 2% 0.6 1% 0.4

Percent Input Percent 1% 0.2 0% 0 20% h 1 Senataxin/WT POLR2A (=1) Control in /POLR2A PRMT5 KD: Senataxin/POLR2A 15% 0.8 SMN KD: Senataxin/POLR2A

Senataxin KD: Senataxin/POLR2A 0.6

10% Senataxin Ratio 0.4 5% 0.2

0% 0 Normalized to to Normalized -72 332 -72 332 1671 3560 3752 4657 5590 Positions along the beta-actin 1671 gene2911 3560 3752 4657 5590

Fig 3| SMN-Senataxin form a complex with RNAPII β-actin polyA c 12% WT POLR2A 9% R1810A POLR2A

6% a pause sites 3% 100% shGFP POLR2A shPRMT5 POLR2A 0% 80% extract in RNAof Percent -72

shSMN POLR2A 332 1671 2911 3560 3752 4657 5590 60% shSETX POLR2A d 4 shPRMT5 POLR2A 3

40% 2

Percent Input Percent 1 20% 0 5 0% shSMN POLR2A 4 -72 332

b 1671 2911 3560 3752 4657 5590 3 2 WT POLR2A 1 40% R1810A POLR2A 0 4 R1810A POLR2A 3 20% 2 Normalized to the Control POLR2A (=1) POLR2A Control the to Normalized Percent Input Percent 1 0% 0 -72 332 1671 2911 3560 3752 4657 5590 -72 332 1671 2911 3560 3752 4657 5590 Positions along the beta-actin gene e

Fig 4| SMN and R1810me2s regulate transcription termination by RNAPII a 25% β-actin polyA R loop RNase H- 20% R loop RNase H+ 15%

10%

b pause sites Input Percent 120% 5% shGFP R loop shPRMT5 R loop 0% -72 90% 332

shSMN R loop 1671 3560 3752 4657 5590 d 4 shSETX R loop shPRMT5 60% 3 R loop

Percent Input Percent 30% 1 0 6 0% 5 shSMN R loop c 4 3 40% WT R loop 2

R1810A R loop 1 30% 0 4 R1810A R loop 20% 3

Normalized to the Control R loop (=1) loop R Control the to Normalized 2

Percent Input Percent 10% 1 0% 0 -72 332 e f 1671 3560 3752 4657 5590 8% 10% shGFP WT

6% shSMN 8% shPRMT5 R1810A 6% 4% 4% Percent Input Percent 2% Input Percent 2%

0% 0%

Positions Along the beta-actin gene

Fig 5| SMN and R1810me2s are important for resolving R-loops created by elongating RNAPII a b WCL c FLAG IP POLR2A Co-IP (N20 Hek293)

Western blots Western blots POLR2A (HA) 250 POLR2A Rme2a (ASYMM24) 1 1.1 Y12 POLR2A (N20) 250 POLR2A Rme2s R1810me2s (SYMM10) 1 1 250 POLR2A (N20) POLR2A Rme2s TUBULIN (Y12) 1 1

d Y12 antibody R1810me2s antibody e CARM1 (qPCR) 1 PRMT5 0.8 CARM1 100 nmol 10 nmol 0.6 TUBULIN 2 nmol 0.4 10 nmol 0.2 0.5 nmol 2 nmol 0

0.5 nmol g WCL

f WCL Western blots Western blots POLR2A (N20)

POLR2A (N20) 1 1 1 POLR2A (8WG16) 1 1 1 1 1 1.1 PRMT5 PRMT5 1 1.1 0.5 1 0.6 0.5 TUBULIN TUBULIN 1 1 1 1 1 1 WCL h WCL i WCL j

Western blots Western blots Western blots POLR2A POLR2A (N20) Senataxin 1 1.2 1.1 1 1 1 1 0.9 1.1 Senataxin PRMT5 Senataxin 1 0.5 1 1 1 1.3 1 0.9 1

SMN SMN PRMT5

1 1 0.5 1 1 0.5 1 1 0.5 TUBULIN TUBULIN TUBULIN 1 1 1 1 1 1 1 1 1 Extended Data Figure 1 | The R1810me2s and R1810me2a modifications on POLR2A depend on PRMT5 and CARM1, respectively. a b 1603 NB SY( R1810p)S( PSPYp)PSSPR(me2s)YTPQSPT kD=120.5±11 1810 kd=430±240 SYS( R1810 pSDM10)PSSPR(me2s)YTPQSPT kD=162.5±18 1810me2a kd=717±200 SYSPSSPR(me2s)YTPQSPT R1810 SDM18 kD=178.5±24 positive control 120 R1810 SDM31 kD=196.9±27 1603me2s kd=150±40140 SY(p)SPSSPR(me2s)YTPQSPT 1810me2s kd=127± 15 100 1603me2a kd=665±272120

80 100

80 60

FP 60 FP 40 40

Fluorescence polarization Fluorescence 20 Fluorescence Fluorescence polarization

0 0

-20 -20 -200 0 200 400 600 800 1000 1200 1400 1600 0 200 400 600 800 SMN concentrationSMN[uM] (μM) SMN concentrationConcenration of SMN[uM] (μM) c 100 100 100 1810 1810 1810 90 1810me2a 1810me2a 90 90 1810me2a 1810me2s 1810me2s 80 1810me2s 80 80 70 70 70 60 60 60 50 50 50

FP 40 FP FP 40 40 30 30 30 20 20 20 10

10 10 0

0 0 -10 -100 0 100 200 300 400 500 600 700 800 900 1000 -20 0 20 40 60 80 100 120 140 0 200 400 600 800 1000 TDRD9[uM] TDRD1[uM] 1810 SMNDC1[uM] 100 1810 100 1810me2a 1810me2a 90 90 1810me2s 1810me2s

80 80

70 70

60 60

50 50 FP FP 40 40

30 30

20 20

10 10

0 0

-20 0 20 40 60 80 100 120 140 0 200 400 600 800 1000 TDRD2[uM] TDRD11[uM] d Time (min) Time (min) -10 0 10 20 30 40 50 60 70 80 -10 0 10 20 30 40 50 60 70 80

0.00 0.00

-2.00 -4.00

-4.00 µWatts µWatts -8.00

-6.00 -12.000.00

-1.00 -2.00

-2.00 -4.00

-3.00 -6.00 Kd =187 ± 32.3 µM Kd =203 ± 21.8 µM -8.00 -4.00 SPSY(p)S(p)PSSPR(me2s)YTPQS SPSYS(p)PSSPR(me2s)YTPQS KJoules/Mole of Injectant KJoules/Mole of Injectant 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Molar Ratio Molar Ratio

Extended Data Figure 2 | In vitro Recognition of R1810me2s by SMN. IP a WCL POLR2A (N20) IP b

Western blots Western blots Western blots SMN POLR2A (N20) POLR2A (N20) POLR2A (N20) 1 0.8 1 0.9 POLR2A SMN Rme2s (Y12) POLR2A-pSer5 (3E8) 1 0.9 1 1.8 Senataxin POLR2A R1810me2s POLR2A-pSer2 (3E10) 1 0.8 1 1.7 SMN PRMT5 POLR2A-no P (8WG16) 1 1.6 1 1.8 Senataxin POLR2A R1810me2a TUBULIN 1 1.3 1 0.9 c WCL POLR2A (N20) IP

Western blots Western blots POLR2A (N20) POLR2A (N20)

1 1.1 1.1 1 1 1 POLR2A Rme2s (Y12) PRMT5 1 0.4 0.9 1 0.5 1.1 POLR2A R1810me2s SMN 1 0.6 1.2 1 1.1 0.5 PRMT5 Senataxin 1 0.4 1 1 1 1 SMN TUBULIN 1 0.6 0.1 1 1 1 Senataxin 1 0.7 0.7 d e SMN SMN

RNAPII pSer5 RNAPII pSer2

Overlap Overlap

Extended Data Figure 3 | Interaction of SMN and Senataxin with RNAPII, depends on PRMT5. polyA

Gapdh

a d pause sites 3.0% 2.4% WT SMN WT Senataxin 2.5% 2.0%

2.0% 1.6% 1.5% 1.2% 1.0% 0.8% Percent Input Percent Input 0.5% 0.4% 0.0% 0.0% 55 1407 2436 3882 4511 5196 55 1407 2436 3882 4511 5196 20% WT SMN/POLR2A 20% WT Senataxin/POLR2A 16% 16%

12% 12%

Ratio 8% Ratio 8% 4% 4% 0% 55 1407 2436 3882 4511 5196 0% 55 1407 2436 3882 4511 5196 b 1 e 1 PRMT5 KD: SMN/POLR2A R1810A: Senataxin/POLR2A

0.8 0.8 =1) =1) ( ( 0.6 0.6

0.4 Control 0.4 Control Control in in 0.2 0.2

0 0 POLR2A POLR2A c 55 1407 2436 3882 4511 5196 /POLR2A f 55 1407 2436 3882 4511 5196 1 SMN/ 1 R1810A: SMN/POLR2A

0.8 Senataxin PRMT5 KD: Senataxin/POLR2A 0.8 SMN KD: Senataxin/POLR2A 0.6 0.6 Normalized Normalized to 0.4 0.4 Normalized Normalized to 0.2 0.2

0 0 55 1407 2436 3882 4511 5196 55 1407 2436 3882 4511 5196 Positions along the gapdh gene Positions along the gapdh gene

Extended Data Figure 4 | Association of SMN and Senataxin with the Gapdh gene depends on R1810 and PRMT5. polyA

β-actin

pause sites

40% 10% WT POLR2A (8WG16) WT POLR2A (H224) 35% R1810A POLR2A (H224) R1810A POLR2A (8WG16) 8% 30%

25% 6% 20% 15% 4% Percent Input Percent Percent Input 10% 2% 5% 0% 0% -72 332 1671 2661 2911 3560 3752 4657 5590 -72 332 1671 2661 2911 3560 3752 4657 5590 20% WT POLR2A (4H8) R1810A POLR2A (4H8) 16%

12%

8% Percent Input 4%

0% -72 332 1671 2661 2911 3560 3752 4687 5590

Extended Data Figure 5 | The R1810 mutation causes RNAPII to accumulate in the termination region of the β-actin gene. polyA Gapdh

pause sites a 40% 2 d R1810A POLR2A WT POLR2A 1.5

30% 1

20% 0.5 Percent Input 10% 0 POLR2A (=1) POLR2A shPRMT5 POLR2A 0% 1.5 55 1407 2436 3882 4511 5196 Control Control b 1 40% WT POLR2A 0.5 shPRMT5 POLR2A 30% 0 shSMN POLR2A Normalized Normalized the to shSMN POLR2A 20% 1.5

PercentInput 1 10%

0.5 0% 55 1407 2436 3882 4511 5196 0 c 40% 55 1407 2436 3882 4511 5196 Control POLR2A R1810A POLR2A 30% e 12% WT POLR2A

20% 10% R1810A POLR2A

8% PercentInput 10% 6%

0% 4% 55 1407 2436 3882 4511 5196

Percent of RNA extract Percentin RNA of 2%

0% 55 1407 2436 3882 4511 5196 Positions along the gapdh gene

Extended Data Figure 6 | R1810, PRMT5, and SMN regulate transcription termination on the Gapdh gene. polyA β-actin

a 20% 30% pause sites shGFP POLR2A shGFP POL2A 16% 25% shCarm1 POLR2A shCARM1 POLR2A

shPRMT5 POLR2A shSMN POLR2A 20% 12% 15% 8%

Percent Input Percent 10% Percent Input Percent 4% 5%

0% 0% -72 1671 2661 2911 3560 3752 4687 5590 -72 1671 2661 3560 3752 4687 5590 b 25% 25% siCtrl POLR2A siCtrl POLR2A 20% siSMN POLR2A 20% siPRMT5 POLR2A

15% 15%

10% 10% Percent Input Percent Percent Input Percent 5% 5%

0% 0% -72 332 1671 2911 3560 3752 4657 5590 -72 332 1671 2911 3560 3752 4657 5590 Positions along the beta-actin gene Positions along the beta-actin gene polyA Gapdh c 30% 14% pause sites shGFP POLR2A shGFP POLR2A 12% 25% shSMN POLR2A

shPRMT5 POLR2A 10% 20% 8% 15% 6% 10% Percent Input Percent Percent Input Percent 4% 5% 2% 0% 0% d 55 1407 2436 3882 4511 5196 55 1407 2436 3882 4511 5196 14% 25% siCONTROL POLR2A 12% siCONTROL POLR2A 20%

siPRMT5 POLR2A 10% siSMN POLR2A 8% 15% 6% 10%

Percent Input Percent 4% Percent Input Percent 5% 2% 0% 0% 55 1407 2436 3882 4511 5196 55 1407 2436 3882 4511 5196 Positions along the gapdh gene Positions along the gapdh gene

Extended Data Figure 7 | PRMT5 and SMN but not CARM1 regulate transcription termination by RNAPII on β-actin and Gapdh. β-actin polyA

a pause sites b c 35% 16% Scramble KO RNAPII (N20) scramble KO GFP-HB 30% 14% SMN KO RNAPII (N20) SMN KO GFP-HB 12% TUB 25%

10% 20% SMN 8% 15%

Percent Input Percent 6% Percent Input Percent 10% 4%

5% 2%

0% 0% -72 332 1671 3560 3752 4657 5590 -72 332 1671 2911 3752 4657 5590 Positions along the beta-actin gene Positions along the beta-actin gene d e 40% Diagnosis Cell Type Origin Affected Family RNAPII (Parents-friboblasts) SMA1 Fibroblast Male (Child) Yes 553 35% SMA1 Fibroblast Female (Parent) No 553 30% RNAPII (SMA-fibroblasts)

SMA1 Fibroblast Male (Parent) No 553 25% 20% SMA1 B-Lymphocyte Female (Child) Yes 3042 15% SMA1 B-Lymphocyte Female (Parent) No 3042 Percent Input 10% SMA1 B-Lymphocyte Male (Parent) No 3042 5% 0% f -72 332 1671 2911 3560 3752 4657 5590 35% 35% R loop (Parents) RNAPII (Parents-B cells) 30% 30% R loop (SMA) RNAPII (SMA-B cells)

25%

20% 20%

15% 15% Percent Input Percent Percent Input 10% 10%

5% 5%

0% 0% -72 332 1671 3560 3752 4657 5590 -72 332 1671 2911 3560 3752 4657 5590 Positions along the beta-actin gene Positions along the beta-actin gene

Extended Data Figure 8 | RNAPII Pausing Defect and R Loop Accumulation observed in SMN CRISPR knock out cells and in SMA Disease Cells. a Western (Raji) b Western (Raji +amanitin)

N20 N20

SMN HA

c B2M

shGFP

shSMN

R1810A

IgG

ATF4 shGFP

shSMN

R1810A

IgG

TUBA1B

shGFP

shSMN

R1810A

IgG

CD40 shGFP

shSMN

R1810A

IgG

JUND shGFP

shSMN

R1810A

IgG

Extended Data Figure 9 | SMN and POLR2A R1810 regulate transcription termination by RNAPII occurs on a genome wide level (RNAPII ChIPseq) β-actin polyA

pause sites b a gammaH2ax/H2ax ratio gammaH2ax/H2ax ratio 100% 100% shGFP shSMN WT 80% 80% shPRMT5 R1810A

60%

60% Ratio Ratio 40% 40%

20% 20%

0% 0% 332 1671 2911 3560 3752 4657 332 1671 2911 3560 3752 4657 c

R loop forming tract RNAPII stalls due to R-loop SMN recruits Senataxin to R1810me2s

XRN2 Methylosome (PRMT5/MEP50) RNA is cleaved at cleavage signal XRN2 begins to degrade RNAPII CTD R1810me2s the downstream RNA at mutated in spinal muscular the cleavage site atrophy (SMA) Senataxin resolves R- SMN loop that stalls RNAPII and enables XRN2 to rare mutations sometimes fully degrade the cause ALS and AOA Senataxin transcript Unresolved R-loops lead to DNA damage R-loop resolution cleavage signal S2-phosphorylated XRN2, PCF11 RNAPII CTD

transcription cleavage factors cleavage termination

Extended Data Figure 10 | Model of the pathway that regulates R-Loop Accumulation to prevent DNA damage Protein-protein interactions Detection method Cell lines used Times observed

PRMT5 methylation of POLR2A RNAPII IP followed by western blot for shRNA or siRNA knock down 6 Rme2s (Y12)

PRMT5 methylation of POLR2A R1810 RNAPII IP followed by western blot using shRNA or siRNA knock down 4 R1810me2s specific antibody

RNAPII and SMN interaction is enhanced RNAPII IP followed by western blot for shRNA or siRNA knock down for 6 by PRMT5 methylation SMN PRMT5 Flag-PRMT5 overexpression cells 3

POLR2A contains R1810me2s HA-POLR2A IP followed by western blot for α-amanitin resistant HA-POLR2A 4 R1810me2s (Y12) WT or R1810A expressing cells HA-POLR2A IP followed by western blot 3 using R1810me2s specific antibody

POLR2A R1810me2s interact with SMN HA-POLR2A IP followed by western blot for α-amanitin resistant HA-POLR2A 5 SMN WT or R1810A expressing cells

RNAPII and Senataxin interaction is RNAPII IP followed by western blot for shRNA or siRNA knock down for 4 enhanced by SMN Senataxin SMN

RNAPII and Senataxin interaction is RNAPII IP followed by western blot for shRNA or siRNA knock down for 3 enhanced by PRMT5 Senataxin PRMT5

RNAPII and Senataxin interaction is HA-POLR2A IP followed by western blot for α-amanitin resistant HA-POLR2A 4 enhanced by R1810me2s Senataxin WT or R1810A expressing cells

SMN-Senataxin interaction is mediated Senataxin IP followed by western blot for shRNA knock down for PRMT5 3 by PRMT5 SMN

Protein-DNA interactions Detection method Cell lines used Times observed

RNAPII R1810A mutant stalls in POLR2A ChIP (4H8, 8WG16, H224 α-amanitin resistant HA-POLR2A WT or 5 termination regions antibodies) on b-actin and gapdh genes R1810A expressing cells

RNAPII R1810A mutant stalls in Nuclear Run-on α-amanitin resistant HA-POLR2A WT or 3 termination regions R1810A expressing cells

RNAPII stalls in termination POLR2A ChIP (4H8, 8WG16 antibodies) shRNA or siRNA knock down for PRMT5 5 regions on b-actin and gapdh genes shRNA or siRNA knock down for SMN 5 CRISPR knock-out for SMN 3

RNAPII R1810A mutant has R loop (S9.6 antibody) DIP α-amanitin resistant HA-POLR2A WT or 4 increased R loop formation R1810A expressed cells

RNAPII has increased R loop R loop (S9.6 antibody) DIP shRNA or siRNA knock down for SMN 5 formation with knock down shRNA or siRNA knock down for PRMT5 3

RNAPII has increased R loop HB-GFP construct CRISPR knock-out for SMN 3 formation with knock-out

detection of DNA damage gamaH2A X and H2AX ChIP α-amanitin resistant HA-POLR2A WT or 4 R1810A expressing cells

detection of DNA damage gamaH2A X and H2AX ChIP shRNA or siRNA knock down for PRMT5 4 shRNA or siRNA knock down for SMN 4

RNAPII and SMN interaction is SMN ChIP shRNA or siRNA knock down for PRMT5 3 enhanced by PRMT5 methylation

RNAPII and SMN interaction is SMN ChIP α-amanitin resistant HA-POLR2A WT or 3 enhanced by R1810me2s R1810A expressing cells

RNAPII and Senataxin interaction Senataxin ChIP α-amanitin resistant HA-POLR2A WT or 3 is enhanced by R1810me2s R1810A expressed cells

RNAPII and Senataxin interaction Senataxin ChIP shRNA or siRNA knock down for SMN and 3 is enhanced by PRMT5 or SMN PRMT5

Extended Data Table 1 | Numbers of times each type of experiment described in the work was performed with the reported result.