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SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export

Müller-McNicoll, Michaela, Botti, Valentina, de Jesus Domingues, Antonio M., Brandl, Holger, Schwich, Oliver D., Steiner, Michaela C., Curk, Tomaz, Poser, Ina, Zarnack, Kathi, Neugebauer, Karla M.·2016·Genes & Development
DOI 10.1101/gad.276477.115PMC PMC4782049
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Abstract

Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1–7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1–7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3′ untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3′ ends.

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Figures

Figure 1. Identification of endogenous mRNA export targets of SR proteins. (A) Scheme illustrating the extraction of candidate mRNA export targets from cytoplasmic and whole-cell RNA-seq data after 36 h of SR protein depletion by RNAi. (B) Numbers of mRNA export targets for each SR protein and overlaps among them. (C) Validation of SRSF3 export targets by RT-qPCR. (Left panel) Knockdown (KD) of SRSF3 in P19 wild-type cells leads to an increased nuclear/cytoplasmic ratio of each mRNA. n = 5. (Right panel) Overexpression (OE) leads to a decreased nuclear/cytoplasmic ratio of each mRNA. n = 3. (*) P < 0.05.
Figure 1. Identification of endogenous mRNA export targets of SR proteins. (A) Scheme illustrating the extraction of candidate mRNA export targets from cytoplasmic and whole-cell RNA-seq data after 36 h of SR protein depletion by RNAi. (B) Numbers of mRNA export targets for each SR protein and overlaps among them. (C) Validation of SRSF3 export targets by RT-qPCR. (Left panel) Knockdown (KD) of SRSF3 in P19 wild-type cells leads to an increased nuclear/cytoplasmic ratio of each mRNA. n = 5. (Right panel) Overexpression (OE) leads to a decreased nuclear/cytoplasmic ratio of each mRNA. n = 3. (*) P < 0.05.CC BY · PubMed Central
Figure 2. Differential association of SR proteins with NXF1 and promotion of NXF1 binding to mRNA. (A) Semiquantitative co-IPs. GFP-tagged SR proteins were pulled down by α-GFP antibodies and probed for interacting NXF1 using α-NXF1 antibodies (forward). Endogenous NXF1 was pulled down using α-NXF1 antibodies and probed for GFP expression (reverse). Lanes marked +RNase show co-IPs carried out after RNase A treatment. PABPN1, which binds polyA tails, served to control successful RNA degradation. (B) Scheme of oligodT capture: P19 cells were UV cross-linked, and mRNA was purified using oligodT beads and probed for NXF1 by Western blot (WB). Stringent conditions were used to strip off non-cross-linked proteins (Castello et al. 2012). (C, left panel) P19 clonal cell lines, selected for high GFP expression by FACS, overexpress SRSF2, SRSF3, or SRSF7 by ∼2.5-fold compared with wild-type (WT) P19 cells (Supplemental Fig. S2F,G). Overexpression (OE) of SRSF2 was quantitated by RT-qPCR because available anti-SRSF2 antibodies were unspecific in our hands. PABPN1 binding served as a loading control. (Right panel) Quantification of NXF1 cross-linking to mRNAs upon SRSF2, SRSF3, and SRSF7 overexpression is expressed relative to wild-type. Mean and SD are shown. n = 3 independent experiments. (**) P < 0.001.
Figure 2. Differential association of SR proteins with NXF1 and promotion of NXF1 binding to mRNA. (A) Semiquantitative co-IPs. GFP-tagged SR proteins were pulled down by α-GFP antibodies and probed for interacting NXF1 using α-NXF1 antibodies (forward). Endogenous NXF1 was pulled down using α-NXF1 antibodies and probed for GFP expression (reverse). Lanes marked +RNase show co-IPs carried out after RNase A treatment. PABPN1, which binds polyA tails, served to control successful RNA degradation. (B) Scheme of oligodT capture: P19 cells were UV cross-linked, and mRNA was purified using oligodT beads and probed for NXF1 by Western blot (WB). Stringent conditions were used to strip off non-cross-linked proteins (Castello et al. 2012). (C, left panel) P19 clonal cell lines, selected for high GFP expression by FACS, overexpress SRSF2, SRSF3, or SRSF7 by ∼2.5-fold compared with wild-type (WT) P19 cells (Supplemental Fig. S2F,G). Overexpression (OE) of SRSF2 was quantitated by RT-qPCR because available anti-SRSF2 antibodies were unspecific in our hands. PABPN1 binding served as a loading control. (Right panel) Quantification of NXF1 cross-linking to mRNAs upon SRSF2, SRSF3, and SRSF7 overexpression is expressed relative to wild-type. Mean and SD are shown. n = 3 independent experiments. (**) P < 0.001.CC BY · PubMed Central
Figure 3. SR proteins and NXF1 bind to mRNA after splicing. (A) Number of cross-link events of individual SR proteins mapping to a window of ±300 nucleotides (nt) around the 5′ splice site (left) and 3′ splice site (right). Each line represents a different SR protein or NXF1. (B, left panel) Scheme of a two-exon gene with iCLIP reads mapping to the 5′ splice site (5′ss). The cross-link site is indicated with a gray arrow, and the remaining peptide is indicated with a green star. Reads that continue from one exon into the neighboring intron (dark green) were counted as unspliced, and reads that span two neighboring exons (light green) were counted as spliced. (Right panel) Plot showing the proportions of spliced and unspliced iCLIP reads mapping near 5′ splice sites. (C) Semiquantitative co-IPs using identical conditions with and without RNase A treatment. Pull-downs of mRNPs containing individual GFP-tagged SR proteins were probed for the presence of PABPN1.
Figure 3. SR proteins and NXF1 bind to mRNA after splicing. (A) Number of cross-link events of individual SR proteins mapping to a window of ±300 nucleotides (nt) around the 5′ splice site (left) and 3′ splice site (right). Each line represents a different SR protein or NXF1. (B, left panel) Scheme of a two-exon gene with iCLIP reads mapping to the 5′ splice site (5′ss). The cross-link site is indicated with a gray arrow, and the remaining peptide is indicated with a green star. Reads that continue from one exon into the neighboring intron (dark green) were counted as unspliced, and reads that span two neighboring exons (light green) were counted as spliced. (Right panel) Plot showing the proportions of spliced and unspliced iCLIP reads mapping near 5′ splice sites. (C) Semiquantitative co-IPs using identical conditions with and without RNase A treatment. Pull-downs of mRNPs containing individual GFP-tagged SR proteins were probed for the presence of PABPN1.CC BY · PubMed Central
Figure 4. SR proteins and NXF1 cobind within exons. (A) Number of significant cross-link events of SR proteins and NXF1 (FDR < 0.05) in different transcript regions normalized to feature length. (B) Hierarchical clustering using distance correlation and Spearman rank correlation of exon cobinding between SR proteins and NXF1 using normalized FDR iCLIP tag counts. (C) Browser shots showing binding profiles for GFP-tagged SRSF1–7, NXF1, and GFP-NLS on the Srsf5 gene. SR proteins of the same group share similar binding patterns. Srsf5 was not detected as export target of any SR protein.
Figure 4. SR proteins and NXF1 cobind within exons. (A) Number of significant cross-link events of SR proteins and NXF1 (FDR < 0.05) in different transcript regions normalized to feature length. (B) Hierarchical clustering using distance correlation and Spearman rank correlation of exon cobinding between SR proteins and NXF1 using normalized FDR iCLIP tag counts. (C) Browser shots showing binding profiles for GFP-tagged SRSF1–7, NXF1, and GFP-NLS on the Srsf5 gene. SR proteins of the same group share similar binding patterns. Srsf5 was not detected as export target of any SR protein.CC BY · PubMed Central
Figure 5. SRSF3 recruits NXF1 to adjacent sites in 3′ UTRs. (A) Numbers of export target candidates with (green) or without (dark gray) significant cross-link sites for their cognate SR proteins. (NA) Transcript ID discrepancies in mm9 and mm10 assemblies. (B) The occurrence of significant SR protein cross-link sites (n > 3) in a small window of 60 nt (±30 nt) around the center of NXF1-binding sites was counted separately in first exons and last exons and over all transcript regions. The ratio of cobinding was calculated with respect to the total number of NXF1-binding sites. (C) Number of NXF1-binding sites where only one SR protein cobinds (significant cross-link sites, greater than three). (D) Examples of SRSF3 export targets with NXF1- and SRSF3-binding sites in close proximity within last exons, including a blow-up with nucleotide resolution. SRSF3-binding motifs are indicated in black. (E) Comparison of in vivo binding motifs (enriched 8-mers) of SRSF3 and NXF1 derived from all transcript regions and separately from 5′ UTRs or 3′ UTRs. (F) Working model for NXF1 recruitment and formation of a trimeric complex with bound mRNAs for export based on NXF1 remodeling by TREX (Viphakone et al. 2012). SRSF3 (bound to the 3′ end of mRNAs) binds to NXF1, opens the loop, and unlocks the sequestered RBD, thus enabling NXF1 to bind to adjacent binding sites on the same mRNA. (m7G) Methylguanosine cap; (p15) NXF1 cofactor; (LRR) leucine-rich domain; (NTF2L) nuclear transport factor 2 (NTF2)-like domain; (UBA) ubiquitin-associated domain.
Figure 5. SRSF3 recruits NXF1 to adjacent sites in 3′ UTRs. (A) Numbers of export target candidates with (green) or without (dark gray) significant cross-link sites for their cognate SR proteins. (NA) Transcript ID discrepancies in mm9 and mm10 assemblies. (B) The occurrence of significant SR protein cross-link sites (n > 3) in a small window of 60 nt (±30 nt) around the center of NXF1-binding sites was counted separately in first exons and last exons and over all transcript regions. The ratio of cobinding was calculated with respect to the total number of NXF1-binding sites. (C) Number of NXF1-binding sites where only one SR protein cobinds (significant cross-link sites, greater than three). (D) Examples of SRSF3 export targets with NXF1- and SRSF3-binding sites in close proximity within last exons, including a blow-up with nucleotide resolution. SRSF3-binding motifs are indicated in black. (E) Comparison of in vivo binding motifs (enriched 8-mers) of SRSF3 and NXF1 derived from all transcript regions and separately from 5′ UTRs or 3′ UTRs. (F) Working model for NXF1 recruitment and formation of a trimeric complex with bound mRNAs for export based on NXF1 remodeling by TREX (Viphakone et al. 2012). SRSF3 (bound to the 3′ end of mRNAs) binds to NXF1, opens the loop, and unlocks the sequestered RBD, thus enabling NXF1 to bind to adjacent binding sites on the same mRNA. (m7G) Methylguanosine cap; (p15) NXF1 cofactor; (LRR) leucine-rich domain; (NTF2L) nuclear transport factor 2 (NTF2)-like domain; (UBA) ubiquitin-associated domain.CC BY · PubMed Central
Figure 6. SR proteins link alternative splicing to mRNA export. (A) Co-IP of NXF1-GFP with or without RNase A (long) using α-GFP. Phosphorylated SRSF3 and SRSF7 were detected with phosphorylation-dependent mAb104 (top panel) and phosphorylation-independent SR protein-specific (bottom panel) antibodies. (B) Gene ontology (GO) term analysis of export targets with significant cross-links of their cognate SR proteins. (C) Quantification of exon usage from cytoplasmic and whole-cell RNA-seq after SR protein depletion. (D, left panel) Changes in exon usage in whole-cell and cytoplasmic RNA samples were calculated using DEX-seq (Anders et al. 2012). P < 0.01. Numbers of changed exons after SR protein knockdown were separated in less inclusion and more inclusion. (Right panel) Venn diagrams showing coregulated splicing events in total and cytoplasmic samples after SRSF1 and SRSF3 depletion. (E) Number of significant cross-link events in changed exons in whole-cell and cytoplasmic RNA. (F) Proportion of exons that changed after SR protein knockdowns separated into first exons (light green), internal exons (dark green), and last exons (dark brown).
Figure 6. SR proteins link alternative splicing to mRNA export. (A) Co-IP of NXF1-GFP with or without RNase A (long) using α-GFP. Phosphorylated SRSF3 and SRSF7 were detected with phosphorylation-dependent mAb104 (top panel) and phosphorylation-independent SR protein-specific (bottom panel) antibodies. (B) Gene ontology (GO) term analysis of export targets with significant cross-links of their cognate SR proteins. (C) Quantification of exon usage from cytoplasmic and whole-cell RNA-seq after SR protein depletion. (D, left panel) Changes in exon usage in whole-cell and cytoplasmic RNA samples were calculated using DEX-seq (Anders et al. 2012). P < 0.01. Numbers of changed exons after SR protein knockdown were separated in less inclusion and more inclusion. (Right panel) Venn diagrams showing coregulated splicing events in total and cytoplasmic samples after SRSF1 and SRSF3 depletion. (E) Number of significant cross-link events in changed exons in whole-cell and cytoplasmic RNA. (F) Proportion of exons that changed after SR protein knockdowns separated into first exons (light green), internal exons (dark green), and last exons (dark brown).CC BY · PubMed Central
Figure 7. SRSF3 and SRSF7 regulate 3′ UTR length in an antagonistic manner and link alternative 3′ end formation to mRNA export. (A) Changes in last exon usage in cytoplasmic samples were mapped to annotated splicing events (ALE and TandemUTR) using MISO (Katz et al. 2010). BF > 5; 20% change. (B) Profiles of significant cross-link sites of SRSF3 and SRSF7 in last exons undergoing APA after depletion of the cognate SR protein. (C) Venn diagram. Thirty-two genes are regulated in an antagonistic manner by SRSF3 and SRSF7 in APA (cutoff FDR < 0.1). (D) Transcripts chosen for validation. Browser shots including affected isoforms (ENSEMBL); RNA-seq read coverage of control RNAi (gray), SRSF3 RNAi (light blue), and SRSF7 RNAi (dark blue); and significant cross-link sites of SRSF3 (light blue), SRSF7 (dark blue), and NXF1 (purple). The transcript isoforms shown are truncated. (Green arrow) Gene-specific forward primer in the penultimate exon for rapid amplification of cDNA 3′ end (3′RACE) RT–PCRs. (E) Validation of APA changes after knockdown (KD) of SRSF3 and SRSF7 by 3′RACE RT–PCR. Orange and black asterisks indicate the transcript isoforms shown in D. (F) Model of how SRSF3 and SRSF7 binding in last exons may affect APA, NXF1 binding, and mRNA export in a splicing-dependent and -independent manner. (G) Length distribution of 3′ UTRs from transcripts, which decrease in cytoplasmic abundance after depletion of SRSF7, SRSF1, and SRSF3.
Figure 7. SRSF3 and SRSF7 regulate 3′ UTR length in an antagonistic manner and link alternative 3′ end formation to mRNA export. (A) Changes in last exon usage in cytoplasmic samples were mapped to annotated splicing events (ALE and TandemUTR) using MISO (Katz et al. 2010). BF > 5; 20% change. (B) Profiles of significant cross-link sites of SRSF3 and SRSF7 in last exons undergoing APA after depletion of the cognate SR protein. (C) Venn diagram. Thirty-two genes are regulated in an antagonistic manner by SRSF3 and SRSF7 in APA (cutoff FDR < 0.1). (D) Transcripts chosen for validation. Browser shots including affected isoforms (ENSEMBL); RNA-seq read coverage of control RNAi (gray), SRSF3 RNAi (light blue), and SRSF7 RNAi (dark blue); and significant cross-link sites of SRSF3 (light blue), SRSF7 (dark blue), and NXF1 (purple). The transcript isoforms shown are truncated. (Green arrow) Gene-specific forward primer in the penultimate exon for rapid amplification of cDNA 3′ end (3′RACE) RT–PCRs. (E) Validation of APA changes after knockdown (KD) of SRSF3 and SRSF7 by 3′RACE RT–PCR. Orange and black asterisks indicate the transcript isoforms shown in D. (F) Model of how SRSF3 and SRSF7 binding in last exons may affect APA, NXF1 binding, and mRNA export in a splicing-dependent and -independent manner. (G) Length distribution of 3′ UTRs from transcripts, which decrease in cytoplasmic abundance after depletion of SRSF7, SRSF1, and SRSF3.CC BY · PubMed Central

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Research works printed but not linked: 61

  1. 10.1101/gr.133744.111 · DOI · Paper page
  2. 10.1016/j.semcdb.2014.03.011 · DOI
  3. 10.1038/nsmb.1862 · DOI
  4. 10.1186/gb-2012-13-3-r17 · DOI
  5. 10.1083/jcb.200806156 · DOI
  6. 10.1261/rna.043893.113 · DOI
  7. 10.1074/jbc.M100400200 · DOI
  8. 10.1101/gad.12.1.55 · DOI
  9. 10.1128/MCB.22.19.6871-6882.2002 · DOI
  10. 10.1016/j.cell.2006.10.044 · DOI
  11. 10.1093/nar/gkr663 · DOI
  12. 10.1016/j.sbi.2008.04.002 · DOI
  13. 10.1261/rna.037044.112 · DOI
  14. 10.1111/j.1742-4658.2010.07992.x · DOI
  15. 10.1038/ncomms1998 · DOI
  16. 10.1261/rna.212106 · DOI
  17. 10.1261/rna.2607011 · DOI
  18. 10.1128/MCB.01117-10 · DOI
  19. 10.1038/sj.emboj.7601385 · DOI
  20. 10.1073/pnas.0709167105 · DOI
  21. 10.1016/j.cub.2009.09.041 · DOI
  22. 10.1002/wrna.1260 · DOI
  23. 10.1016/S1097-2765(01)00233-7 · DOI
  24. 10.1016/j.molcel.2005.02.020 · DOI
  25. 10.1016/S1097-2765(03)00089-3 · DOI · Paper page
  26. 10.1073/pnas.0403533101 · DOI · Paper page
  27. 10.1038/emboj.2009.5 · DOI
  28. 10.1038/nmeth.1528 · DOI · Paper page
  29. 10.1016/S0960-9822(02)00840-0 · DOI
  30. 10.1074/jbc.C400173200 · DOI
  31. 10.1093/emboj/19.24.6860 · DOI
  32. 10.1101/gad.939502 · DOI
  33. 10.1038/nature05107 · DOI
  34. 10.1016/j.molcel.2005.09.015 · DOI
  35. 10.1128/MCB.18.9.4977 · DOI
  36. Maslon MM , Heras SR , Bellora N , Eyras E , Caceres JF . 2014. The translational landscape of the splicing factor SRSF1 and its role in mitosis. Elife: e02028. · DOI
  37. 10.1101/gad.1302205 · DOI
  38. 10.1111/j.1365-2443.2004.00774.x · DOI
  39. 10.1016/j.molcel.2008.03.013 · DOI
  40. 10.1038/nrg3434 · DOI
  41. 10.1074/jbc.M700629200 · DOI
  42. 10.1016/j.molcel.2013.03.001 · DOI
  43. 10.1038/nsmb759 · DOI
  44. 10.1073/pnas.0507827102 · DOI
  45. 10.1371/journal.pone.0003369 · DOI
  46. 10.1016/j.molcel.2009.02.031 · DOI
  47. 10.1016/j.molcel.2007.12.009 · DOI
  48. 10.1016/S1097-2765(04)00025-5 · DOI
  49. 10.1186/gb-2009-10-10-242 · DOI
  50. 10.1016/j.molcel.2008.12.028 · DOI
  51. 10.1016/j.cell.2012.10.007 · DOI
  52. 10.1074/jbc.M701660200 · DOI
  53. 10.1128/MCB.01341-07 · DOI
  54. 10.1038/nsmb.2094 · DOI
  55. 10.1038/sj.embor.7401031 · DOI
  56. 10.1074/jbc.M109.040113 · DOI
  57. 10.1038/ncomms2005 · DOI
  58. 10.1093/nar/gkv070 · DOI
  59. 10.1042/BST0380232 · DOI
  60. 10.1371/journal.pbio.1000530 · DOI
  61. 10.1038/nsmb.1545 · DOI · Paper page

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Metadata

Authors
Müller-McNicoll, M., Botti, V., de Jesus Domingues, A. M., Brandl, H., Schwich, O. D., Steiner, M. C., Curk, T., Poser, I., Zarnack, K., & Neugebauer, K. M.
Venue
Genes & Development
Publisher
Cold Spring Harbor Laboratory
Published
2016-03-01
Volume
30
Issue
5
Pages
553-566
ISSN
  • 0890-9369
  • 1549-5477
DOI
10.1101/gad.276477.115
PMC
PMC4782049
Cited by
313
References
3
Language
en
Funders
  • Deutsche Forschungsgemeinschaft (NE 909/3-1)
  • European Commission (FP7-PEOPLE-2010-IEF)
  • LOEWE program Ubiquitin Networks (Ub-Net)
  • European Molecular Biology Organization (ALTF455-2010)
  • Cluster of Excellence Frankfurt (CEF-MC)

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