A multi-factor tracking site on the spliceosomal remodeling enzyme, BRR2, recruits C9ORF78 to regulate alternative splicing

Alexandra Bergfort Freie Universität Berlin Marco Preussner Freie Universität Berlin Benno Kuropka Freie Universität Berlin https://orcid.org/0000-0001-5088-6346 İbrahim Ilik Max Planck Institute for Molecular Genetics Tarek Hilal https://orcid.org/0000-0002-7833-8058 Gert Weber Helmholtz-Zentrum Berlin für Materialien und Energie https://orcid.org/0000-0003-3624-1060 Christian Freund Freie Universität Berlin https://orcid.org/0000-0001-7416-8226 Tugce Aktas Max Planck Institute for Molecular Genetics https://orcid.org/0000-0003-1599-9454 Florian Heyd Freie Universitä Markus Wahl (  [email protected] ) Freie Universität Berlin https://orcid.org/0000-0002-2811-5307

Article

Keywords: electron microscopy, alternative splicing, BRR2, C9ORF78

Posted Date: July 19th, 2021

DOI: https://doi.org/10.21203/rs.3.rs-639521/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 A multi-factor trafficking site on the spliceosomal

2 remodeling enzyme, BRR2, recruits C9ORF78 to regulate

3 alternative splicing

4

5 Alexandra Bergfort1, Marco Preußner2, Benno Kuropka3,4, İbrahim Avşar Ilik5, Tarek Hilal1,4,6,

6 Gert Weber7, Christian Freund3, Tuğçe Aktaş5, Florian Heyd2, Markus C. Wahl1,7,*

7

8 1 Freie Universität Berlin, Institute of Chemistry and Biochemistry, Laboratory of Structural

9 Biochemistry, Takustrasse 6, D-14195 Berlin, Germany

10 2 Freie Universität Berlin, Institute of Chemistry and Biochemistry, Laboratory of RNA

11 Biochemistry, Takustrasse 6, D-14195 Berlin, Germany

12 3 Freie Universität Berlin, Institute of Chemistry and Biochemistry, Laboratory of Protein

13 Biochemistry, Thielallee 63, D-14195, Berlin, Germany

14 4 Freie Universität Berlin, Institute of Chemistry and Biochemistry, Core Facility BioSupraMol,

15 Thielallee 63, D-14195, Berlin, Germany

16 5 Max Planck Institute for Molecular Genetics, Ihnestr. 63, D-14195 Berlin, Germany

17 6 Freie Universität Berlin, Institute of Chemistry and Biochemistry, Research Center of

18 Electron Microscopy and Core Facility BioSupraMol, Fabeckstr. 36a, 14195 Berlin,

19 Germany

20 7 Helmholtz-Zentrum Berlin für Materialien und Energie, Macromolecular Crystallography,

21 Albert-Einstein-Straße 15, D-12489 Berlin, Germany

22

23 * Correspondence to: [email protected]

24

1 25 Abstract

26 The complete inventory of regulatory factors in human spliceosomes remains unknown, and

27 many flexibly bound components are not revealed in present spliceosome structures. The

28 intrinsically unstructured C9ORF78 protein was detected in C complex spliceosomes but is not

29 contained in present spliceosome structures. We found a tight interaction between C9ORF78

30 and the key spliceosome remodeling factor, BRR2, in a large-scale yeast two-hybrid screen,

31 validated by targeted in vitro assays. Affinity purification/mass spectrometry and RNA UV-

32 crosslinking analyses identified several additional C9ORF78 interactors in spliceosomes.

33 High-resolution cryogenic electron microscopy structures revealed how C9ORF78 and the

34 spliceosomal B complex protein FBP21 wrap around the C-terminal helicase cassette of BRR2

35 in a mutually exclusive manner. Knock-down of C9ORF78 led to global alternative splicing

36 changes, including a substantial usage of alternative NAGNAG 3’-splice sites, at least in part

37 dependent on BRR2. Comparison of our structure to C* complex spliceosomes shows that

38 C9ORF78 could contact several detected interactors from its BRR2 “home base”, in particular

39 the RNA helicase PRPF22, a suggested 3’-splice site regulator. Together our data firmly

40 establish C9ORF78 as a novel, late-stage splicing regulatory protein that takes advantage of

41 a multi-factor trafficking site on BRR2, providing one explanation for the suggested, but

42 puzzling, roles of BRR2 during splicing catalysis and alternative splicing.

43

44 Introduction

45 Splicing of precursor messenger RNAs (pre-mRNAs) is a crucial step in eukaryotic gene

46 expression, and more than 95 % of all human multi-exon genes give rise to pre-mRNAs that

47 undergo alternative splicing, thereby massively increasing the genome’s coding capacity1. The

48 regulation of alternative splicing is highly complex, with many protein and RNA components

49 being involved, and aberrations are often linked to human disease2. Splicing is mediated by a

50 complex and dynamic RNA-protein (RNP) molecular machine, the spliceosome. A single round

51 of splicing is characterized by numerous distinct, sequential stages of the spliceosome; each

52 transition between these stages involves profound remodeling of the spliceosome’s molecular

2 53 composition and conformation3,4. At least eight conserved RNA helicases/RNP remodeling

54 enzymes drive and regulate key transitions in this splicing cycle, one of which is BRR2

55 (SNRNP200)5. BRR2 is a specific subunit of the U5 small nuclear (sn) RNP that plays a pivotal

56 role during spliceosome activation, the step during which the most substantial rearrangements

57 occur6,7. During this transition, BRR2 unwinds the initially extensively base-paired U4/U6 di-

58 snRNA, facilitating the release of U4 snRNA and of all U4/U6 di-snRNP-associated proteins,

59 and allowing U6 to form a catalytically important stem-loop and to engage in alternative

60 interactions with the 5’-splice site (ss) and U2 snRNA6-8. However, BRR2 has been suggested

61 to be also involved in the catalytic and disassembly phases of splicing, but the underlying

62 mechanisms are not understood, as these stages do not present known RNA/RNP substrates

63 on which the remodeling factor might act, and as BRR2’s ATPase/helicase activity does not

64 seem to be required during these stages9-11.

65 BRR2 is structurally and functionally unique among the spliceosomal RNA helicases/RNP

66 remodeling factors. It is the only Ski2-like helicase of the spliceosome and it comprises not a

67 single, but two closely associated and structurally similar helicase units (cassettes)12. Only the

68 N-terminal cassette (NC) is an active ATPase and RNA helicase, while the C-terminal cassette

69 (CC) is catalytically inactive, but can regulate the activity of the NC12,13. Both cassettes

70 comprise two RecA-like domains, followed by a winged helix (WH) domain and a Sec63

71 homology module, consisting of helical bundle (HB), helix-loop-helix (HLH) and

72 immunoglobulin-like (IG) domains12,14,15.

73 While all other helicases join the spliceosome only transiently, BRR2 is recruited to the pre-

74 catalytic B complex as part of the U4/U6-U5 tri-snRNP and stays associated during all

75 remaining phases of splicing. Furthermore, it encounters its U4/U6 di-snRNA substrate already

76 prior to spliceosome activation within the U4/U6-U5 tri-snRNP, and, consequently, its

77 enzymatic activity needs to be precisely regulated16. Apart from several intra-molecular

78 regulatory mechanisms17-19, the BRR2 activity depends on multiple trans-acting factors20. Most

79 importantly, BRR2 is regulated by PRPF8, the largest and most conserved spliceosomal

80 protein that forms a scaffold for the catalytic RNA core of the spliceosome4,21. PRPF8

3 81 encompasses C-terminal RNase H-like and Jab1/MPN-like (Jab1) domains, both of which can

82 regulate BRR222-25. The Jab1 domain can inhibit BRR2 helicase activity by inserting its flexible,

83 C-terminal 16 residues into the BRR2 RNA-binding tunnel; upon removal of the C-terminal

84 Jab1 tail from the tunnel, Jab1 is converted into a strong BRR2 activator 20,24.

85 The largely intrinsically disordered formin-binding protein 21 (FBP21) is one of the non-

86 snRNP accessory proteins, that enters the spliceosomal B complex prior to activation together

87 with eight other B-specific proteins26,27. FBP21 and other B-specific proteins are displaced

88 again during the BRR2-dependent conversion of the B complex to the Bact complex26,27. FBP21

89 stably interacts with the Sec63 module of the BRR2 CC and inhibits the BRR2-mediated U4/U6

90 di-snRNA unwinding by functionally uncoupling NC and CC28,29. Two additional, largely

91 intrinsically disordered proteins (IDPs), NTR2 and SNU66, interact with the BRR2 CC in

92 yeast30-32. Although the human SNU66 and FBP21 interactions with BRR2 are not entirely

93 visible in recent cryogenic electron microscopy (cryoEM) structures of the human pre-B and B

94 complexes33-35, there might be a pattern for the BRR2 CC to act as a landing pad for IDPs that

95 can have a splicing regulatory function.

96 The 34 kDa C9ORF78 protein is predicted to be largely intrinsically disordered. It has been

97 described as a tumor antigen in hepatocellular carcinoma36 and is considered a marker for a

98 favorable clinical development of renal cancer37, but its cellular functions are essentially

99 uncharacterized. A C9ORF78 homolog, TLS1, of Schizosaccharomyces pombe has been

100 described as a BRR2 interactor that modulates splicing of shelterin components and thereby

101 affects telomeric heterochromatin assembly and telomere length 38. Human C9ORF78 has

102 been found associated with the spliceosomal C complex26, but whether the protein directly

103 interacts with BRR2 and which role it might play in pre-mRNA splicing is presently unknown.

104 Here, we demonstrate a direct C9ORF78-BRR2 interaction in human and show mutually

105 exclusive binding of C9ORF78 to BRR2 with FBP21. CryoEM-based structural analyses

106 elucidated details underlying this binding competition and allowed targeted disruption of the

107 C9ORF78-BRR2 interaction. System-wide protein and RNA interaction studies revealed

108 additional C9ORF78-interacting components of the spliceosome. SiRNA-mediated knock-

4 109 down (KD), RNA sequencing (RNA-seq) and radioactive RT-PCR analyses in combination with

110 a C9ORF78 variant that is dysfunctional in BRR2 binding demonstrated a role of C9ORF78 in

111 alternative splicing, in particular with respect to alternative 3’-ss selection, which is at least in

112 part dependent on its interaction with BRR2.

113

114 Results

115 C9ORF78 interacts with BRR2 in vitro and in HEK293 cells

116 We found the human C9ORF78 protein as a putative novel interactor of BRR2 in a large-

117 scale yeast two-hybrid screen. To test whether C9ORF78 binds BRR2 directly, we produced

118 recombinant His6-GST-C9ORF78 (hereafter referred to as GST-C9ORF78) in Escherichia coli

119 cells. Cleavage of the tag led to degradation of the protein and was therefore omitted for

120 functional analyses in vitro. GST-C9ORF78 stably interacted with full-length BRR2 (BRR2FL)

121 in analytical size exclusion chromatography (SEC; Fig. 1a). Testing of BRR2 truncation

122 variants revealed that its helicase region (BRR2HR, residues 395-2129), encompassing both

123 helicase cassettes but lacking a ~ 400-residue, auto-regulatory N-terminal region17, is sufficient

124 for GST-C9ORF78 binding (Supplementary Fig. 1a). However, binding efficiency was reduced

125 for the isolated BRR2 CC (BRR2CC, residues 1282-2136), and the BRR2 NC (BRR2NC,

126 residues 395-1324) did not co-migrate with GST-C9ORF78 (Supplementary Fig. 1a),

127 suggesting that the interaction takes place predominantly via the CC and is supported by the

128 NC. We further validated an interaction of BRR2 and Flag-C9ORF78 in HEK293 cells via Flag-

129 immunoprecipitation (Flag-IP) followed by Western blot (WB). BRR2 was strongly enriched in

130 the C9ORF78 Flag-IP, suggesting that the interaction also takes place in vivo (Fig. 1b).

131 In recent years, cryoEM structures of the human spliceosome revealed persistent BRR2

132 binding by the PRPF8 Jab1 domain (PRPF8Jab1; residues 2064-2335) throughout phases of

133 the splicing cycle that encompass both proteins8,33-35,39-41. We therefore tested if BRR2HR

134 binding by PRPF8Jab1 and GST-C9ORF78 can occur simultaneously. Both proteins together

135 co-migrated with BRR2HR in analytical SEC, while no BRR2-independent interaction of

136 PRPF8Jab1 and GST-C9ORF78 was detected (Supplementary Fig. 1b).

5 137

138 CryoEM structure of a BRR2-PRPF8Jab1-C9ORF78 complex

139 Co-purification of BRR2 and C9ORF78 allowed for His6/GST-tag cleavage without

140 degradation of the C9ORF78 protein. To better characterize the interaction of C9ORF78 with

141 BRR2HR, we determined a cryoEM structure of a BRR2HR-PRPF8Jab1-C9ORF78 complex at a

142 nominal resolution of 2.76 Å (Fig 1c; Supplementary Fig. 2 a-e; Supplementary Fig. 3a;

143 Supplementary Table 1). The globular part of PRPF8Jab1 resides at its known binding site on

144 the BRR2 NC, with the PRPF8Jab1 C-terminal tail penetrating the BRR2HR RNA-binding tunnel

145 (Fig 1c). C9ORF78 residues 5-58 run along one entire flank of the BRR2 CC in an extended

146 conformation. BRR2 binding by the N-terminal part of C9ORF78 is supported by data we

147 obtained via limited proteolysis and peptide spot array (Supplementary Fig. 1c,d). Upon

148 interaction, C9ORF78 residues 14-18, 20-41 and 49-54 adopt α-helical structures (α1-α3; Fig.

149 2a). It has been shown that salt bridges formed between oppositely charged residues spaced

150 at positions i and i+3/i+4 (ER/K motifs) can stabilize isolated α-helices42,43 and can reduce loss

151 of conformational entropy upon interactions44. Such motifs are frequently found in long, single

152 α-helices in spliceosomal proteins44. The long central helix α2 of C9ORF78 exhibits five such

153 motifs, suggesting that it constitutes an important docking device on BRR2.

154 C9ORF78 residues 5-19 bind to a groove between the HLH and IG domains of the C-

155 terminal Sec63 module. The long central helix α2 runs along the C-terminal HB domain, with

156 its tip linking up to the WH domain of the CC. Residues 42-58, including α3, interconnect the

157 CC and NC, explaining partially reduced binding of BRR2 CC as compared to BRR2HR. Residues

158 42-48 line a groove between the C-terminal WH and N-terminal IG domain. The short helix α3

159 additionally reinforces the interaction between the globular part of the PRPF8 Jab1 domain

160 and the N-terminal IG domain of BRR2. The four N-terminal and 231 C-terminal residues of

161 C9ORF78, while contained in the complex, could not be located in the cryoEM map, suggesting

162 that they remain unbound and flexible. The limited contacts of C9ORF78 to the NC and to the

163 PRPF8 Jab1 domain are consistent with these interactions not being stable in isolation, but

164 the NC reinforcing C9ORF78 interaction with the CC (Supplementary Fig. 1a,b).

6 165 At the N-terminus of C9ORF78, F8 stacks on F1983 of BRR2 and the following R9 engages

166 in ionic interactions with two glutamic acid residues on BRR2 (E1944 and E2119; Fig. 2a). At

167 the tip of the long central helix, R41 of C9ORF78 engages in a particularly large number of

168 polar contacts with BRR2 residues Q1798, S1799 and E1830 (Fig. 3a). In the following linker

169 region, R43 and N45 maintain polar contacts to Q1791 and E1201 of BRR2, respectively.

170 Mixed hydrophobic and hydrophilic contacts ensue in the interaction between the C-terminal

171 49-58 residues of C9ORF78 and BRR2. F8, R9, R41, R43 and N45 as well as neighboring

172 regions of C9ORF78 are highly conserved (Fig. 2b).

173 Analysis of the structure with the PISA server45 revealed a small interaction interface of

174 C9ORF78 with PRPF8Jab1 (59 Å2 of interface area), consistent with this interaction not being

175 stable in isolation. In contrast, C9ORF78 exhibits 2,195 Å2 of interface area with BRR2HR,

176 comparable to the interface area seen for the BRR2 HR-PRPF8Jab1 interaction (2,698 Å2).

177 Despite the similarly sized interfaces, the calculated solvation free energy gain (ΔiG) upon

178 interface formation (reflecting hydrophobic interactions) is much more favorable for the

179 BRR2HR-PRPF8Jab1 compared to the BRR2HR-C9ORF78 interaction (estimated -16.9 vs. -3.9

180 kcal/mol). Consistently, BRR2HR-PRPF8Jab1 is suggested to represent a much more “likely”

181 interaction compared to BRR2HR-C9ORF78 (ΔiG p-value of 0.42 vs. 0.72). The BRR2HR-

182 C9ORF78 interaction relies relatively more on electrostatic interactions; 38 interface residues

183 of C9ORF78 engage in 40 hydrogen bonds and salt bridges to BRR2, contributing an

184 estimated -17.8 kcal/mol to ΔiG, while 58 interface residues of PRPF8Jab1 engage in 39

185 electrostatic contacts to BRR2 (estimated -17.9 kcal/mol). Therefore, despite similar size

186 interfaces, we expect the BRR2HR-C9ORF78 interaction to be significantly weaker and more

187 transient, than the BRR2HR-PRPF8Jab1 interaction, for which a Kd of about 8 nM has been

188 estimated in yeast46. Taken together, the mode of BRR2 binding observed for C9ORF78

189 reflects its intrinsically unstructured nature47; it relies to a relatively large part on electrostatic

190 interactions; it buries a large interaction surface per residue, likely giving rise to a highly specific

191 interaction; yet it exhibits moderate stability, presumably due to loss of conformational entropy

192 as a consequence of local folding and immobilization upon binding47.

7 193

194 C9ORF78 only marginally affects BRR2 helicase activity

195 The U4/U6 di-snRNA is the only known BRR2 helicase substrate in the spliceosome.

196 Although C9ORF78 has only been detected in spliceosomes after U4/U6 unwinding26, we

197 resorted to U4/U6 di-snRNA as a known BRR2 substrate to test whether C9ORF78 has the

198 potential to affect the BRR2 helicase activity. To this end, we performed radioactive, gel-based

199 U4/U6 unwinding assays in the absence or presence of C9ORF78, PRPF8Jab1 (BRR2 inhibitor)

200 and/or PRPF8Jab1ΔC (residues 2064-2319; activator lacking the C-terminal tail). Only a very

201 moderate inhibitory effect was observed upon C9ORF78 binding, which was relatively stronger

202 in the presence of PRPF8Jab1 or PRPF8Jab1ΔC (Supplementary Fig. 4). These findings suggest

203 that C9ORF78 may aid in keeping BRR2 in an inactive form, but argues against a modulation

204 of BRR2 helicase activity constituting a major C9ORF78 function.

205

206 BRR2 binding by C9ORF78 and FBP21 is mutually exclusive

207 The FBP21 C-terminal region is known to interact with the BRR2 CC.28,29 Therefore, we

208 addressed the question if FBP21 and C9ORF78 occupy a common BRR2 binding site, by

209 determining a BRR2HR-FBP21200-376 cryoEM structure at a nominal resolution of 3.3 Å (Fig. 1d;

210 Supplementary Fig. 2f-j; Supplementary Fig. 3b; Supplementary Table 1). The structure

211 revealed that residues 357-375 of FBP21200-376 wrap around the C-terminal Sec63 module of

212 BRR2HR, as previously suggested by NMR and cross-linking analyses28,29, with the binding site

213 overlapping a C9ORF78 interaction surface (Fig. 2a,c, top). In particular, FBP21 exhibits an

214 L369-R370 motif that engages the same glutamic acid and phenylalanine residues of BRR2HR

215 (F1983, E1944, E2119) as seen for the F8-R9 motif in C9ORF78 (Fig. 2a,c, bottom).

216 To test mutually exclusive binding biochemically, we conducted a GST-C9ORF78-BRR2HR

217 pull-down assay, while adding increasing amounts of FBP21200-376, and analyzed bound (bead)

218 and unbound (elution) fractions. Increasing concentrations of FBP21200-376 led to increasing

219 release of BRR2HR from bead-bound GST-C9ORF78, and, as expected, FBP21200-376 and

220 C9ORF78 did not bind BRR2HR simultaneously (Fig. 2d). These findings suggest that

8 221 C9ORF78 can only bind BRR2 upon conversion of the B to the Bact complex, when FBP21 and

222 other B-specific proteins are released.

223

224 C9ORF78 plays a role in alternative splicing of specific target pre-mRNAs

225 Together with previous observations of C9ORF78 in the C complex26, our above findings

226 firmly establish C9ORF78 as a component of the human spliceosome. To elucidate which

227 function C9ORF78 might elicit during splicing, we conducted an siRNA-mediated C9ORF78

228 KD in HEK293 cells. 72 hours after transfection we obtained a KD efficiency of 75-80 % based

229 on RT-qPCR (Fig. 4a). RNA-seq yielded 35 M reads for each of triplicate KD and control

230 samples. RMATS analysis of differential alternative splicing revealed 667 significantly affected

231 alternative splicing events upon C9ORF78 KD, with skipped exons (SE) being the most

232 dominant form, followed by mutually exclusive exons (MX) and alternative 3’-ss usage (A3ss;

233 Fig. 4b; Supplementary Table 2). While exon skipping events were up- and down-regulated

234 upon C9ORF78 KD to a similar extent (Fig. 4b; Fig. 4c, left), upstream alternative 3’-ss were

235 predominantly skipped upon C9ORF78 KD, leading to the preferred usage of the downstream

236 alternative 3’-ss and thus shorter products (Fig. 4b; Fig. 4c, middle).

237 Further analysis of regulated, alternative 3’-ss events revealed particularly pronounced

238 upstream 3’-ss skipping upon C9ORF78 KD when the two alternative 3’-ss were separated by

239 a very small number of nucleotides (nts). We found a median distance of 3 nts between

240 alternative 3’-ss affected upon C9ORF78 KD. Such alternative 3’-ss pairs are referred to as

241 NAGNAG splice sites48. In the regulated events, we found the upstream 3’-ss to be generally

242 weaker, indicating a requirement of C9ORF78 to promote usage of weak upstream 3’-ss

243 (Supplementary Fig. 5). An analysis of all HEK293 NAGNAG splice sites revealed that a

244 substantial number of these alternative 3’-ss pairs is regulated via C9ORF78 (Fig. 4c, right).

245 Enhanced skipping of PTBP2 exon 10 and skipping of upstream NAGNAG splice sites in

246 SMARCA4 and C1ORF131 upon C9ORF78 KD were validated via radioactive RT-PCR (Fig.

247 5a). PTBP2 exon 10 skipping results in a frame shift, generation of a pre-mature termination

248 codon and nonsense-mediated decay49,50. As PTBP2 is a splicing factor itself, we compared

9 249 genes regulated by C9ORF78 with known PTBP2 target genes, as suggested by published

250 cross-linking/IP (CLIP) data51. Based on these data, only 26 of the 392 C9ORF78 exon

251 skipping targets are bound by PTBP2. Thus, the majority of exon skipping events affected by

252 C9ORF78 KD cannot be explained by an effect of C9ORF78 on PTBP2 exon 10 skipping.

253 Taken together, our data establish C9ORF78 as a novel splicing factor that regulates

254 alternative splicing of specific target pre-mRNAs. In addition to regulating inclusion of certain

255 exon, C9ORF78 seems to be an important regulator of NAGNAG splice sites.

256

257 C9ORF78 function in alternative splicing at least partially depends on its interaction

258 with BRR2

259 To test whether the function of C9ORF78 in alternative splicing is mediated through its

260 interaction with BRR2, we inspected our BRR2HR-PRPF8Jab1-C9ORF78 cryoEM structure to

261 pinpoint key interacting residues whose alteration might abolish the BRR2-C9ORF78

262 interaction. Multiple sequence alignment of human C9ORF78 with homologs from other

263 species revealed highly conserved residues within epitopes of C9ORF78 that contact BRR2,

264 including the C9ORF78 F8-R9 motif, which competes with a similar motif in FBP21 for a

265 common BRR2 binding site, and the region around R41 in the long central helix of C9ORF78,

266 which engages in multiple polar contacts with BRR2 (Fig. 2b; Supplementary Fig. 6). We

267 exchanged C9ORF78 F8 and R41 individually for alanine residues, and tested BRR2 binding

268 of the C9ORF78 variants via analytical SEC. While C9ORF78F8A showed reduced binding to

269 BRR2 in analytical SEC, BRR2 binding by C9ORF78R41A in vitro was completely abolished

270 (Fig. 3b). We also tested the C9ORF78R41A-BRR2 interaction in HEK293 cells via Flag-IP

271 followed by WB. The results suggest that, contrary to wild-type (wt) C9ORF78, C9ORF78R41A

272 does not stably interact with BRR2 in vivo (Fig. 3c).

273 We employed the C9ORF78R41A variant in KD/rescue experiments in HEK293 cells. We

274 again knocked down endogenous C9ORF78 via siRNAs, and after 24 hours transfected with

275 siRNA-resistant genes encoding either C9ORF78wt or C9ORF78R41A. Enhanced skipping of

276 PTBP2 exon 10 could be reversed by re-expression of C9ORF78wt, while the rescue efficiency

10 277 with C9ORF78R41A was significantly reduced (Fig. 5b, left), pointing towards a regulatory

278 mechanism that is dependent on the BRR2-C9ORF78 interaction. On the other hand, while

279 we also observed rescue with C9ORF78wt for C9ORF78 KD-dependent alteration of

280 SMARCA4 or C1ORF131 NAGNAG splice sites, we could not detect a significant difference

281 to the rescue with C9ORF78R41A in this experimental setup (Fig. 5b, middle and right). These

282 findings confirm that the observed alternative splicing changes upon C9ORF78 KD are indeed

283 specific to C9ORF78 and that they at least partially depend on the interaction of C9ORF78

284 with BRR2.

285

286 C9ORF78 UV-crosslinks with U5 snRNA

287 To test if C9ORF78 regulates alternative splicing through direct interactions with target pre-

288 mRNAs, we generated a Flp-In™ T-REx™ 293 cell line that stably expresses C9ORF78, and

289 performed UV-crosslinking followed by enrichment of C9ORF78-coupled RNAs via Fast

290 Ligation of RNA after some sort of Affinity Purification for High-throughput Sequencing

291 (FLASH)52. Green fluorescent protein (GFP)-expressing cells were used as a negative control.

292 RNA-seq yielded 1.8 M reads after filtering PCR duplicates and low quality reads, among which

293 we searched for pre-mRNAs that exhibited C9ORF78-dependent skipped exons or alternative

294 3’-ss via RMATS analysis using deepTools53 (Supplementary Fig. 7). No enrichment of specific

295 pre-mRNAs was observed in the C9ORF78 cross-linking experiment compared to the GFP

296 control. This observation argues against regulatory mechanisms for C9ORF78-dependent

297 alternative splicing that rely on direct C9ORF78-pre-mRNA interactions. However,

298 snakePipes54 analysis revealed a significant enrichment of U5 snRNA (log2-fold change 2.25;

299 p-value 4.290826e-31) and L13AP, a retrotransposon of the LINE-1 family (log2-fold change

300 0.59; p-value 5.475290e-11) among C9ORF78-cross-linked RNAs (Fig. 6a). Together with the

301 direct C9ORF78-BRR2 interaction we established, a direct C9ORF78-U5 snRNA contact, as

302 suggested by this finding, supports the notion that C9ORF78 functions in alternative splicing

303 depend on interactions with U5 snRNP components.

304

11 305 C9ORF78 interacts with additional spliceosomal proteins

306 To identify possible additional spliceosomal protein interactors of C9ORF78, we generated

307 HEK293 cell lines stably expressing Flag-C9ORF78wt or Flag-C9ORF78R41A, and conducted

308 Flag-IPs with nuclear extracts, followed by mass spectrometric identification of the recovered

309 proteins. Enrichment was calculated in comparison to a control IP (cells not expressing a Flag-

310 C9ORF78 variant). In total, we quantified 2,411 IP-enriched proteins (Supplementary Table 3).

311 Spliceosomal proteins enriched in both IPs are shown in Fig. 6b. While C9ORF78wt and

312 C9ORF78R41A were enriched at almost equal levels, enrichment of BRR2, PRPF8, PRPF6,

313 CWC27 and CWC22 was strongly reduced in the Flag-C9ORF78R41A IP compared to the Flag-

314 C9ORF78wt IP. All these putative C9ORF78wt interactors are present at the C complex

315 stage26,27, supporting the notion that C9ORF78 is associated with the spliceosome during this

316 phase of splicing. In addition, the observations further confirm an effective disruption of the

317 BRR2 interaction via an R41A exchange in C9ORF78. However, as R41 is completely buried

318 in the C9ORF78-BRR2HR interface in our BRR2HR-PRPF8Jab1-C9ORF78 cryoEM structure (Fig.

319 3a), a direct effect of the R41A exchange on the enrichment of PRPF8, PRPF6, CWC27 and

320 CWC22 is unlikely. While direct contacts of C9ORF78 to these proteins through other

321 C9ORF78 regions are certainly possible, our findings of reduced enrichment of the proteins in

322 the Flag-C9ORF78R41A IP suggest that such putative binary interactions would be relatively

323 weak and dependent on concomitant stable interaction of C9ORF78 with BRR2.

324 On the other hand, the PRPF19/CDC5L-related protein, aquarius (AQR), and the second

325 step factor, PRPF22 (DHX8), were enriched at comparable levels in both IPs (a slight reduction

326 in the Flag-C9ORF78R41A IP is most likely due to a slight difference in the enrichment of Flag-

327 C9ORF78wt and Flag-C9ORF78R41A themselves). As again, both proteins are present in the

328 spliceosome at the C complex stage26,27, these data suggest that C9ORF78 contacts AQR and

329 PFPF22 within the spliceosome directly or indirectly, but not dependent on its interaction with

330 BRR2. As AQR is recruited to the spliceosome already during the Bact stage and as PRPF22

331 is also present in a remodeled C complex (C*) before exon ligation, presence of C9ORF78

332 may also not be exclusively restricted to the C complex.

12 333 Finally, protein DDX23, the ortholog of the Prp28 RNA helicase in yeast, was less enriched

334 in the C9ORF78wt IP compared to the C9ORF78R41A IP. Notably, a long N-terminal helix and

335 neighboring loop regions in DDX23 cross-strut BRR2 and the PRPF8 Jab1 domain in the

336 human U4/U6-U5 tri-snRNP8. This helix runs across the binding site for the short helix α3 and

337 neighboring regions of C9ORF78 that establish interactions to BRR2HR and PRPF8Jab1 in our

338 BRR2HR-PRPF8Jab1-C9ORF78 structure; furthermore, the CC of BRR2 and PRPF8 Jab1 are

339 slightly further apart in our BRR2HR-PRPF8Jab1-C9ORF78 complex than in the U4/U6-U5 tri-

340 snRNP, suggesting that they are pried apart by C9ORF78 or contracted by DDX23 (Fig. 3d).

341 These observations again argue for a mutually exclusive binding of C9ORF78 and the N-

342 terminus of DDX23 to BRR2HR-PRPF8Jab1. Together with our pull-down results, they suggest

343 that weakening of the C9ORF78-BRR2 interaction allows DDX23, which is normally

344 significantly reduced after the B complex stage26, to remain bound or re-bind to some extent

345 during the C complex stage. In our assays, DDX23 is most likely pulled down via C9ORF78R41A

346 by virtue of its interaction with other C complex proteins (and possibly U5 snRNA).

347 The 231 C-terminal residues of the 289-residue C9ORF78 protein are not visible in our

348 BRR2HR-PRPF8Jab1-C9ORF78 cryoEM structure (Fig. 1c). Many of these residues are highly

349 conserved, pointing towards a functional importance (Supplementary Fig. 6). Obviously, this

350 region could harbor binding sites for other spliceosome components, such as the proteins

351 enriched in our pull-down experiments. As the entire C9ORF78 protein is predicted to be

352 mostly unstructured, these 231 C-terminal residues could extend far away from the N-terminal

353 anchor on BRR2 and reach distal regions of the spliceosome. Furthermore, one can expect

354 interactions to be maintained through short C9ORF78 epitopes, as is frequently observed in

355 IDPs47, such that C9ORF78 could engage in direct contacts to multiple additional spliceosome

356 components at the same time. We aligned our BRR2HR-PRPF8Jab1-C9ORF78 structure

357 according to the BRR2HR/BRR2 subunits with a structure of the human C* complex, the

358 spliceosome intermediately prior to exon-ligation (PDB 5XJC)40 (Fig. 7). From the overlay, it is

359 easily conceivable how C9ORF78 C-terminal regions could reach proteins that we find

13 360 enriched in our pull-down assays, in particular PRPF22 and CWC22 that reside in immediate

361 vicinity of BRR2.

362

363 Discussion

364 Mechanisms underlying roles of BRR2 during the catalytic phase of splicing and in

365 alternative splicing

366 While requirements for BRR2, beyond U4/U6 unwinding, during the catalytic and

367 disassembly stages of splicing have been suggested, at least in yeast,9,10 the mechanistic

368 basis for these observations is not clear, as BRR2 does not seem to resort to its

369 ATPase/helicase activities to exert these additional roles9,11. Here, we show how the ~ 60 N-

370 terminal residues of an IDP, C9ORF78, bind along the BRR2 CC, and that this interaction is

371 required for PTBP2 exon 10 inclusion and, most likely, also for other alternative splicing events

372 we see affected upon C9ORF78 KD. These observations suggest a blueprint for how BRR2

373 might exert ATPase/helicase-independent functions during splicing, i.e., by serving as a

374 landing pad or “home base” for other splicing factors, including stage-specifically recruited

375 splicing regulators. In addition, our findings reveal an unexpected role of BRR2, a constitutive

376 splicing factor, in alternative splicing. This role in alternative splicing is brought about by BRR2

377 recruiting a hitherto poorly characterized, splicing regulatory IDP, C9ORF78.

378

379 Multi-factor trafficking sites on BRR2 may support ordered transitions in the splicing

380 cycle

381 The BRR2 C-terminal Sec63 module has been suggested to serve as a binding platform for

382 other spliceosomal proteins that could regulate the helicase’s unwinding activity. E.g., the IDP

383 FBP21 is a component of the spliceosomal B complex26,27, and an N-terminal α-helix of FBP21

384 can bind the BRR2 NC, as observed in cryoEM structures of B complex spliceosomes 33,35.

385 However, these structures did not reveal the location of the C-terminal unstructured tail of

386 FBP21, which was suggested to wrap around the C-terminal Sec63 module of BRR2 by NMR

387 and cross-linking studies28,29. This latter interaction is validated by the BRR2HR-FBP21200-376

14 388 cryoEM structure presented here. FBP21 indeed shows a strong inhibitory effect of BRR2

389 helicase activity28, consistent with a suggested role in preventing BRR2 from pre-maturely

390 unwinding U4/U633. In contrast, the BRR2-modulatory activity of C9ORF78 we report here is

391 very moderate, consistent with C9ORF78 associating with the spliceosome only at stages

392 when BRR2 has already unwound U4/U6.

393 The docking site on BRR2HR of FBP21200-376 outlined here is also used by C9ORF78 for

394 BRR2 binding. Indeed, comparison of our BRR2HR-PRPF8Jab1-C9ORF78 and BRR2HR-

395 FBP21200-376 structures showed that both proteins use a conserved FR/LR-motif to intimately

396 interact with the same glutamate and phenylalanine residues on the C-terminal Sec63 module

397 of BRR2HR (Fig. 2a,c). This common binding mode to BRR2 explains the observed mutually

398 exclusive binding of C9ORF78 and FBP21 to BRR2. Moreover, it supports the notion of a

399 highly specific, yet marginally stable C9ORF78-BRR2 interaction, as displacement of only one

400 short binding epitope of C9ORF78 by FBP21 is sufficient to destabilize the entire BRR2-

401 C9ORF78 complex.

402 Based on these findings, we suggest that the observed binding competition on a multi-factor

403 trafficking site of BRR2 represents an important principle in the spliceosome, which facilitates

404 ordered, stage-specific recruitment of splicing factors. E.g., the FBP21/C9ORF78 competition

405 may at least in part underlie the ordered recruitment of FBP21 to the B complex and of

406 C9ORF78 to the C complex; C9ORF78 can only bind BRR2 after conversion of the B to the

407 Bact complex, when FBP21 and other B-specific proteins are displaced26,27. While C9ORF78

408 has so far been found associated with the C complex, our observation that C9ORF78 regulates

409 exon skipping, a process which is thought to be decided before the C complex stage, provides

410 indirect evidence that C9ORF78 is already present in the Bact complex, when FBP21 has been

411 displaced. Our observed interaction of C9ORF78 with AQR, a protein present during the Bact

412 stage, is consistent with this notion. Initial docking of C9ORF78 during B-to-Bact conversion

413 may, thus, even facilitate concomitant displacement of FBP21.

414 Structural comparison between the present BRR2HR-PRPF8Jab1-C9ORF78 structure and a

415 structure of the human U4/U6-U5 tri-snRNP, together with our co-IP analyses with C9ORF78wt

15 416 and a C9ORF78R41A variant defective in BRR2 binding, suggest that a similar principle is at

417 work for excluding the RNA helicase DDX23, which is required for assembly of the B complex

418 through a phosphorylation-based mechanism55, from later stages of splicing. C9ORF78 and

419 DDX23 again appear to engage BRR2HR-PRPF8Jab1 in a mutually exclusive manner, but they

420 compete at a different site than C9ORF78 and FBP21 (Fig. 3d). Thus, apart from using BRR2

421 as a home base from which to exert effects on alternative splicing, C9ORF78 seems to keep

422 the C complex clean of factors that act at earlier stages. Taken together, our analyses suggest

423 that multi-factor trafficking sites on BRR2 represent a recurring principle for ordering factor

424 recruitment during splicing.

425

426 C9ORF78-dependent splicing regulation at late stages of the splicing cycle

427 Comprehensive studies of the effects of splicing factor KD on alternative splicing have

428 previously suggested that alternative splicing decisions are not necessarily fixed during initial

429 exon/intron definition56. Our findings provide another example of alternative splicing regulation

430 comparatively late in the splicing cycle. Specifically, we observe a strong effect of the KD of

431 the C complex-associated protein, C9ORF78, on alternative usage of NAGNAG 3’-ss, with

432 C9ORF78 strongly favoring usage of the upstream 3’-ss. Alternative NAGNAG splicing has

433 been shown to be of physiological importance in various cellular processes57-59. However, the

434 molecular mechanisms underlying alternative NAGNAG splicing in human are presently poorly

435 understood, and have been approached predominantly through in silico methods60. Consistent

436 with our finding of C9ORF78 as an important regulator of these alternative splice sites,

437 alternative NAGNAG splice site choice was suggested to take place during the second step of

438 the splicing reaction48.

439 Our observed interaction of C9ORF78 with the second step factor, PRPF22, suggests that

440 C9ORF78 remains associated with the C*-complex. Comparison of our BRR2HR-PRPF8Jab1-

441 C9ORF78 structure with the structure of a human C* complex revealed that the C-terminal 231

442 residues of C9ORF78 could easily reach and directly contact PRPF22, which resides in

443 immediate vicinity of BRR2 in the C* complex (Fig. 7). Another C9ORF78 interactor detected

16 444 here, CWC22, also resides close to BRR2 and PRPF22 in the C* complex (Fig. 7). PRPF22

445 has been shown to be involved in 3’-ss selection and exon ligation in yeast61,62. Thus, one

446 possible mechanism for C9ORF78 to regulate alternative 3’-ss usage may be a modulation of

447 PRPF22 motor activity, which is thought to reposition alternative 3’-ss in the spliceosome’s

448 active site from a distance62, via direct protein-protein interactions. This interpretation is

449 consistent with our observation that presence of C9ORF78 leads to preferential use of

450 upstream alternative 3’-ss.

451 While our rescue experiments do not reveal a BRR2-dependence of C9ORF78’s function

452 in alternative 3’-ss usage, our structural comparisons show that C9ORF78-BRR2 and

453 C9ORF78-PRPF22 interactions could easily take place concomitantly. Therefore, we suggest

454 that functions of C9ORF78 in alternative splicing generally depend on its interaction with

455 BRR2, but that our rescue experiments were not sensitive enough to detect all of these

456 dependencies, as the C9ORF78-BRR2 interaction is most likely strongly buffered by

457 C9ORF78’s contacts to other regions of BRR2, which are unaffected by the mutation, and to

458 multiple additional subunits within spliceosomes as detected in our UV-cross-linking and IP

459 experiments.

460

461 Roles of C9ORF78 in human diseases

462 Although C9ORF78 has been associated with hepatocellular carcinoma 36 and renal

463 cancer37, specific functions of this protein in cancer metabolism have not yet been investigated.

464 In our RNA-seq upon C9ORF78 KD data, at least two of the skipped exon events altered in

465 the C9ORF78 KD, affecting SLIT2 and IRF3, are known aberrations that occur in certain

466 cancers63. For instance, C9ORF78 KD leads to enhanced levels of SLIT2 exon 15 skipping.

467 Inclusion of SLIT2 exon 15 was shown to promote lung cancer progression, pointing towards

468 a tumor-promoting function of C9ORF78. However, in renal cancer, high expression levels of

469 C9ORF78 correlate with a favorable clinical outcome37, suggesting that a potential C9ORF78

470 function in cancer metabolism might be dependent on tissue and cancer type. A further cancer-

471 relevant C9ORF78 target is SMARCA4. SMARCA4 is a chromatin remodeler that is often

17 472 mutated or silenced in tumors64. C9ORF78 KD led to suppression of an alternative SMARCA4

473 3’-ss and a potential impact of this splice event on cancer progression might be of interest for

474 future research.

475 Taken together, our findings shed light on the previously poorly characterized C9ORF78

476 protein and its function in pre-mRNA splicing. Furthermore, we demonstrate how BRR2

477 interacts with the IDPs FBP21 and C9ORF78 via its CC, which in case of the latter protein has

478 a demonstrated or likely impact on numerous alternative splicing events. These observations

479 point towards hitherto under-appreciated, additional functions of human BRR2 during the

480 catalytic phase of splicing, and to novel functions of BRR2 in the regulation of alternative

481 splicing. Our findings underscore the importance for revealing the structural organization

482 among the non-resolved “dark matter” surrounding more stably folded core regions of

483 spliceosomes in cryoEM structures.

484

18 485 Online methods

486 Protein production

487 BRR2 and PRPF8 constructs were produced and purified as described before12,24.

488 C9ORF78 variants were produced as His6-GST-tagged proteins from pETM30 plasmids in a

489 BL21 RIL E. coli strain. The cell pellet was resuspended in lysis buffer (20 mM Tris-HCl, pH

490 7.5, 200 mM NaCl, 1 mM DTT), supplemented with protease inhibitors (Roche) and DNaseI,

491 and lysed by sonication. The GST-tagged protein was subjected to affinity chromatography

492 using Glutathione Sepharose 4 Fast Flow resin (GE Healthcare). To reduce chaperone

493 contamination, a wash with 50 mM Tris-HCl, pH 7.5, 10 mM ATP, 10 mM MgCl2, 150 mM KCl

494 before elution. The protein was further purified by SEC on a 16/60 Superdex 200 column (GE

495 Healthcare), equilibrated in 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM DTT.

496 For reconstitution of the BRR2HR-C9ORF78 complex, lysates of the individually expressed

497 proteins (His10-BRR2HR in insect cells and His6-GST-C9ORF78 in E. coli) were mixed after

498 sonication. Complexes were subjected to affinity chromatography using Glutathione

499 Sepharose 4 Fast Flow resin as described above, followed by overnight dialysis against 20

500 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM DTT and TEV protease at a 1:20 ratio. Cleaved

501 proteins were recycled on Ni2+-NTA Sepharose (GE Healthcare) and further purified by gel

502 filtration on a 16/60 Superdex 200 column equilibrated in 20 mM Tris-HCl, pH 7.5, 200 mM

503 NaCl, 1 mM DTT.

504 For cryoEM, 1.8 nmol of the BRR2HR-C9ORF78 complex were incubated with a 1.5-fold

505 molar excess of PRPF8Jab1 for 10 minutes on ice. Subsequently the complex was purified by

506 size exclusion chromatography on a Superdex 200 increase 3.2/300 column (GE Healthcare),

507 equilibrated in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT, and then concentrated to 5

508 mg/ml using ultrafiltration (Amicon, Merck Chemicals GmbH).

509 FBP21 was purified as described before28. For cryoEM 1.8 nmol BRR2HR were incubated

510 with a two-fold molar excess of FBP21200-376 for 10 minutes on ice. Subsequently, the complex

511 was purified as described above for BRR2HR-PRPF8Jab1-C9ORF78.

512

19 513 Analytical size exclusion chromatography

514 Analytical SEC was performed on a Superdex 200 increase 3.2/300 column, equilibrated in

515 20 mM Tris-HCl, pH 8.0, 200 mM NaCl and 1 mM DTT. Proteins or protein mixtures were pre-

516 incubated on ice for 10 minutes in 50 µl reaction volumes, containing 75 µg of the respective

517 individual protein, or 75 µg of BRR2 construct or PRPF8Jab1 and a 1.5-fold molar excess of

518 putative interaction partner, prior to injection on the column. 60 µL elution fractions were

519 collected and analyzed by SDS-PAGE.

520

521 GST pull-down assay

522 Glutathione Sepharose 4 Fast Flow resin was equilibrated in binding buffer (20 mM Tris-

523 HCl, pH 8.0, 150 mM NaCl, 1 mM DTT). For each of 5 reactions, 20 µl resin slurry were

524 transferred to 1.5 ml Eppendorf tubes and incubated with 50 µg GST-C9ORF78 and equal

525 molar amounts of BRR2HR in a total volume of 500 µl (dilution in binding buffer) for 1 hour at 4

526 °C on a rotating wheel. Subsequently, the resin was washed three times with 1 ml binding

527 buffer (centrifugation at 500g for 3 minutes) and then incubated with a 0-10-fold molar excess

528 of FBP21200-376 in 50 µl binding buffer for 20 minutes at 4 °C on a shaker (600 rpm). The

529 supernatants were transferred to new tubes and kept on ice, while the resin was washed three

530 times with 1 ml binding buffer. Elution from the beads was carried out by incubating the resin

531 in 50 µl SDS-PAGE loading dye at 96 °C for 5 minutes, followed by 2-minute centrifugation at

532 maximum speed in a table-top centrifuge. 10 µl elution (beads) and supernatant were used for

533 SDS-PAGE analysis.

534

535 Limited proteolysis of BRR2HR-C9ORF78

536 26 µM complex were digested with 0.052 µg elastase in a 50 µl reaction in 50 mM Tris-HCl,

537 pH 8.0, 200 mM NaCl, 10 % (v/v) glycerol, 1 mM DTT for 45 minutes at room temperature.

538 Subsequently, the reaction was loaded on a Superdex increase 3.2/300 column, equilibrated

539 in the same buffer. 60 µl elution fractions were collected, of which 15 µl were analyzed via

20 540 SDS-PAGE. Bands of interest were excised, in-gel digested with trypsin and analyzed by mass

541 spectrometry.

542

543 Peptide SPOT array

544 Membranes with spots of peptides (25 residues, overlap of 20 residues) of C9ORF78 with

545 were obtained from Dr. Rudolf Volkmer, Charité – Universitätsmedizin Berlin. Membranes were

546 pre-washed once with 100 % ethanol and three times with phosphate-buffered saline (PBS),

547 supplemented with 1 mM DTT. The remaining binding capacity of the membranes was blocked

548 by a 3-hour incubation with blocking buffer (5 % (w/v) BSA in PBS, supplemented with 1 mM

549 DTT). Subsequently, the membranes were incubated overnight at 4 °C with His10-BRR2NC,

550 His10-BRR2CC, His10-BRR2HR or His10-BRR2FL at a concentration of 25 µg/ml (His10-BRR2NC,

551 His10-BRR2CC), 50 µg/ml (His10-BRR2HR) or 60 µg/ml (His10-BRR2FL) in binding buffer (10 mM

552 Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM DTT). As a negative control, one membrane was

553 incubated with binding buffer without added protein. The membranes where then washed three

554 times with PBS, supplemented with 0.05 % (v/v) Tween 20, 1 mM DTT (PBST) and incubated

555 with an HRP-coupled anti-His antibody (Miltenyi Biotech) diluted 1:5000 in PBS with 5 % (w/v)

556 BSA for 1 hour at room temperature. After washing the membranes three times in PBST, the

557 peptide SPOT arrays were developed with HRP juice (p.j.k. GmbH) on an Intas Advanced

558 Fluorescence and ECL imager.

559

560 U4/U6 snRNA unwinding assays

561 Yeast U4 and U6 snRNA production (commonly used also in unwinding assays with human

562 BRR2), purification, labeling and assembly were carried out as described before65. All U4/U6

563 unwinding assays were performed at 30 °C. Unwinding of 0.6-1.5 nM radioactive U4/U6 di-

564 snRNA was compared for 100 nM BRR2 (FL or HR) alone with 100 nM BRR2 (FL or HR) in

565 complex with 150 nM GST-C9ORF78 in the presence or absence of 250 nM PRPF8Jab1 or

566 PRPF8Jab1ΔC. All reactions were pre-incubated for 3 minutes in unwinding buffer (40 mM Tris-

567 HCl, pH 7.5, 50 mM NaCl, 8 % (v/v) glycerol, 0.5 mM MgCl2, 15 ng/μl acetylated BSA, 1 U/μl

21 568 RNase inhibitor, 1.5 mM DTT) in a total volume of 120 μl. Unwinding was initiated by addition

569 of 1.7 mM ATP/MgCl2, and 10 μl samples were withdrawn at the indicated time points and

570 mixed with 10 μl of stop buffer (40 mM Tris-HCl, pH 7.4, 50 mM NaCl, 25 mM EDTA, 1 % (w/v)

571 SDS, 10 % (v/v) glycerol, 0.05 % (w/v) xylene cyanol, 0.05 % (w/v) bromophenol blue). The

572 samples were run on a 6 % non-denaturing PAGE gel for 1 hour at 200 V and 4 °C. RNA bands

573 were visualized by autoradiography using a phosphoimager and quantified using the Image

574 Quant 5.2 software (GE Healthcare). Data were fit to a single exponential equation (fraction

575 unwound = A (1 – exp[- kut])); A, amplitude of the reaction; ku, apparent first-order rate constant

576 of unwinding; t, time) using GraphPad Prism.

577

578 CryoEM analysis

579 Complexes were prepared freshly in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT

580 and concentrated to 5 mg/ml using ultrafiltration. Immediately before grid preparation, the

581 samples were supplemented with 0.15 % (w/v) n-octylglucoside to overcome preferred particle

582 orientation. 3.8 µl of the final mixtures were applied to plasma-treated R1.2/1.3 holey carbon

583 grids (Quantifoil Micro Tools GmbH). Grids were plunged into liquid ethane after blotting, using

584 a Vitrobot Mark IV device (FEI) at 10 °C/100 % humidity. Image acquisition was conducted on

585 a FEI Titan Krios G3i (300 kV) with a Falcon 3EC camera, operated in counting mode using

586 EPU software (Thermo Fisher Scientific). The BRR2HR-PRPF8Jab1-C9ORF78 dataset was

587 acquired with a nominal magnification of 120,000x, resulting in a pixel size of 0.657 Å/px. The

588 BRR2HR-FBP21200-376 dataset was acquired with a nominal magnification of 92,000x, resulting

589 in a pixel size of 0.832 Å/px. A total electron dose of 40 e/Å2 was accumulated over an

590 exposure time of 31 s and 40 s, respectively.

591 All image analysis steps were done with cryoSPARC. Movie alignment was done with patch

592 motion correction, CTF estimation was conducted by Patch CTF. For the BRR2HR-FBP21200-376

593 dataset, class averages of manually selected particles were used to generate an initial

594 template for reference-based particle picking from 1,877 micrographs. Particle images were

595 extracted with a box size of 336 px and binned to 84 px for initial analysis. Ab initio

22 596 reconstruction using a small subset of particles was conducted to generate an initial 3D

597 reference for 3D heterogeneous refinement. 271,493 selected particle images were re-

598 extracted with a box of 168 px (1.664 Å/px) and subjected to non-uniform refinement followed

599 by heterogeneous refinement. Finally, 165,220 particle images were re-extracted using local

600 motion correction at full spatial resolution (box size 336 px). After per-particle CTF correction,

601 non-uniform refinement was applied to generate the final reconstruction at a resolution of 3.3

602 Å.

603 The BRR2HR-PRPF8Jab1-C9ORF78 dataset was refined similarly with only minor

604 differences. References generated from the BRR2 HR-FBP21200-376 dataset were used for

605 picking and refinement. To improve the density for C9ORF78, 3D variability analysis was

606 applied using a mask generously enclosing the N-terminal region of C9ORF78. From 542,565

607 particle images, a total of 370,493 particle images were selected for the final non-uniform

608 refinement, yielding a reconstruction at 2.76 Å resolution.

609

610 Model Building and Refinement

611 The final cryoEM map for the BRR2HR-PRPF8Jab1-C9ORF78 complex was used for

612 placement of the BRR2HR model in complex with PRPF8Jab1 24 employing PHENIX Dock in

613 Map66. The additional density observed after model placement was of sufficient quality to

614 manually and unequivocally build the N-terminal residues 5-58 of C9ORF78. The region

615 spanning residues 12-23 exhibited poorer cryoEM density and was modelled with less

616 reliability. An additional refined map focussing on C9ORF78 displayed an improved density for

617 this area providing more confidence in modelling. The structure of BRR2HR was placed into the

618 final cryoEM map of the BRR2HR-FBP21200-376 complex. Parts of FBP21200-376 could be

619 unequivocally built into additional density that was not covered by the BRR2HR model. Models

620 were completed and manually adjusted residue-by-residue, supported by real space

621 refinement in Coot. The manually built models were refined against the cryoEM maps using

622 the real space refinement protocol in PHENIX. The structures were evaluated with

623 Molprobity67. Structure figures were prepared using PyMOL (Version 1.8 Schrödinger, LLC).

23 624

625 Culturing HEK293T cells and transient transfection

626 HEK293T cells were grown in DMEM supplemented with 10 % (v/v) fetal bovine serum and

627 1 % (w/v) penicillin/streptomycin (Invitrogen). Transient transfection was performed using

628 Rotifect (Carl Roth) according to the manufacturer’s instructions. Briefly, 4.5 x 105 cells were

629 seeded on 6-well plates 24 hours prior to transfection. For each well to be transfected, 2 µg

630 plasmid and 5 µl Rotifect were mixed with 250 µl Optimum, and incubated for 5 minutes at

631 room temperature. Reactions were mixed, incubated for 20 more minutes at room temperature

632 and then added to the cells. Cells were harvested 48 hours after transfection.

633

634 Nuclear extract preparation

635 HEK293T cells grown in T75 flasks were harvested in cold PBS and pelleted by

636 centrifugation (1000 x g, 1 minute). Cell pellets were resuspended in 600 µl cold CTX buffer

637 (10 mM HEPES-NaOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl), supplemented with proteinase

638 inhibitors, and incubated for 5 minutes on ice. 600 µl CTX supplemented with 0.2 (v/v) % NP-

639 40 were added, and the reactions, after gentile mixing, were incubated for another 5 minutes

640 on ice. Nuclei were pelleted by centrifugation at 4000 x g in a table-top centrifuge for 3 minutes,

641 and the supernatant (cytosolic fraction) was discarded. Nuclei were resuspended in 240 µl NX

642 buffer (20 mM HEPES-NaOH, pH 7.9, 1.5 mM MgCl2, 420 mM KCl, 0.2 mM EDTA, 25 % (v/v)

643 glycerol), supplemented with proteinase inhibitors, and subsequently three times frozen (-80

644 °C) and thawed (37 °C), followed by 1 minute of vortexing. After a final centrifugation step at

645 maximum speed for 20 minutes at 4 °C, the supernatant (nuclear extract) was stored at -20 °C

646 until further use. Protein concentrations were determined via Bradford assay.

647

648 Flag-IP combined with Western blot

649 For Flag-IP combined with Western blot, 100 µg nuclear extract were incubated with 400 µl

650 RIPA lysis buffer, including 100 mM NaCl, 2 % (w/v) BSA and proteinase inhibitors, for 1 hour

651 at 4 °C on a rotating wheel. Subsequently, 15 µl Flag M2 affinity gel (Sigma Aldrich),

24 652 equilibrated in RIPA buffer, were added to the reactions and incubated overnight at 4 °C on a

653 rotating wheel. The resin was washed four times with RIPA buffer (without BSA) with

654 centrifugation at 4000 x g for 1 minute, then resuspended in 40 µl SDS-loading dye (2-fold

655 concentrated). Samples were boiled at 96 °C for 5 minutes, centrifuged at maximum speed for

656 2 minutes, and the supernatant was then analyzed by SDS-PAGE and Western blot using

657 monoclonal anti-Flag M2 antibody (Sigma Aldrich) and rabbit serum containing a polyclonal

658 anti-human BRR2 antibody.

659

660 Generating stable cell lines

661 Stable Flp-In™ T-REx™ 293 cells for expression of C9ORF78 and C9ORF78R41A with C-

662 terminal 3xFlag-His6-Biotin-His6 (3xFlag-HBH) tags were generated as described before52.

663 Transfection of the cell lines were done using Lipofectamine 2000 (Thermo Fisher Scientific).

664 After hygromycin selection, expression of the tagged proteins was confirmed by Western Blot

665 using monoclonal anti-Flag M2 antibody.

666

667 Flag-IP followed by mass spectrometry

668 For mass spectrometric analysis of C9ORF78 interactors, Flp-In™ T-REx™ 293 cells stably

669 expressing C-terminally 3xFlag-HBH-tagged C9ORF78 or C9ORF78R41A were grown in T75

670 flasks, and nuclear extracts were prepared as described above. As control, unmodified Flp-

671 In™ T-REx™ 293 cells were used. 500 µg nuclear extract were incubated with 800 µl IP buffer

672 (10 mM HEPES-NaOH, pH 7.3, 150 mM NaCl,10 mM MgCl2, 10 mM KCl, 0.5 mM EGTA),

673 supplemented with 3 U/ml benzonase and proteinase inhibitors, for 1 hour on a rotating wheel.

674 For each IP, 50 µl Flag M2 affinity gel (Sigma Aldrich), equilibrated in IP buffer, were added to

675 the reactions and incubated overnight at 4 °C on a rotating wheel. The resin was washed four

676 times with IP buffer (without supplements) by centrifugation at 4000 x g for 1 minute. Bound

677 proteins were eluted by incubation with 50 µl 3xFlag Peptide (Sigma Aldrich) at a concentration

678 of 0.5 µg/µl in TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) for 30 minutes on ice. The

679 supernatant was run on a SDS-PAGE gel until entrance into the separating gel. Bands were

25 680 excised and digested with trypsin using a standard protocol68. After digestion, peptides were

681 extracted and dried for LC-MS analysis.

682 Peptides were reconstituted in 10 µl of 0.05 % (w/v) TFA, 2 % (v/v) acetonitrile, and 7 µl

683 were applied to an Ultimate 3000 reversed-phase capillary nano liquid chromatography system

684 connected to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Samples were

685 injected and concentrated on a PepMap100 C18 trap column (3 µm, 100 Å, 75 µm i.d. x 2 cm;

686 Thermo Fisher Scientific) equilibrated with 0.05 % (w/v) TFA in water. After switching the trap

687 column inline, LC separations were performed on an Acclaim PepMap100 C18 capillary

688 column (2 µm, 100 Å, 75 µm i.d. x 25 cm; Thermo Fisher Scientific) at an eluent flow rate of

689 300 nl/min. Mobile phase A contained 0.1 % (v/v) formic acid in water, and mobile phase B

690 contained 0.1 % (v/v) formic acid in 80 % (v/v) acetonitrile. The column was pre-equilibrated

691 with 5 % mobile phase B followed by an increase to 44 % mobile phase B over 100 minutes.

692 Mass spectra were acquired in a data-dependent mode, utilizing a single MS survey scan (m/z

693 350-1650) with a resolution of 60,000 in the Orbitrap, and MS/MS scans of the 15 most intense

694 precursor ions with a resolution of 15,000. The dynamic exclusion time was set to 20 seconds,

695 and the automatic gain control was set to 3x106 and 1x105 for MS and MS/MS scans,

696 respectively.

697 MS and MS/MS raw data were analyzed using the MaxQuant software package with

698 implemented Andromeda peptide search engine69. Data were searched against the human

699 reference proteome downloaded from Uniprot (77,027 proteins, taxonomy 9606, last modified

700 January 29, 2021) using label-free quantification, and the match between runs option was

701 enabled. Filtering and statistical analysis was carried out using Perseus software70. Only

702 proteins which were identified with LFQ intensity values in all three replicates (within at least

703 one of the 3 experimental groups) were used for downstream analysis. Missing values were

704 replaced from normal distribution (imputation), using the default settings (width 0.3, down shift

705 1.8). Mean log2-fold differences between Flag-C9ORF78 IP (wt or R41A) against control IP

706 were calculated in Perseus, using Student’s t-tests with permutation-based FDR of 0.05.

26 707 siRNA KD and rescue

708 For siRNA KD of C9ORF78, 2.25x105 HEK293T cells per well were seeded on 6-well-plates

709 in 1.5 ml/well DMEM supplemented with 10 % (v/v) fetal bovine serum (without antibiotics) one

710 day prior to transfection. For each KD three technical replicates were prepared. Transfection

711 was carried out as described above, using 10 µl of 20 µM siRNA instead of plasmid (1:1 mix

712 of siRNA_1 5’GCA ACU GAU GAC UAU CAU UTT3’ and siRNA_2 5’CCA GAG AGG UAC

713 AGA ACU UTT3’; Eurofins Genomics). Cells were harvested in cold PBS 72 hours post

714 transfection, pelleted and resuspended in 1 ml RNA Tri-flüssig (Bio&Sell) for RNA extraction.

715 RNA was extracted following the manufacturer’s instructions. RNA pellets were dissolved in

716 88 µl water and subsequently digested with DNase I for 20 minutes at 37 °C, followed by RNA

717 extraction with ROTI-Aqua-P/C/I (Carl Roth). RNA concentrations were quantified on a

718 Nanodrop spectrophotometer and adjusted to 0.5 µg/µl. For rescue experiments, cells were

719 transfected with siRNA resistant C9ORF78 (wt or R41A) genes in pcDNA3.1(+) vectors

720 (ThermoFisher) 24 hours post transfection with siRNAs, and harvested after an additional 48

721 hours.

722

723 RT-PCR, quantitative PCR and radioactive PCR

724 For reverse transcription-PCR (RT-PCR), 1 µg RNA was used with 1 ng gene-specific

725 reverse primer (combining up to 3 gene specific primers in one RT reaction; Supplementary

726 Table 4). The reaction was carried out using M-MuLV reverse transcriptase (Enzymatics)

727 according to the manufacturer’s protocol.

728 Quantitative PCR (qPCR) was performed in 96 well format using the PowerUp SYBR Green

729 Master Mix (Thermo Fisher) on a Stratagene Mx3000P instruments. qPCR reactions were

730 performed in duplicates. Mean values were used to normalize expression to mRNA of

731 GAPDH(ΔCT). Values shown are means +/-standard deviation, p-values were calculated using

732 Student’s unpaired t-Test (Microsoft Office, Excel). Significance is indicated by asterisks (*, p

733 < 0.05; **, p < 0.01; ***, p < 0.001).

27 734 Low-cycle PCR with a [32P]-labeled forward primer (Supplementary Table 4) was performed

735 using Q5 High Fidelity DNA Polymerase (NEB). Products were separated by denaturing 5 %

736 PAGE and quantified using a Phosphoimager and ImageQuantTL software. Quantifications

737 are given as mean values of several technical and biological replicates, error bars represent

738 standard deviation, p-values were calculated using Student’s unpaired t-test (Microsoft Office,

739 Excel). Significance is indicated by asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p <

740 0.0001).

741

742 RNA sequencing and data analysis of siRNA KD

743 RNA sequencing was performed in biological triplicates using DNAse I-digested RNA

744 samples for library preparation. Libraries were prepared using the mRNA enrichment method

745 at BGI Genomics and sequenced using DNBSeq PE150 sequencing. This yielded ~ 35 million

746 paired-end 150 nt reads for C9ORF78 and control samples. Reads were aligned to the human

747 hg38 genome, using STAR, yielding ~ 95 % uniquely aligned reads. Alternative splicing

748 changes were calculated using RMATS. To obtain only high confidence targets, only targets

749 with a p-value < 0.01 and a deltaPSI > 0.15 were considered alternatively spliced. Additionally,

750 to filter out splicing events in weakly expressed genes or gene regions with low expression,

751 events with less than 100 combined junction reads in all samples were excluded.

752 To analyze the splice site strength of altered exons, the respective coordinates were

753 extracted from the RMATS output table in bed format (-20 to +3 for 3'-ss; -3 to +6 for 5'-ss).

754 Bedtools getfasta was then used to extract splice site sequences. Sequences were input to

755 MaxEntScan71, using the Maximum Entropy Model to score. As a control, all RMATS-quantified

756 exons were treated identically.

757

758 FLASH

759 FLASH was carried out essentially as described before52. Briefly, Flp-In™ T-REx™ 293

760 cells expressing either 3xFlag-BIO tagged C9ORF78 or 3xFlag-BIO tagged GFP were induced

761 with 0.1 µg/mL doxycycline for ~ 16 hours, and UV-cross-linked with 0.2 mJ/cm2 UV-C light.

28 762 Target proteins were purified using MyONE C1 streptavidin beads (ThermoFisher Scienific).

763 After a partial RNase digestion using RNase I and end-repair with T4 PNK, uniquely barcoded

764 s-oligos were ligated to each sample (two biological replicates). After several stringent washes

765 (up to 1 % (w/v) SDS), biological replicates were merged, bead-bound RNA was released with

766 proteinase K and purified with Oligo Clean and concentrator columns (Zymo Research). RNA

767 was then reverse-transcribed and treated with RNase H to phosphorylate 5’-ends of cDNA.

768 The cDNA was circularized with CircLigaseII (Lucigen), amplified with Q5 polymerase and

769 sequenced on an Illumina platform (100 bp, PE sequencing).

770 The resulting fastq files were first merged with bbmerge, replicates were then split using

771 flexbar and mapped to hg38 assembly of the human genome using bowtie2 and bbmap, after

772 which umi-tools was used to remove PCR duplicates. Enrichment plots were generated using

773 the snakePipes “noncoding-RNA-seq” workflow, which uses TEtools to calculate specific

774 enrichment of non-coding RNAs against a background dataset (GFP in this case). Cumulative

775 coverage plots were generated with computeMatrix scale-regions and plotHeatmap from the

776 deepTools2 package, using alternatively spliced exons and 3’-ss detected with RMATS.

777

778 Data availability

779 CryoEM maps have been deposited in the Electron Microscopy Data Bank

780 (https://www.ebi.ac.uk/pdbe/emdb) under accession codes EMD-13046 (BRR2HR-PRPF8Jab1-

781 C9ORF78; https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-13046) and EMD-13045 (BRR2HR-

782 FBP21200-376; https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-11105). Structure coordinates

783 have been deposited in the RCSB Protein Data Bank (https://www.rcsb.org) with accession

784 codes 7OS2 (BRR2HR-PRPF8Jab1-C9ORF78) and 7OS1 (BRR2HR-FBP21200-376).

785 RNA sequencing data from the siRNA KDs have been deposited to GEO under accession

786 number GSE176517. FLASH data are available under GSE176464. Other data are available

787 from the corresponding author upon reasonable request.

788

29 789 Acknowledgements

790 Large-scale yeast two-hybrid analyses were conducted in collaboration with the group of

791 Ulrich Stelzl, University of Graz, Austria, in collaboration with Camilla von Alvensleben,

792 Laboratory of Structural Biochemistry, Freie Universität Berlin. We acknowledge the

793 assistance of the core facility BioSupraMol supported by the Deutsche

794 Forschungsgemeinschaft in mass spectrometric and electron microscopic analyses. This work

795 was supported by grants from the Deutsche Forschungsgemeinschaft (TRR186-A15 to F.H.

796 and M.C.W.; INST 130/1064-1 FUGG to Freie Universität Berlin) and the Berlin University

797 Alliance (501_BIS-CryoFac to M.C.W.).

798

799 Author contributions

800 A.B. cloned genes, purified proteins, generated stable cell lines with help of T.A. and I.A.I.

801 and performed all experiments, except for sample preparation for mass spectrometry (B.K.),

802 grid freezing for cryoEM (T.H.) and FLASH sample preparation of UV-crosslinked cells (I.A.I.).

803 M.P. analyzed RNA-seq data derived from the siRNA KD eperiments and contributed to

804 experimental design. B.K. acquired and processed proteomics data. I.A.I. analyzed RNA-seq

805 data derived from the FLASH experiments. T.H. acquired, processed and refined cryoEM data.

806 M.C.W. built atomic models and G.W. refined structures. All authors contributed to the analysis

807 of the data and the interpretation of the results. A.B. wrote the manuscript with contributions

808 from the other authors. C.F., T.A., F.H. and M.C.W. supervised work in their respective groups.

809 M.C.W. conceived and coordinated the project.

810

811 Competing interests

812 The authors declare no competing interests.

813

30 814 References

815 1. Barbosa-Morais, N.L. et al. The evolutionary landscape of alternative splicing in vertebrate 816 species. Science 338, 1587-1593 (2012). 817 2. Kelemen, O. et al. Function of alternative splicing. Gene 514, 1-30 (2013). 818 3. Wahl, M.C., Will, C.L. & Luhrmann, R. The spliceosome: design principles of a dynamic 819 RNP machine. Cell 136, 701-718 (2009). 820 4. Wilkinson, M.E., Charenton, C. & Nagai, K. RNA Splicing by the Spliceosome. Annu Rev 821 Biochem 89, 359-388 (2020). 822 5. Cordin, O. & Beggs, J.D. RNA helicases in splicing. RNA biology 10, 83-95 (2013). 823 6. Laggerbauer, B., Achsel, T. & Lührmann, R. The human U5-200kD DEXH-box protein 824 unwinds U4/U6 RNA duplices in vitro. Proc. Natl. Acad. Sci. USA 95, 4188-4192 (1998). 825 7. Raghunathan, P.L. & Guthrie, C. RNA unwinding in U4/U6 snRNPs requires ATP 826 hydrolysis and the DEIH-box splicing factor Brr2. Curr Biol 8, 847-855 (1998). 827 8. Charenton, C., Wilkinson, M.E. & Nagai, K. Mechanism of 5' splice site transfer for human 828 spliceosome activation. Science 364, 362-367 (2019). 829 9. Hahn, D., Kudla, G., Tollervey, D. & Beggs, J.D. Brr2p-mediated conformational 830 rearrangements in the spliceosome during activation and substrate repositioning. Genes 831 Dev 26, 2408-2421 (2012). 832 10. Small, E.C., Leggett, S.R., Winans, A.A. & Staley, J.P. The EF-G-like GTPase Snu114p 833 regulates spliceosome dynamics mediated by Brr2p, a DExD/H box ATPase. Mol Cell 23, 834 389-399 (2006). 835 11. Fourmann, J.B. et al. Dissection of the factor requirements for spliceosome disassembly 836 and the elucidation of its dissociation products using a purified splicing system. Genes 837 Dev 27, 413-428 (2013). 838 12. Santos, K.F. et al. Structural basis for functional cooperation between tandem helicase 839 cassettes in Brr2-mediated remodeling of the spliceosome. Proc Natl Acad Sci U S A 109, 840 17418-17423 (2012). 841 13. Kim, D.H. & Rossi, J.J. The first ATPase domain of the yeast 246-kDa protein is required 842 for in vivo unwinding of the U4/U6 duplex. RNA 5, 959-971 (1999). 843 14. Zhang, L. et al. Structural evidence for consecutive Hel308-like modules in the 844 spliceosomal ATPase Brr2. Nat. Struct. Mol. Biol. 16, 731-739 (2009). 845 15. Pena, V. et al. Common design principles in the spliceosomal RNA helicase Brr2 and in 846 the Hel308 DNA helicase. Mol. Cell 35, 454-466 (2009). 847 16. Absmeier, E., Santos, K.F. & Wahl, M.C. Functions and regulation of the Brr2 RNA 848 helicase during splicing. Cell cycle 15, 3362-3377 (2016). 849 17. Absmeier, E. et al. The large N-terminal region of the Brr2 RNA helicase guides productive 850 spliceosome activation. Gene Dev 29, 2576-2587 (2015). 851 18. Absmeier, E., Santos, K.F. & Wahl, M.C. Molecular Mechanism Underlying Inhibition of 852 Intrinsic ATPase Activity in a Ski2-like RNA Helicase. Structure 28, 236-243 e233 (2020). 853 19. Vester, K., Santos, K.F., Kuropka, B., Weise, C. & Wahl, M.C. The inactive C-terminal 854 cassette of the dual-cassette RNA helicase BRR2 both stimulates and inhibits the activity 855 of the N-terminal helicase unit. J Biol Chem 295, 2097-2112 (2020). 856 20. Absmeier, E., Becke, C., Wollenhaupt, J., Santos, K.F. & Wahl, M.C. Interplay of cis- and 857 trans-regulatory mechanisms in the spliceosomal RNA helicase Brr2. Cell cycle 16, 100- 858 112 (2017).

31 859 21. Grainger, R.J. & Beggs, J.D. Prp8 protein: at the heart of the spliceosome. RNA 11, 533- 860 557 (2005). 861 22. Maeder, C., Kutach, A.K. & Guthrie, C. ATP-dependent unwinding of U4/U6 snRNAs by 862 the Brr2 helicase requires the C terminus of Prp8. Nat. Struct. Mol. Biol. 16, 42-48 (2009). 863 23. Mozaffari-Jovin, S. et al. The Prp8 RNase H-like domain inhibits Brr2-mediated U4/U6 864 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA. Genes Dev. 26, 2422- 865 2434 (2012). 866 24. Mozaffari-Jovin, S. et al. Inhibition of RNA helicase Brr2 by the C-terminal tail of the 867 spliceosomal protein Prp8. Science 341, 80-84 (2013). 868 25. Nguyen, T.H. et al. Structural basis of Brr2-Prp8 interactions and implications for U5 869 snRNP biogenesis and the spliceosome active site. Structure 21, 910-919 (2013). 870 26. Agafonov, D.E. et al. Semiquantitative proteomic analysis of the human spliceosome via 871 a novel two-dimensional gel electrophoresis method. Mol Cell Biol 31, 2667-2682 (2011). 872 27. Wahl, M.C. & Luhrmann, R. SnapShot: Spliceosome Dynamics I. Cell 161, 1474-e1471 873 (2015). 874 28. Henning, L.M. et al. A new role for FBP21 as regulator of Brr2 helicase activity. Nucleic 875 Acids Res 45, 7922-7937 (2017). 876 29. Sticht, J. et al. FBP21's C-Terminal Domain Remains Dynamic When Wrapped around 877 the c-Sec63 Unit of Brr2 Helicase. Biophys J 116, 406-418 (2019). 878 30. Wollenhaupt, J. et al. Intrinsically Disordered Protein Ntr2 Modulates the Spliceosomal 879 RNA Helicase Brr2. Biophys J 114, 788-799 (2018). 880 31. Nguyen, T.H. et al. Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 A resolution. 881 Nature 530, 298-302 (2016). 882 32. Wan, R.X. et al. The 3.8 angstrom structure of the U4/U6.U5 tri-snRNP: Insights into 883 spliceosome assembly and catalysis. Science 351, 466-475 (2016). 884 33. Bertram, K. et al. Cryo-EM Structure of a Pre-catalytic Human Spliceosome Primed for 885 Activation. Cell 170, 701-713 e711 (2017). 886 34. Zhan, X., Yan, C., Zhang, X., Lei, J. & Shi, Y. Structure of a human catalytic step I 887 spliceosome. Science 359, 537-545 (2018). 888 35. Zhan, X., Yan, C., Zhang, X., Lei, J. & Shi, Y. Structures of the human pre-catalytic 889 spliceosome and its precursor spliceosome. Cell Res 28, 1129-1140 (2018). 890 36. Wang, Y. et al. Large scale identification of human hepatocellular carcinoma-associated 891 antigens by autoantibodies. J Immunol 169, 1102-1109 (2002). 892 37. Uhlen, M. et al. A pathology atlas of the human cancer transcriptome. Science 357, 893 aan2507 (2017). 894 38. Wang, J. et al. Tls1 regulates splicing of shelterin components to control telomeric 895 heterochromatin assembly and telomere length. Nucleic Acids Res 42, 11419-11432 896 (2014). 897 39. Zhang, X. et al. Structure of the human activated spliceosome in three conformational 898 states. Cell Res 28, 307-322 (2018). 899 40. Zhang, X. et al. An Atomic Structure of the Human Spliceosome. Cell 169, 918-929 e914 900 (2017). 901 41. Zhang, X. et al. Structures of the human spliceosomes before and after release of the 902 ligated exon. Cell Res 29, 274-285 (2019).

32 903 42. Sivaramakrishnan, S., Spink, B.J., Sim, A.Y., Doniach, S. & Spudich, J.A. Dynamic charge 904 interactions create surprising rigidity in the ER/K alpha-helical protein motif. Proc Natl 905 Acad Sci U S A 105, 13356-13361 (2008). 906 43. Suveges, D., Gaspari, Z., Toth, G. & Nyitray, L. Charged single alpha-helix: a versatile 907 protein structural motif. Proteins 74, 905-916 (2009). 908 44. Ulrich, A.K., Seeger, M., Schütze, T., Bartlick, N. & Wahl, M.C. Scaffolding in the 909 Spliceosome via Single alpha Helices. Structure 24, 1972-1983 (2016). 910 45. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. 911 J Mol Biol 372, 774-797 (2007). 912 46. Mozaffari-Jovin, S. et al. Novel regulatory principles of the spliceosomal Brr2 RNA 913 helicase and links to retinal disease in humans. RNA biology 11, 298-312 (2014). 914 47. Dyson, H.J. & Wright, P.E. Intrinsically unstructured proteins and their functions. Nat Rev 915 Mol Cell Biol 6, 197-208 (2005). 916 48. Busch, A. & Hertel, K.J. Extensive regulation of NAGNAG alternative splicing: new tricks 917 for the spliceosome? Genome Biol 13, 143 (2012). 918 49. Spellman, R., Llorian, M. & Smith, C.W. Crossregulation and functional redundancy 919 between the splicing regulator PTB and its paralogs nPTB and ROD1. Mol Cell 27, 420- 920 434 (2007). 921 50. Boutz, P.L. et al. A post-transcriptional regulatory switch in polypyrimidine tract-binding 922 proteins reprograms alternative splicing in developing neurons. Genes Dev 21, 1636-1652 923 (2007). 924 51. Vuong, J.K. et al. PTBP1 and PTBP2 Serve Both Specific and Redundant Functions in 925 Neuronal Pre-mRNA Splicing. Cell Rep 17, 2766-2775 (2016). 926 52. Ilik, I.A., Aktas, T., Maticzka, D., Backofen, R. & Akhtar, A. FLASH: ultra-fast protocol to 927 identify RNA-protein interactions in cells. Nucleic Acids Res 48, e15 (2020). 928 53. Ramirez, F., Dundar, F., Diehl, S., Gruning, B.A. & Manke, T. deepTools: a flexible 929 platform for exploring deep-sequencing data. Nucleic Acids Res 42, W187-191 (2014). 930 54. Bhardwaj, V. et al. snakePipes: facilitating flexible, scalable and integrative epigenomic 931 analysis. Bioinformatics 35, 4757-4759 (2019). 932 55. Mathew, R. et al. Phosphorylation of human PRP28 by SRPK2 is required for integration 933 of the U4/U6-U5 tri-snRNP into the spliceosome. Nat Struct Mol Biol 15, 435-443 (2008). 934 56. Papasaikas, P., Tejedor, J.R., Vigevani, L. & Valcarcel, J. Functional splicing network 935 reveals extensive regulatory potential of the core spliceosomal machinery. Mol Cell 57, 7- 936 22 (2015). 937 57. Tadokoro, K. et al. Frequent occurrence of protein isoforms with or without a single amino 938 acid residue by subtle alternative splicing: the case of Gln in DRPLA affects subcellular 939 localization of the products. J Hum Genet 50, 382-394 (2005). 940 58. Vogan, K.J., Underhill, D.A. & Gros, P. An alternative splicing event in the Pax-3 paired 941 domain identifies the linker region as a key determinant of paired domain DNA-binding 942 activity. Mol Cell Biol 16, 6677-6686 (1996). 943 59. Condorelli, G., Bueno, R. & Smith, R.J. Two alternatively spliced forms of the human 944 insulin-like growth factor I receptor have distinct biological activities and internalization 945 kinetics. J Biol Chem 269, 8510-8516 (1994). 946 60. Mironov, A., Denisov, S., Gress, A., Kalinina, O.V. & Pervouchine, D.D. An extended 947 catalogue of tandem alternative splice sites in human tissue transcriptomes. PLoS Comput 948 Biol 17, e1008329 (2021).

33 949 61. Strittmatter, L.M. et al. psiCLIP reveals dynamic RNA binding by DEAH-box helicases 950 before and after exon ligation. Nat Commun 12, 1488 (2021). 951 62. Semlow, D.R., Blanco, M.R., Walter, N.G. & Staley, J.P. Spliceosomal DEAH-Box 952 ATPases Remodel Pre-mRNA to Activate Alternative Splice Sites. Cell 164, 985-998 953 (2016). 954 63. Bonnal, S.C., Lopez-Oreja, I. & Valcarcel, J. Roles and mechanisms of alternative splicing 955 in cancer - implications for care. Nat Rev Clin Oncol 17, 457-474 (2020). 956 64. Guerrero-Martinez, J.A. & Reyes, J.C. High expression of SMARCA4 or SMARCA2 is 957 frequently associated with an opposite prognosis in cancer. Sci Rep 8, 2043 (2018). 958 65. Theuser, M., Hobartner, C., Wahl, M.C. & Santos, K.F. Substrate-assisted mechanism of 959 RNP disruption by the spliceosomal Brr2 RNA helicase. Proc Natl Acad Sci U S A 113, 960 7798-7803 (2016). 961 66. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and 962 electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol 75, 861-877 963 (2019). 964 67. Williams, C.J. et al. MolProbity: More and better reference data for improved all-atom 965 structure validation. Protein Sci 27, 293-315 (2018). 966 68. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of 967 proteins silver-stained polyacrylamide gels. Anal Chem 68, 850-858 (1996). 968 69. Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass 969 spectrometry-based shotgun proteomics. Nat Protoc 11, 2301-2319 (2016). 970 70. Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of 971 (prote)omics data. Nat Methods 13, 731-740 (2016). 972 71. Yeo, G. & Burge, C.B. Maximum entropy modeling of short sequence motifs with 973 applications to RNA splicing signals. J Comput Biol 11, 377-394 (2004). 974 72. Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence 975 alignments using Clustal Omega. Mol Syst Biol 7, 539 (2011). 976 73. Barton, G.J. ALSCRIPT: a tool to format multiple sequence alignments. Protein Eng 6, 37- 977 40 (1993). 978

34 979 Figures and figure legends

980

981 982 Fig. 1. C9ORF78 stably interacts with BRR2

983 a, SDS-PAGE gels showing elution fractions from analytical SEC, monitoring interaction of

984 GST-C9ORF78 with BRR2FL. Elution direction is indicated by an arrow. The same elution

985 fractions from runs under identical conditions are shown. Protein bands are identified on the

986 right. M, molecular mass marker. In the third and fifth panel, upper and lower regions of the

987 same gels were spliced together. Dotted lines, splice positions.

988 b, C9ORF78 interacts with BRR2 in HEK293 cells. Flag-IP with HEK293 nuclear extract

989 followed by WB and antibody staining against BRR2 (top) or Flag-tag (middle and bottom).

990 Flag-CLK1 protein was taken as negative control (Flag-control). Protein bands are identified

991 on the right.

35 992 c, CryoEM map of a BRR2HR-PRPF8Jab1-C9ORF78 complex at 2.76 Å resolution, contoured at

993 6 root-mean-square-deviation (RMSD). Color coding in this and the following figures: BRR2

994 NC, gray; BRR2 CC), slate blue; PRPF8Jab1, gold; C9ORF78, orange.

995 d, CryoEM map of a BRR2HR-FBP21200-376 complex at 3.3 Å resolution, contoured at 6 RMSD.

996 Color coding in this and the following figures: FBP21 200-376, yellow.

997

36 998

999

37 1000 Fig. 2. BRR2 binding by FBP21 and C9ORF78 is mutually exclusive

1001 a, Cartoon model of the BRR2HR-PRPF8Jab1-C9ORF78 complex (top) and close-up of the

1002 C9ORF78 binding site at the C-terminal Sec63 module of BRR2HR (bottom; boxed in the top

1003 image). Selected interacting residues are shown as sticks and labeled. In this and the following

1004 structure images: dashed lines, hydrogen bonds or salt bridges.

1005 b, Multiple sequence alignment of C9ORF78 residues 5-58 (BRR2HR-binding region) from

1006 different species, as indicated on the left. Residues are colored light to dark according to

1007 increasing level of conservation (54 % conservation, light yellow; 100 % conservation, orange).

1008 Residue numbering according to human C9ORF78 is indicated above the sequences.

1009 Secondary structure elements observed in the BRR2HR-PRPF8Jab1-C9ORF78 cryoEM

1010 structure are indicated below the sequence. Residues R9 (reduced BRR2 binding upon alanine

1011 exchange) and R41 (abrogation of BRR2 binding upon alanine exchange) are highlighted by

1012 triangles. The alignment was prepared with Homologene (NCBI) employing Clustal Omega 72

1013 and shaded with ALSCRIPT73.

1014 c, Cartoon model of the BRR2HR-FBP21200-376 complex (top) and close-up of the FBP21200-376

1015 binding site at the C-terminal Sec63 module of BRR2HR (bottom; boxed in the top image).

1016 Selected interacting residues are shown as sticks and labeled.

1017 d, SDS PAGE analysis monitoring results from a GST pull-down assay, revealing competitive

1018 binding of C9ORF78 and FBP21200-376 to BRR2HR. BRR2HR-GST-C9ORF78 complex was

1019 immobilized on glutathione beads, and increasing amounts of FBP21200-376 were added.

1020 Protein bands are identified on the right. M, molecular mass marker; [FBP21200-376], increasing

1021 molar excess of FBP21200-376; b, bead fractions; e, elution fractions. Different regions of the

1022 same gel were spliced together. Dashed lines, splice positions.

1023

38 1024

1025

1026 Fig. 3. Mutational disruption of the C9ORF78-BRR2HR interaction and comparison to

1027 DDX23 binding.

1028 a, Close-up view of C9ORF78 residue R41 interacting with BRR2 residues Y1826, D1795,

1029 E1830 and Q1798.

1030 b, SDS-PAGE gels showing elution fractions from analytical SEC, monitoring interactions of

1031 GST-C9ORF78wt and GST-C9ORF78R41A with BRR2HR. The same elution fractions from runs

1032 under identical conditions are shown. Protein bands are identified on the right. M, molecular

1033 mass marker. In the third and fifth panel, upper and lower regions of the same gels were spliced

1034 together. Dotted lines, splice positions.

1035 c, Flag-IP followed by Western blot with C9ORF78wt or C9ORF78R41A. Immunostaining against

1036 BRR2 and Flag-tag.

1037 d, Superposition of the present BRR2HR-PRPF8Jab1-C9ORF78 complex on the corresponding

1038 components of the human U4/U6-U5 tri-snRNP (PDB ID 6QW6) according to the BRR2 CC.

39 1039 Components of the tri-snRNP are shown in darker colors; BRR2 NC, dark gray; BRR2 CC,

1040 dark blue; PRPF8Jab1, brown; DDX23, violet.

1041

40 1042

1043

1044 Fig. 4. C9ORF78 regulates alternative splicing

1045 a, Results from qPCR analyses, showing normalized C9ORF78 expression levels relative to

1046 GAPDH expression, in cells transfected with control siRNA (si control) or C9ORF78-targeting

1047 siRNA (si C9ORF78). Bars represent means ± SD, n = 3.

1048 b, Alternative splicing changes upon siRNA-mediated C9ORF78 KD, as determined by

1049 RMATS. MXE, mutually exclusive exons; SE, skipped exon; RI, retained intron; A5’/3’ss,

1050 alternative 5’/3’-splice sites. More inclusion upon C9ORF78 KD is indicated in light gray (up),

1051 skipping in black (dn).

1052 c, Volcano plots of significantly changed skipped exons (SE; left), alternative 3’-splice sites

1053 (A3’ss, middle) and splicing changes of all NAGNAG splice sites annotated in HEK293 cells

1054 (right) upon C9ORF78 KD. Targets selected for validation PCR (PTBP2, C9ORF131,

1055 SMARCA4) are indicated by large data points and labeled.

41 1056

1057

1058 Fig. 5. Regulation of PTBP2 exon 10 depends on the BRR2-C9ORF78 interaction.

1059 a, Validation PCRs confirm C9ORF78 KD-induced changes in alternative splicing. Top,

1060 quantifications (n = 3). Horizontal lines, medians; whiskers, minimum/maximum values.

1061 Statistical significance was determined by unpaired t-tests; **p < 0.01; ****p < 0.0001. Bottom,

1062 representative gels.

1063 b, Quantification of radioactive RT-PCRs after siRNA mediated KD of C9ORF78 and rescue

1064 with C9ORF78wt or C9ORF78R41A for the indicated targets. Bars represent means ± SD; n = 9

1065 (3 biological replicates with 3 technical replicates each). Statistical significance was

1066 determined by unpaired t-tests; **p < 0.01; ***p < 0.001; ns, not significant.

1067

42 1068 1069

1070 Fig. 6. C9ORF78 interacts with additional spliceosomal components.

1071 a, Heat map displaying the top 20 differentially enriched non-coding RNAs in GFP (negative

1072 control) and C9ORF78 pull-down after RNA-protein UV-cross-linking and FLASH52, analyzed

1073 with snakePipes54. Two biological replicates each are shown. Colored by Z-score as indicated.

1074 Negatively enrichment RNAs (blue) in the C9ORF78 pull-down are mainly tRNAs, which are

1075 commonly found as background in such analyses.

1076 b, Spliceosomal proteins enriched in Flag-IP from nuclear extract of HEK293 cells stably

1077 expressing Flag-C9ORF78wt (orange) or Flag-C9ORF78R41A (light orange). Other co-

43 1078 precipitated proteins are listed in Supplementary Table 3. Data represent log2-fold changes in

1079 the mean difference of Flag-C9ORF78 IP (wt or R41A) vs. control Flag-IP, n = 3.

1080

44 1081

1082

1083 Fig. 7. Putative C8ORF78 neighborhood in the C* complex.

1084 Model for the positioning of C9ORF78 in the C* complex. The BRR2HR-PRPF8Jab1-C9ORF78

1085 structure was aligned with a cryoEM structure of the spliceosomal C* complex (PDB ID 5XJC)

1086 according to the common parts of BRR2HR/BRR2. Color code: BRR2 NC, gray; BRR2 CC,

1087 slate blue; C9ORF78, orange; PRPF8, gold; PRPF22, forest green; CWC22, pink; SLU7, sky

1088 blue. The 231 C-terminal C9ORF78 residues that are not visible in the BRR2HR-PRPF8Jab1-

1089 C9ORF78 cryoEM map are indicated by a dotted line.

1090

45 1091 Supplementary information

1092

1093 A multi-factor trafficking site on the spliceosomal

1094 remodeling enzyme, BRR2, recruits C9ORF78 to regulate

1095 alternative splicing

1096

1097 Alexandra Bergfort1, Marco Preußner2, Benno Kuropka3,4, İbrahim Avşar Ilik5, Tarek Hilal1,4,6,

1098 Gert Weber7, Christian Freund3, Tuğçe Aktaş5, Florian Heyd2, Markus C. Wahl1,7,*

1099

1100 1 Freie Universität Berlin, Institute of Chemistry and Biochemistry, Laboratory of Structural

1101 Biochemistry, Takustrasse 6, D-14195 Berlin, Germany

1102 2 Freie Universität Berlin, Institute of Chemistry and Biochemistry, Laboratory of RNA

1103 Biochemistry, Takustrasse 6, D-14195 Berlin, Germany

1104 3 Freie Universität Berlin, Institute of Chemistry and Biochemistry, Laboratory of Protein

1105 Biochemistry, Thielallee 63, D-14195, Berlin, Germany

1106 4 Freie Universität Berlin, Institute of Chemistry and Biochemistry, Core Facility BioSupraMol,

1107 Thielallee 63, D-14195, Berlin, Germany

1108 5 Max Planck Institute for Molecular Genetics, Ihnestr. 63, D-14195 Berlin, Germany

1109 6 Freie Universität Berlin, Institute of Chemistry and Biochemistry, Research Center of

1110 Electron Microscopy and Core Facility BioSupraMol, Fabeckstr. 36a, 14195 Berlin,

1111 Germany

1112 7 Helmholtz-Zentrum Berlin für Materialien und Energie, Macromolecular Crystallography,

1113 Albert-Einstein-Straße 15, D-12489 Berlin, Germany

1114

1115 * Correspondence to: [email protected]

1116

46 1117 Supplementary Tables

1118 Supplementary Table 1. CryoEM data collection and refinement statistics

BRR2HR-PRPF8Jab1- BRR2HR- Complex C9ORF78 FBP21200-376 Data collection Microscope FEI Titan Krios G3i Voltage [keV] 300 Camera Falcon 3EC Magnification 120,000 96,000 Pixel size [Å/px] 0.657 0.832 Total electron exposure [e–/Å2] 40 40 Exposure rate [e–/px/s] 0.6 0.7 No. of frames collected during exposure 33 33 Defocus range [μm] 0.8 -1.8 0.8 -1.8 Automation software EPU2.10 EPU2.09 Micrographs collected 5,160 1,986 Micrographs used 4,986 1,877 Data processing Total extracted particles 936,716 787,610 Refined particles 542,565 271,493 Final particles 370,493 57,854 Point-group or helical symmetry parameters C1 C1 Global resolution [Å] 2.72 3.30 FSC0.143 unmasked/masked [Å] 3.1 / 2.7 3.7 / 3.0 Local resolution range [Å] 30.0 - 2.2 24.6 - 2.4 Map sharpening B factor [Å2] / (B factor range) -97 -120.3 Map sharpening method Local B-factor Local B-factor Model refinement CC mask 0.89 0.86 CC volume 0.89 0.85 Model Non-H atoms 16,485 14,054 Protein residues 2,046 1,746 RMSD1 Bond lengths [Å] 0.004 0.005 Bond angles [°] 0.564 0.602 Ramachandran plot Favored [%] 97.84 97.18 Allowed [%] 2.16 2.82 Outliers [%] 0 0 Model quality2 Clash score 7.93 10.12 Rotamer outliers [%] 0.93 0.96 Overall score 1.47 1.68 Data availability EMDB ID 13046 13045 PDB ID 7OS2 7OS1 1119 1120 1 RMSD, root-mean-square deviation from ideal geometry 1121 2 Assessed using MolProbity67 1122

47 1123 Supplementary Table 2. RNA-seq results upon C9ORF78 KD

1124 See attached Excel file.

1125

1126 Supplementary Table 3. Proteins enriched in Flag-C9ORF78wt IP or Flag-C9ORF78R41A IP

1127 See attached Excel file.

1128

1129 Supplementary Table 4. PCR primers

C9ORF78 (RT-qPCR) Forward 5‘-TCCTTCGTGCCTACCAACAT-3‘ Reverse 5‘-GCTTCTCCGTGTCACCTACT-3‘ GAPDH (RT-qPCR) Forward 5‘-CTTCGCTCTCTGCTCCTCCTGTTCG-3‘ Reverse 5‘-ACCAGGCGCCCAATACGACCAAAT-3‘ PTBP2 (radioactive RT-PCR) Forward 5‘-CTGAGTCCTTTGGCCATTCC-3‘ Reverse 5‘-CACGCTGCACATCTCCATAA-3‘ SMARCA4 (radioactive RT-PCR) Forward 5‘-AAACTCTCCCCTAACCCACC-3‘ Reverse 5‘-TGGATGAAGACCTCGCTGAG-3‘ C1ORF131 (radioactive RT-PCR) Forward 5‘-ACAAGTAGAAGTGGTAGAATTTCAC-3‘ Reverse 5‘-TCCACATCTCTCTCCAAAACAC-3‘ 1130

48 1131 Supplementary Figures

1132

49 1133

1134 Supplementary Fig. 1. Protein interaction studies in vitro

1135 a, SDS-PAGE gels showing elution fractions from analytical SEC, monitoring interaction of

1136 GST-C9ORF78 with BRR2 truncation variants (BRR2HR, BRR2NC, BRR2CC). Elution direction

1137 is indicated by an arrow. The same elution fractions from runs under identical conditions are

1138 shown. Protein bands are identified on the right. M, molecular mass marker. In the second

1139 panel, upper and lower regions of the same gels were spliced together. Dotted line, splice

1140 position.

1141 b, Like a, monitoring simultaneous interaction of PRPF8Jab1ΔC and C9ORF78 with BRR2HR

1142 (upper panels), and interaction of C9ORF78 with PRPF8Jab1ΔC in the absence of BRR2HR (lower

1143 panels). Elution direction is indicated by an arrow. The same elution fractions from runs under

1144 identical conditions are shown. Protein bands are identified on the right. M, molecular mass

1145 marker. In the first, second and fourth panel, different regions of the same gels were spliced

1146 together. Dotted lines, splice positions.

1147 c, Left, SDS-PAGE gels showing elution fractions from analytical SEC after digest of the

1148 BRR2HR-C9ORF78 complex with elastase. Elution direction is indicated by an arrow. Protein

1149 bands are identified on the right. M, molecular mass marker. IS, injected sample. Gel bands

1150 subjected to mass spectrometric analysis are numbered and highlighted by red boxes. Right,

1151 C9ORF78 peptides and sequence coverage identified in the analyzed gel bands by mass

1152 spectrometry.

1153 d, C9ORF78 peptide SPOT arrays incubated with His-tagged BRR2 constructs and probed by

1154 anti-His antibody. Peptides 1 and 2 (red boxes) are highly reactive with BRR2FL, BRR2HR and

1155 BRR2CC, but not with BRR2NC. Sequences for peptides 1 and 2 are indicated in the lower left

1156 box. The conserved BRR2-interacting F8-R9 motif, contained in these peptides, is shown in

1157 bold and underlined. Positively charged residues, blue; negatively charged residues, red.

1158

50 1159 51 1160 Supplementary Fig. 2. CryoEM analysis of BRR2HR-PRP8Jab1-C9ORF78 and BRR2HR-

1161 FBP21200-376 complexes

1162 a, Representative cryoEM micrographs of the BRR2HR-PRP8Jab1-C9ORF78 complex. Scale

1163 bar, 50 nm.

1164 b, 2D class averages of BRR2HR-PRP8Jab1-C9ORF78 particle images after reference-free

1165 classification.

1166 c, Viewing direction distribution plot of the particle images used for the final reconstruction of

1167 the BRR2HR-PRP8Jab1-C9ORF78 cryoEM map as obtained during NU refinement with

1168 cryoSPARC.

1169 d, Global resolution estimation for the BRR2HR-PRP8Jab1-C9ORF78 cryoEM map by gold-

1170 standard Fourier shell correlation (FSC). Dashed line, FSC0.143.

1171 e, Local resolution estimation as determined with cryoSPARC, ranging from 2.2 Å to 5.4 Å for

1172 the BRR2HR-PRP8Jab1-C9ORF78 cryoEM map.

1173 f, Representative cryoEM micrographs of the BRR2HR-FBP21200-376 complex. Scale bar, 50 nm.

1174 g, 2D class averages of BRR2HR-FBP21200-376 particle images after reference-free

1175 classification.

1176 h, Viewing direction distribution plot of the particle images used for the final reconstruction of

1177 the BRR2HR-FBP21200-376 cryoEM map as obtained during NU refinement with cryoSPARC.

1178 i, Global resolution estimation for the BRR2HR-FBP21200-376 cryoEM map by gold-standard

1179 Fourier shell correlation (FSC). Dashed line, FSC0.143.

1180 j, Local resolution estimation as determined with cryoSPARC, ranging from 2.4 Å to 6.4 Å for

1181 the BRR2HR-FBP21200-376 cryoEM map.

1182

52 1183

1184

1185 Supplementary Fig. 3. CryoEM data refinement.

1186 a, Reconstruction of the BRR2HR-PRPF8Jab1-C9ORF78 cryoEM map. 936,716 particle images

1187 were picked from 4,986 micrographs and subjected to reference-free 2D classification.

1188 836,442 particle images were selected for heterogeneous 3D refinement into 3 classes. The

1189 best appearing class, consisting of 545,045 particle images, was selected for re-extraction with

53 1190 a box size of 384 px, Fourier-cropped to 192 px (pixel size 1.314 Å). Homogeneous NU

1191 refinement yielded a reconstruction at 2.79 Å resolution that could be improved to 2.71 Å

1192 resolution by CTF refinement. To further improve the reconstruction, local motion correction

1193 was applied and particle images were re-extracted at full spatial resolution with a box size of

1194 384 px (0.657 Å/px). NU refinement yielded a reconstruction at 2.64 Å resolution that showed

1195 only fragmented density for C9ORF78. 3D variability analysis using a generous mask covering

1196 the N-terminal part of C9ORF78 was applied to remove sub-stoichiometric complexes.

1197 370,493 particle images were selected for final NU refinement yielding a reconstruction at 2.76

1198 Å resolution.

1199 b, Reconstruction of the BRR2HR-FBP21200-376 cryoEM map. 787,610 particle images were

1200 picked from 1,877 micrographs and subjected to reference-free 2D classification. 349,113

1201 particle images were selected for heterogeneous 3D refinement into 3 classes. Two classes

1202 were merged, the 271,494 particle images were re-extracted with a box size of 336 px, Fourier-

1203 cropped to 168 px (pixel size 1.664 Å). Homogeneous refinement yielded a reconstruction of

1204 3.47 Å that was subjected to another round of heterogeneous 3D refinement. 165,229 particle

1205 images were selected for local motion correction and un-binned re-extraction (box size 336 px,

1206 0.832 Å/px), which yielded a map at a resolution of 3.0 Å. 3D variability analysis using a mask

1207 around the FBP21200-376-binding region was applied to select the final 57,854 particle images

1208 for NU refinement, yielding a reconstruction at 3.30 Å resolution.

1209

54 1210

1211

1212 Supplementary Fig. 4. C9ORF78 has a moderate inhibitory effect on the BRR2 helicase

1213 activity.

1214 a, Exemplary non-denaturing PAGE, monitoring BRR2HR-PRPF8Jab1-mediated U4/U6

1215 unwinding in the absence (black outline) or presence (orange outline) C9ORF78. U4*, radio-

1216 labeled U4 snRNA.

55 1217 b, Quantification of data shown in a. Data points represent means ± SD; n = 3. Data were fit

1218 to a single exponential equation (fraction unwound = A (1 – exp[- kut])); A, amplitude of the

1219 reaction; ku, apparent first-order rate constant of unwinding; t, time).

1220 c, Change of unwinding rate of the indicated BRR2 variants alone or in complex with

1221 PRPF8Jab1ΔC or PRPF8Jab1 in the absence (black bars) or presence (orange bars) C9ORF78.

1222 Gray bar, control with GST instead of C9ORF78 added. Bars indicate means ± SD; n = 3.

1223

56 1224

1225

1226 Supplementary Fig. 5. Features of alternative 3’-ss regulated by C9ORF78.

1227 a, Scheme for alternative 3’-ss usage; us5’-ss, upstream 5’-ss; us3’-ss, upstream 3’-splice site;

1228 ds3’-ss, downstream 3’-splice site; ds5’-ss, downstream 5’-ss.

1229 b-h, Features of alternative 3’-ss regulated by C9ORF78.

1230 b, Lengths of the upstream introns.

1231 c, Distances between the us3’-ss and the ds3’-ss (3’-ss gap).

1232 d, Lengths of the exon containing the alternative 3’-ss.

1233 e, Strengths of the 5’-splice sites of the upstream intron (us5’-ss).

1234 f, Strengths of the upstream 3’-splice sites (us3’-ss).

1235 g, Strengths of the downstream 3’-splice sites (ds3’-ss).

1236 h, Strengths of the 5’-splice sites of the downstream intron (ds5’ss).

1237 In each panel, all alternative 3’-ss quantified by RMATS (black) are compared to alternative 3’-

1238 ss showing C9ORF78 KD-induced skipping (red) and alternative 3’-ss showing C9ORF78 KD-

1239 induced inclusion (blue). Horizontal lines, medians; whiskers minimum and maximum values;

57 1240 boxes, upper and lower quartile. Statistical significance was determined by unpaired Student’s

1241 t-tests (*, p <0.05; **, p < 0.01; ****, p < 0.0001; ns, not significant). Upstream 3’-ss requiring

1242 C9ORF78 for usage are characterized by a very short distance to the alternative downstream

1243 3’-ss (c), a comparatively long exon that contains the alternative 3’-ss (d), and they are

1244 comparatively weak (f).

1245

58 1246

1247

1248 Supplementary Fig. 6. C9ORF78 conservation.

1249 Multiple sequence alignment of C9ORF78 from different species, as indicated on the left.

1250 Residues are colored light to dark according to increasing level of conservation (54 %

1251 conservation, light yellow; 100 % conservation, orange). Residue numbering according to

1252 human C9ORF78 is indicated above the sequences. Secondary structure elements observed

1253 in the BRR2HR-PRPF8Jab1-C9ORF78 cryoEM structure are indicated below the sequences. The

59 1254 alignment was prepared with Homologene (NCBI) employing Clustal Omega 72 and shaded

1255 with ALSCRIPT73.

1256

60 1257

1258

1259 Supplementary Fig. 7. Comparison of siRNA KD and FLASH data.

1260 a, Cumulative coverage plots obtained by deepTools, displaying RNAs that show changes in

1261 alternative 3’-ss usage upon C9ORF78 KD and UV-cross-linked to GFP (negative control) or

1262 C9ORF78 (FLASH-derived sequence coverage of C9ORF78 splicing targets detected by

61 1263 RMATS analysis). Heat maps are colored by normalized coverage average (n = 2). Sequences

1264 of upstream exons as well as long and short exons harboring the alternative 3’-ss (schemes

1265 on the left) plus 50 base pairs upstream and downstream of the exons (illustrated below the

1266 heat maps) were analyzed. Top, targets with 3’-ss more skipped upon C9ORF78 KD; bottom,

1267 targets with 3’-ss more skipped upon C9ORF78 KD.

1268 b, Like a, but for skipped exon events.

62