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. 2015 Jul 14;112(28):8608-13.
doi: 10.1073/pnas.1506282112. Epub 2015 Jun 29.

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

Yong Yu  1 , Robin Reed  2
Affiliations

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

Yong Yu et al. Proc Natl Acad Sci U S A. .

Abstract

Pre-mRNA splicing is coupled to transcription by RNA polymerase II (RNAP II). We previously showed that U1 small nuclear ribonucleoprotein (snRNP) associates with RNAP II, and both RNAP II and U1 snRNP are also the most abundant factors associated with the protein fused-in-sarcoma (FUS), which is mutated to cause the neurodegenerative disease amyotrophic lateral sclerosis. Here, we show that an antisense morpholino that base-pairs to the 5' end of U1 snRNA blocks splicing in the coupled system and completely disrupts the association between U1 snRNP and both FUS and RNAP II, but has no effect on the association between FUS and RNAP II. Conversely, we found that U1 snRNP does not interact with RNAP II in FUS knockdown extracts. Moreover, using these extracts, we found that FUS must be present during the transcription reaction in order for splicing to occur. Together, our data lead to a model that FUS functions in coupling transcription to splicing via mediating an interaction between RNAP II and U1 snRNP.

Keywords: ALS; RNA polymerase II; U1 snRNP; coupling transcription to splicing.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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 Western blot 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.
Fig. S1.
Fig. S1.
An AMO targeting U1 snRNA inhibits splicing and spliceosome assembly in the coupled txn/splicing system. (A) Schematic of CMV–Ftz DNA template. The black boxes indicate the exons, and the line indicates the intron. The sizes of the exons and introns are shown. (B) The indicated amounts of control (Cntl; lanes 1–6) or U1 AMOs (lanes 7–12) were added to HeLa cell nuclear extract before carrying out the coupled txn/splicing reaction. (C and D) Cntl or U1 AMO (12 μM) were incubated in nuclear extract for 10 (lanes 1 and 2) or 30 (lanes 3 and 4) minutes and then analyzed on a nondenaturing gel (C) or on a native agarose gel (D). The asterisks in D indicate the faster-migrating complex that was generated in the presence of the U1 AMO.
Fig. S2.
Fig. S2.
The U1 AMO inhibits splicing and spliceosome assembly when added before, but not after, transcription. (A) No AMO (lanes 1 and 2), control AMO (Cntl, lanes 2–5) or U1 AMO (lanes 7–10) was added to HeLa cell nuclear extract before or after 10 min of transcription of CMV–Ftz, followed by continued incubation for 20 min. The splicing intermediates and products are indicated. The line below the lariat intron marks breakdown of the lariat intron. (B) Same as A, except that spliceosome assembly (10-min time point) was analyzed on an agarose gel. The asterisk indicates the faster migrating complex that was generated in the presence of the U1 AMO. (C) The CMV–Ftz DNA template was transcribed in the presence of the Cntl or U1 AMO, followed by a 10-fold dilution in normal nuclear extract to allow a chase for 20 additional minutes.
Fig. 2.
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.
Fig. 3.
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 RNAP II. (B) Western blot showing KD efficiency of FUS, using scrambled shRNA as a control. Tubulin was a loading control. (C) IP/Westerns were carried out with control (Cntl; lanes 3 and 4) or FUS (lanes 5 and 6) KD small-scale nuclear extracts. The respective inputs are shown in lanes 1 and 2. (D) Same as C, except that total RNA was isolated from the IPs with the indicated antibodies and end-labeled with 32pCp. Bands were detected by phosphorimager.
Fig. 4.
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.
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References

    1. Greenleaf AL. Positive patches and negative noodles: Linking RNA processing to transcription? Trends Biochem Sci. 1993;18(4):117–119. - PubMed
    1. Maniatis T, Reed R. An extensive network of coupling among gene expression machines. Nature. 2002;416(6880):499–506. - PubMed
    1. Prasanth KV, Spector DL. Eukaryotic regulatory RNAs: An answer to the ‘genome complexity’ conundrum. Genes Dev. 2007;21(1):11–42. - PubMed
    1. Buratowski S. Progression through the RNA polymerase II CTD cycle. Mol Cell. 2009;36(4):541–546. - PMC - PubMed
    1. Margueron R, Reinberg D. Chromatin structure and the inheritance of epigenetic information. Nat Rev Genet. 2010;11(4):285–296. - PMC - PubMed

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