FUS functions in coupling to splicing by mediating an interaction between RNAP II and U1 snRNP

Yong Yu and Robin Reed1

Department of Biology, Harvard Medical School, Boston, MA 02115

Edited by Tom Maniatis, Columbia University Medical Center, New York, NY, and approved May 27, 2015 (received for review March 30, 2015) Pre-mRNA splicing is coupled to transcription by RNA polymerase II (28–32). How FUS is involved in these processes and whether (RNAP II). We previously showed that U1 small nuclear ribonu- FUS is directly involved have not been established. cleoprotein (snRNP) associates with RNAP II, and both RNAP II and Because robust in vitro systems for the coupled txn/splicing U1 snRNP are also the most abundant factors associated with the reaction are now routinely used (14, 15, 17, 19, 33), we can di- fused-in-sarcoma (FUS), which is mutated to cause the rectly test the roles of U1 snRNP and FUS in this coupled re- neurodegenerative disease amyotrophic lateral sclerosis. Here, action. In recent work, an antisense morpholino we show that an antisense morpholino that base-pairs to the 5′ (AMO) that base-pairs to the 5′ end of U1 snRNA was used to end of U1 snRNA blocks splicing in the coupled system and com- functionally inactivate U1 snRNP (34, 35). Here, we report that pletely disrupts the association between U1 snRNP and both FUS this U1 AMO disrupts the association between U1 snRNP and and RNAP II, but has no effect on the association between FUS and both FUS and RNAP II, whereas FUS remains associated with RNAP II. Conversely, we found that U1 snRNP does not interact RNAP II. However, in FUS knockdown (KD) nuclear extracts, with RNAP II in FUS knockdown extracts. Moreover, using these we found that U1 snRNP can no longer interact with RNAP II. extracts, we found that FUS must be present during the transcrip- Furthermore, our data reveal that FUS must be present during tion reaction in order for splicing to occur. Together, our data lead transcription in order for splicing to take place in the coupled to a model that FUS functions in coupling transcription to splicing txn/splicing system. Together, our data indicate that FUS func- via mediating an interaction between RNAP II and U1 snRNP. tions in coupling transcription to splicing via mediating an as- sociation between U1 snRNP and RNAP II. ALS | coupling transcription to splicing | RNA polymerase II | U1 snRNP Results and Discussion t is now well established that the steps in expression are U1 AMO Blocks Spliceosome Assembly and Splicing in the Coupled Iextensively coupled to one another, including both physical and Txn/Splicing System. An extensively characterized U1 AMO functional coupling between RNA polymerase II (RNAP II) (Fig. 1A) is known to inhibit splicing in vivo and also in vitro in transcription and pre-mRNA processing (1–11). Moreover, the uncoupled splicing systems, and to disrupt the essential base- majority of nascent transcripts are spliced cotranscriptionally, pairing interaction between U1 snRNA and the 5′ splice site (34, while the transcripts are still tethered to RNAP II. In vitro sys- 35). In our study, we used this U1 AMO to investigate U1 tems have been developed for the coupled transcription/splicing snRNP in the coupled txn/splicing reaction. A size-matched (txn/splicing) reaction as well as for cotranscriptional splicing, scrambled AMO was used as a negative control. The well-char- and these systems have been used to investigate the mechanisms acterized CMV–Ftz DNA template was used to examine the underlying these processes (12–17). In the case of coupled txn/ effects of the AMOs on coupled txn/splicing (Fig. S1) (13). This splicing, studies using the in vitro system revealed that tran- scription potently enhances spliceosome assembly, which in turn Significance leads to a strong enhancement of the splicing reaction (13). Additional studies revealed that the only essential splicing fac- The protein fused-in-sarcoma (FUS) is mutated to cause the tors that copurify with RNAP II are U1 small nuclear ribonu- neurodegenerative disease amyotrophic lateral sclerosis, but cleoprotein (snRNP) and its associated factors, the serine/ its normal cellular role remains to be understood. Previous arginine-rich (SR) (18–20). This observation is partic- work showed that FUS associates with both RNA polymerase II ularly noteworthy because U1 snRNP/SR proteins are the first (RNAP II) and the essential splicing factor U1 small nuclear ri- splicing factors that bind to pre-mRNA during spliceosome as- bonucleoprotein (snRNP). Here we were able to directly in- sembly (21). Although functional studies indicate that SR pro- vestigate the functional significance of these interactions using teins play a key role in coupling transcription to splicing (19), the an in vitro system. We show that FUS is essential for the in- role of U1 snRNP in this coupling event has not been examined. teraction between U1 snRNP and RNAP II and that FUS must be It is also not known how U1 snRNP interacts with RNAP II. U1 present during the RNAP II transcription reaction in order for snRNA is known to base-pair to the 5′ splice site during the splicing to occur. Together, these data indicate that FUS me- earliest steps in spliceosome assembly, and this interaction is diates an interaction between RNAP II and U1 snRNP, thereby physically and functionally coupling transcription to splicing. essential for splice-site recognition (21). In addition, we and

others found that U1 snRNP/SR and RNAP II are among the Author contributions: Y.Y. and R.R. designed research; Y.Y. performed research; Y.Y. and main factors that associate with the protein fused-in-sarcoma R.R. analyzed data; and Y.Y. and R.R. wrote the paper. (FUS) (19, 22–27). Understanding the normal roles of FUS and The authors declare no conflict of interest. the pathways in which it functions are of great importance be- This article is a PNAS Direct Submission. cause FUS is mutated to cause the fatal motor neuron disease Freely available online through the PNAS open access option. – amyotrophic lateral sclerosis (ALS) (28 32). In vivo studies in- 1To whom correspondence should be addressed. Email: [email protected]. dicate that FUS plays numerous roles in the nucleus, including This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. transcription, splicing, microRNA processing, and DNA repair 1073/pnas.1506282112/-/DCSupplemental.

8608–8613 | PNAS | July 14, 2015 | vol. 112 | no. 28 www.pnas.org/cgi/doi/10.1073/pnas.1506282112 Downloaded by guest on September 24, 2021 IP/ Western A U1-70K C A C AMO:

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AUAAUUUGUGGUAGU

GCU CGA

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- U FUS

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AAAGGG UUUCCC

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G SmB/B’ 1 2 3 456 IP/Coomassie D B Cntl AMO U1 AMO IP/Ethidium bromide 10% Input Cntl AMO U1 AMO

IgG RNAP II IgG RNAP II

Cntl AMOU1 AMO IgG RNAP II IgG RNAP II -180

-115 7S- FUS- U1-70K- -82 U2- U1- BIOCHEMISTRY -64 U4- -49 U1A- U6- -37

U1C- -26 tRNA- -19 Sm D1, D2, D3- -15 1234 12 34 5 6

Fig. 1. U1 AMO disrupts the interaction between U1 snRNP and RNAP II. (A) Schematic showing the U1 AMO (red line) base paired to U1 snRNA in U1 snRNP. The Sm core and the U1 snRNP-specific proteins (U1-70K, U1A, and U1C) are indicated. (B) Coomassie-stained gel showing the proteins immunoprecipitated by RNAP II (lanes 2 and 4) or control (lanes 1 and 3) antibody after nuclear extract was incubated with the control (lanes 1 and 2) or U1 (lanes 3 and 4) AMO. FUS, the U1 snRNP proteins, and molecular-mass markers (in kDa) are indicated. (C and D) Same as B except proteins (C) or RNA (D) was analyzed by using the indicated antibodies or on an ethidium bromide-stained gel, respectively. In D, we note that samples 1–6 were prepared side-by-side, but samples 1 and 2 and samples 3–6 were run on two separate gels, as indicated by the white space between the two gels.

analysis revealed that the U1 AMO specifically inhibited splic- (21), the data lead to a model in which U1 snRNP/SR proteins ing, but not transcription, and in a dose-dependent manner (Fig. bound to RNAP II are recruited to promoters, and then, while S1). Moreover, the U1 AMO inhibited spliceosome assembly, the nascent pre-mRNA is being synthesized, these splicing fac- and, instead, a faster migrating complex was detected (Fig. S1). tors are recruited to the 5′ splice site and adjacent to ini- Continued incubation did not allow proper spliceosome assem- tiate spliceosome assembly. This close physical and functional bly from this complex (Fig. S1). Furthermore, the U1 AMO relationship between RNAP II and U1 snRNP/SR proteins blocked both splicing and spliceosome assembly only when the provides a likely explanation for the potent enhancement of AMO was added to the coupled system before, but not after, spliceosome assembly and splicing observed both in vivo and in transcription (Fig. S2). Finally, when CMV–Ftz was transcribed vitro when pre-mRNAs are transcribed by RNAP II vs. other in the presence of the U1 AMO, followed by dilution into a polymerases (12–15, 36, 37). chase reaction in normal nuclear extract, splicing did not occur (Fig. S2). Together, these data using the U1 AMO indicate that The U1 AMO Disrupts RNAP II Interaction with U1 snRNP, but Not with U1 snRNP must be loaded onto pre-mRNA during the tran- FUS. To further investigate the significance of the association scription reaction in order for efficient spliceosome assembly and between U1 snRNP and RNAP II, we next asked whether the U1 splicing to occur. In previous work, we found that SR proteins, AMO affected this interaction. In addition, we investigated the which associate with U1 snRNP, also must be recruited to pre- effect of the U1 AMO on the association of FUS with RNAP II, mRNA during the RNAP II transcription reaction in order for becauseFUSinteractswithbothU1snRNPandRNAPII.Nuclear splicing to occur (19). In addition, we and others found that U1 extracts were treated with the control or U1 AMO, followed by snRNP/SR proteins are the only essential splicing factors that immunoprecipitations (IPs) with a control antibody or an antibody associate with RNAP II, and this association occurs even when against RNAP II. As expected, analysis of the proteins revealed RNAP II is not present at transcription promoters (18–20). that both FUS and U1 snRNP components (U1-70K, U1A, and Because U1 snRNP/SR proteins are recruited to pre-mRNA U1C) were specifically coimmunoprecipitated with RNAP II from during the earliest steps in the spliceosome assembly pathway extracts treated with the control AMO (Fig. 1B,lanes1and2).

Yu and Reed PNAS | July 14, 2015 | vol. 112 | no. 28 | 8609 Downloaded by guest on September 24, 2021 A IP/Coomassie D IP/Coomassie Cntl AMO U1 AMO Cntl AMO U1 AMO

IgG U1C IgG U1C IgG FUS IgG FUS

-180 180 -115 - -115 FUS- -82 FUS- U1-70K- -82 U1-70K- -64 -64 -49 U1A- -49 -37 U1A- SmB, B’ -37 -26 U1C- -26 -19 -19 Sm D1, D2, D3- -15 Sm D1, D2, D3- -15 Sm E, F- Sm E, F- -6 12 34 12 34 B IP/Western E IP/Western Cntl AMO U1 AMO Cntl AMO U1 AMO

5% Input IgG FUS IgG FUS IgG U1C IgG U1C FUS- RNAP II U1-70K- FUS

U1A- U1 70K U1A U1C- SmB/B’ 12345 1234

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Cntl U1 AMO IgG FUS IgG FUS Cntl AMOU1 AMO IgG U1C IgG U1C

7S- 7S- U2- U1- U2- U4- 5S- U1- U5- U4- 5S- U6- U5 U6- 12 34 56 12 3 4 56

Fig. 2. U1 AMO disrupts the interaction between FUS and U1 snRNP. (A) Coomassie-stained gel showing the proteins immunoprecipitated by a control antibody (lanes 1 and 3) or an antibody against U1C (lanes 2 and 4) after nuclear extract was incubated with the control (lanes 1 and 2) or U1 (lanes 3 and 4) AMO. FUS, the U1 snRNP proteins, and molecular-mass markers (in kDa) are shown. (B and C) Same as A, except that proteins were analyzed by Western (B)or RNA was analyzed by ethidium bromide staining (C). snRNAs and 5S rRNA are indicated. (D–F) Same as A–C, except that FUS antibody was used for the IPs.

However, when the same IPs were carried out by using extracts association between RNAP II and U1 snRNP. In contrast, the treated with the U1 AMO, the U1 snRNP components were interaction between RNAP II and FUS remains intact in the specifically lacking (Fig. 1B,lanes3and4).Incontrast,FUSlevels presence of the U1 AMO. were the same in nuclear extract treated with either the control or U1 AMOs (Fig. 1B, lanes 2 and 4). These results, showing loss of Interaction of FUS with U1 snRNP Is Blocked by the U1 AMO. We next U1 snRNP components in the presence of the U1 AMO and no used the AMOs to investigate the interaction between U1 effect on FUS, were also observed on Western blots (Fig. 1C). snRNP and FUS. To undertake this investigation, we first used Moreover, U1 snRNA was present in the RNAP II IP from the antibodies against U1C to carry out an IP of U1 snRNP, fol- control, but not the U1, AMO-treated extract (Fig. 2D). Together, lowed by analysis of the proteins on a Coomassie-stained gel these data indicate that the U1 AMO specifically disrupts the (Fig. 2A). This analysis revealed that the U1 snRNP proteins and

8610 | www.pnas.org/cgi/doi/10.1073/pnas.1506282112 Yu and Reed Downloaded by guest on September 24, 2021 FUS were immunoprecipitated in extracts treated with the con- A C trol AMO (Fig. 2A, lane 2). In marked contrast, FUS was not IP/Western immunoprecipitated when extracts were treated with the U1 knockdown: Cntl FUS Cntl FUS U1 snRNP AMO (Fig. 2A, lane 4). We also observed a decrease in levels of U1-70K and U1A when U1 snRNP was immunoprecipitated in FUS the U1 AMO-treated extracts, but no effect was detected on the 10% Input10%Input IgG RNAP II IgG RNAP II levels of the Sm snRNP core components. The same results were RNAP II observed on Western blots of the U1 snRNP IP in the AMO- RNAP II treated extracts. Importantly, these data revealed that FUS was FUS associated with U1 snRNP when the control AMO was used, but B knockdown: Cntl FUS U1 70K was specifically and completely dissociated from U1 snRNP FUS when the U1 AMO was used (Fig. 2B, compare lanes 3 and 5). Tubulin U1A As expected from the Coomassie data (Fig. 2A), U1 snRNA was 12 12 3 4 56 detected on an ethidium bromide-stained gel in the U1C IP 32 when extracts were treated with either the control or U1 AMO D IP/ pCp-labeled RNA (Fig. 2C). knockdown: Cntl FUS Cntl FUS To further examine the effect of the U1 AMO on the U1 snRNP–FUS interaction, we carried out FUS IPs from extracts treated with the AMOs and examined the IPs on a Coomassie- 5% Input5% Input IgG U1A FUS RNAP IIIgG U1A FUS RNAP II stained gel. This analysis also revealed that the U1 AMO causes a striking loss of association between FUS and U1 snRNP (Fig. 2D). The same results were obtained by Western analysis of the FUS IPs (Fig. 2E). Moreover, U1 snRNA was present in the FUS IP when carried out in the extract treated by the control 7S- U2- AMO, but was completely lost when the FUS IPs were carried U1- out in extracts treated with the U1 AMO (Fig. 2F, lanes 4 and 6). U5- We conclude that the interaction between FUS and U1 snRNP is BIOCHEMISTRY completely blocked in extracts treated with the U1 AMO. This AMO base-pairs to the 5′ portion of U1 snRNA (Fig. 1A), and it is possible that this base pairing affects the conformation of U1 tRNA- snRNP. This conformational change may be responsible for the 12 3 4 567 8 910 complete dissociation of FUS from U1 snRNP, as well as lead to the decreased levels of U1-70K and U1A that we observed after Fig. 3. FUS is required for the interaction between U1 snRNP and RNAP II. (A) Schematic showing FUS mediating the interaction between U1 snRNP and treatment with this AMO. Whether the splicing block observed RNAP II. (B) Western blot showing KD efficiency of FUS, using scrambled shRNA with the U1 AMO in the previous work (34, 35) and in our work as a control. Tubulin was a loading control. (C) IP/Westerns were carried out reported here is due to disruption of base-pairing between U1 with control (Cntl; lanes 3 and 4) or FUS (lanes 5 and 6) KD small-scale nuclear snRNA and the 5′ splice site and/or due to the disruption of the extracts. The respective inputs are shown in lanes 1 and 2. (D)SameasC,ex- proteins associated with U1 snRNP remains to be established. cept that total RNA was isolated from the IPs with the indicated antibodies and end-labeled with 32pCp. Bands were detected by phosphorimager. FUS Is Required for the Interaction Between RNAP II and U1 snRNP. Our data presented above reveal that FUS still interacts with A Role for FUS in Coupling Transcription to Splicing. We next used RNAP II in the presence of the U1 AMO, whereas U1 snRNP the FUS KD nuclear extracts to investigate the role of FUS in does not interact with either FUS or RNAP II in the presence of the coupled txn/splicing reaction. When CMV–Ftz (Fig. S1A) the U1 AMO. Thus, we next investigated the possibility that FUS was transcribed in the FUS or control KD extracts, pre-mRNA might mediate the interaction between RNAP II and U1 snRNP A A was efficiently generated by the 10-min time point (Fig. 4 , lanes (Fig. 3 ). To test this hypothesis, we knocked down FUS in 1 and 3). By 30 min of incubation, the pre-mRNA was efficiently HeLa cells with shRNA, using a scrambled shRNA as a control. converted into spliced mRNA in the control nuclear extract Western blot analysis showed that FUS was efficiently knocked (lane 2). In contrast, in the FUS KD extract, splicing was B down (Fig. 3 ). These cells were used to prepare small-scale inhibited, which can be seen by comparing the level of unspliced nuclear extracts by using a method that we established for pre-mRNA to spliced mRNA (Fig. 4A, lane 4). In addition, the making extracts active in the coupled txn/splicing system (17, 33). splicing intermediates (exon 1 and lariat–exon 2) accumulate to When the control KD extracts were used for IP/Westerns, we higher levels in the FUS KD extract than in the control extract found that U1 snRNP proteins coimmunoprecipitated with (Fig. 4A, compare lanes 2 and 4), suggesting that the low levels of RNAP II (Fig. 3C, lane 4). Strikingly, however, the co-IP of U1 FUS remaining in the KD extract allow some splicing, but with snRNP proteins with RNAP II was lost in the FUS KD extracts delayed kinetics. To further investigate the role of FUS in the (Fig. 3C, lane 6). Likewise, the co-IP of RNAP II with U1 coupled txn/splicing reaction, we performed a chase assay in snRNA was lost in the FUS KD extracts (Fig. 3D, lane 10). In which the CMV–Ftz DNA template was first transcribed in the contrast, U1 snRNA was immunoprecipitated by the U1 snRNP control or FUS KD nuclear extracts, followed by an eightfold antibody with the same efficiency in the FUS and control KD dilution into normal nuclear extract and continued incubation. extracts (Fig. 3D, compare lanes 4 and 8). As expected, U1 As shown in Fig. 4B, when pre-mRNA was transcribed in the snRNA was coimmunoprecipitated by the FUS antibody in the control KD extract, it was efficiently spliced when chased in control, but not the FUS KD extract (Fig. 3D, lanes 5 and 9). normal nuclear extract (lanes 1 and 2). In contrast, when pre- Together, these data indicate that FUS is required for the in- mRNA was transcribed in FUS KD nuclear extract, it was not teraction between RNAP II and U1 snRNP. To our knowledge, spliced when chased in normal nuclear extract (lanes 3 and 4). FUS is the first protein shown to tether an essential splicing We also carried out the reciprocal experiment in which pre- factor (U1 snRNP) to RNAP II. mRNA was transcribed in normal nuclear extract (Fig. 4C, lane

Yu and Reed PNAS | July 14, 2015 | vol. 112 | no. 28 | 8611 Downloaded by guest on September 24, 2021 A B Chase assay Cntl KD FUS KD 10’ txn in KD NE: Cntl FUS 10’ 30’ 10’ 30’ Chase in normal NE 0’ 20’ 0’ 20’ pre-mRNA- mRNA- pre-mRNA- lariat-exon 2 mRNA- exon1- lariat-exon 2 exon1-

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pre-mRNA- pre-mRNA- pre-mRNA mRNA- mRNA-- -lariat exon 2 mRNA lariat-exon 2 exon1- exon 1- -exon 1

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Fig. 4. FUS is required during the transcription reaction to promote splicing. (A) CMV–Ftz was incubated under txn/splicing conditions in the control (Cntl; lanes 1 and 2) or FUS (lanes 3 and 4) KD nuclear extracts for 10 and 30 min. Splicing intermediates and products are indicated. (B) CMV–Ftz was incubated under txn/splicing conditions for 10 min to allow transcription in control (lane 1) or FUS (lane 3) KD nuclear extracts followed by addition of α-amanitin. An aliquot of the transcription reaction was then diluted eightfold into fresh nuclear extract and incubated under txn/splicing conditions for an additional 20 min to allow splicing (lanes 2 and 4). (C) Same as B, except that transcription was carried out for 10 min in the normal nuclear extract (lanes 1 and 3) followed by dilution and continued incubation in the control (lane 2) or FUS (lane 4) knockdown nuclear extracts. (D) CMV–Ftz DNA template (lanes 1–4) or naked T7–Ftz pre-mRNA (lanes 5–8) was incubated under identical conditions in control or FUS KD nuclear extract as indicated. Splicing intermediates and products are designated (note that the Ftz–CMV pre-mRNA has a longer first exon owing to the CMV promoter sequence). Ftz–CMV DNA template was incubated for 5 min to allow transcription, followed by continued incubation for 20 min to allow splicing, whereas naked T7–Ftz pre-mRNA was incubated for 0 and 20 min.

1and 3) and chased in the control or FUS KD nuclear extract. which leads to mislocalization of FUS to the in ALS This analysis revealed that the pre-mRNA was efficiently spliced patient cells (29, 38, 39). Moreover, we recently obtained evi- when chased in either the control or FUS KD nuclear extract dence that the U1 snRNP core complex (U1 snRNA and the Sm (Fig. 4C, lanes 2 and 4). Thus, if the pre-mRNA is transcribed in proteins) is comislocalized to the cytoplasm with FUS in ALS the presence of available FUS, then splicing occurs efficiently in patient fibroblasts harboring mutations in the NLS (40). Thus, normal nuclear extract. However, if the pre-mRNA is tran- decreased levels of both FUS and U1 snRNP in the nucleus may scribed in the absence of available FUS, then it does not splice in lead to the aberrant splicing that has been reported in trans- normal nuclear extract. These data indicate that the function of fected cells and ALS patient cells expressing FUS with NLS FUS in splicing has to take place during the transcription re- mutations (29, 30, 38, 39). In previous work, FUS was shown to action. This conclusion is further supported by the observation bind to the C-terminal domain of RNAP II and regulate phos- that FUS KD has no significant effect on uncoupled splicing phorylation of serine-2 (23, 25, 27). Moreover, when FUS is using naked T7–Ftz pre-mRNA, whereas coupled splicing of knocked down, RNAP II accumulates at the start site of tran- CMV–Ftz is inhibited in the same FUS KD extract (Fig. 4D). scription, and premature polyadenylation is observed (23, 25). The data presented here, together with previous observations Notably, U1 snRNP has been found to play other important roles showing that FUS associates with both RNAP II and U1 snRNP, in addition to its function in splicing. One of these roles is known lead to the model that FUS functions in coupling transcription to as telescripting, in which binding of U1 snRNP to nascent pre- splicing via mediating an interaction between RNAP II and U1 mRNAs is required to prevent premature polyadenylation during snRNP. Considering that transcription-coupled splicing is such a transcription (34, 41). The lack of telescripting, which was found fundamental cellular process, it is possible that its disruption by to occur on antisense transcripts due to the presence of more mutant FUS contributes to the pathogenesis of ALS. In support polyA sites and fewer 5′ splice sites, is thought to be a mecha- of this possibility, many of the mutations in FUS that are ALS- nism for suppressing transcription of antisense (35). Thus, causative are located in the nuclear localization signal (NLS), the observation that premature polyadenylation occurs with FUS

8612 | www.pnas.org/cgi/doi/10.1073/pnas.1506282112 Yu and Reed Downloaded by guest on September 24, 2021 KD (23, 25), together with our observation that FUS mediates an naked T7–Ftz pre-mRNA shown in Fig. 4D was carried out in control or FUS KD interaction between U1 snRNP and RNAP II, suggests that the nuclear extracts side-by-side under identical conditions as used for txn/splicing RNAP II–FUS–U1 snRNP complex is the entity that functions in with the CMV–DNA template. telescripting. The data also raise the possibility that defective telescripting may contribute to ALS pathogenesis. IPs. Antibodies were coupled to protein A Sepharose and then covalently cross-linked to the beads by using dimethylpimelimidate (Sigma). A 500-μL μ μ Materials and Methods mixture containing 150 L of nuclear extract, 150 L of splicing dilution buffer (20 mM Hepes, pH 7.9, 100 mM KCl), 500 μM ATP, 3.2 mM MgCl2, and – and Antibodies. The encoding CMV Ftz was described (13) 20 mM creatine (di-Tris salt) was incubated for 30 min and 30°. The templates for RNAP II transcription were prepared as described (13, 16) After incubation, mixtures were spun at 4° for 5 min at 16,000 × g. Super- Antibodies to U1-70K (9C4.1) were from Millipore; U1C (4H12) was from natants were added to 250 mL of IP buffer [1X PBS, 0.1% Triton X-100, 0.2 Sigma; U1A (BJ-7), HA, and tubulin were from Santa Cruz; RNAP II (8WG16) mM PMSF, protease inhibitor EDTA-free (Roche)], spun at 4 °C for 5 min, and ′ were from Covance; FUS (293A) was from Bethyl; and SmB/B (Y12), mouse added to 40 mL antibody–cross-linked beads. After rotation overnight at IgG, and rabbit IgG were from Abcam. Mouse IgG, rabbit IgG, and HA were 4 °C, six washes (1.5 mL each) were performed by using wash buffer (1X PBS used as negative controls for polyclonal and monoclonal antibodies, re- containing 0.1% Triton X-100, 0.2 mM PMSF, pH 8.0). Proteins were eluted spectively. The FUS rabbit polyclonal antibody was described (22). by adding 60 μL of SDS sample loading buffer, followed by incubation for 20 min at room temperature. DTT was added to a final concentration of 5 mM, – RNAP II Transcription and Splicing. CMV Ftz DNA was incubated under txn/ and samples were boiled for 10 min and loaded on 4–12% SDS gradient gels. splicing conditions as described (13, 17). For AMO assays, control or U1 AMOs For RNA IPs, total RNA was isolated and analyzed on 6.5% denaturing gels (34, 35) were added to HeLa cell nuclear extract at a final concentration of stained with ethidium bromide. 12 μM. Control or FUS KD nuclear extracts were prepared by using the small- scale nuclear extract procedure (17, 33). For spliceosome assembly, the CMV–Ftz ACKNOWLEDGMENTS. We thank Binkai Chi, Shanye Yin, Edward Adams, DNA template was incubated under txn/splicing conditions for 10 min. G-50 and other members of the R.R. laboratory for useful discussion. This work 32 μ columns were used to remove unincorporated P-UTP. A total of 1 Lof was supported by National Institutes of Health Grant GM043375 (to R.R.) heparin(6.5g/L)wasaddedto10μL of G-50 column-purified reactions before and ALS Therapy Alliance Grant 2013-S-006 (to R.R.). HeLa cells were loading on 1.2% low-melting-point agarose gels (42). Uncoupled splicing of obtained from the National Center Biovest International.

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