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Insight into the role of alternative splicing within the RBM10v1 exon 10 tandem donor site.

Tessier SJ, Loiselle JJ, McBain A, Pullen C, Koenderink BW, Roy JG, Sutherland LC - BMC Res Notes (2015)

Bottom Line: We then show 2-fold elevated expression of the transcripts encoding the minus valine RBM10v1 isoform in GLC20 cells, compared to those encoding the plus valine isoform.This expression correlates with preferential expression of the lung cancer-associated NUMB exon 11 inclusion variant.A model is presented.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Laurentian University, 935 Ramsey Lake Road, Sudbury, ON, P3E 2C6, Canada. sarah.tessier@live.ca.

ABSTRACT

Background: RBM10 is an RNA binding protein involved in the regulation of transcription, alternative splicing and message stabilization. Mutations in RBM10, which maps to the X chromosome, are associated with TARP syndrome, lung and pancreatic cancers. Two predominant isoforms of RBM10 exist, RBM10v1 and RBM10v2. Both variants have alternate isoforms that differ by one valine residue, at amino acid 354 (RBM10v1) or 277 (RBM10v2). It was recently observed that a novel point mutation at amino acid 354 of RBM10v1, replacing valine with glutamic acid, correlated with preferential expression of an exon 11 inclusion variant of the proliferation regulatory protein NUMB, which is upregulated in lung cancer.

Findings: We demonstrate, using the GLC20 male-derived small cell lung cancer cell line - confirmed to have only one X chromosome - that the two (+/-) valine isoforms of RBM10v1 and RBM10v2 result from alternative splicing. Protein modeling of the RNA Recognition Motif (RRM) within which the alteration occurs, shows that the presence of valine inhibits the formation of one of the two α-helices associated with RRM tertiary structure, whereas the absence of valine supports the α-helical configuration. We then show 2-fold elevated expression of the transcripts encoding the minus valine RBM10v1 isoform in GLC20 cells, compared to those encoding the plus valine isoform. This expression correlates with preferential expression of the lung cancer-associated NUMB exon 11 inclusion variant.

Conclusions: Our observations suggest that the ability of RBM10v1 to regulate alternative splicing depends, at least in part, on a structural alteration within the second RRM domain, which influences whether RBM10v1 functions to support or repress splicing. A model is presented.

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Related in: MedlinePlus

Conformation of RBM10v1 RRM2. The RBM10 RRM2 conformation was modeled using SwissProtKB (A) or Phyre2(B). (i) Isoforms V354. (ii) Isoforms V354E. (iii) Isoforms V354del. Arrow indicates the position of the +/− valine. Yasara structure colors indicate beta-sheets (red), alpha-helixes (dark blue), turns (green) and random coils (cyan).
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Fig2: Conformation of RBM10v1 RRM2. The RBM10 RRM2 conformation was modeled using SwissProtKB (A) or Phyre2(B). (i) Isoforms V354. (ii) Isoforms V354E. (iii) Isoforms V354del. Arrow indicates the position of the +/− valine. Yasara structure colors indicate beta-sheets (red), alpha-helixes (dark blue), turns (green) and random coils (cyan).

Mentions: To better understand how a change to this residue might contribute to RBM10 functional alterations we compared the structures of a valine-retaining, a glutamic acid-substituting and a valine-lacking amino acid within RRM2. We uploaded these altered RBM10v1 RRM2 sequences into SwissProtKB/Swiss-Prot (www.expasy.org), a program that predicts a two-dimensional configuration and ranks it against similar configurations of previously crystalized structures. A crystal structure for a minus-valine RBM10 RRM2 (designated 2m2d) [17,18] was the reference structure for the V354, V354E and V354del RBM10v1 RRM2 predictions. We also uploaded the RRM2 sequences into Phyre2 (the Protein Homology/analogY Recognition Engine v2.0, www.sbg.bio.ic.ac.uk). To visualize a rotatable three-dimensional structure, the structure predictions for V354, V354E and V354del from both SwissProtKB and Phyre2 were uploaded into the Yasara modeling program (Yet Another Scientific Artificial Reality Application, www.yasara.org). A comparison of all the predictions (Figure 2) revealed that the addition of valine did, as anticipated, disrupt the α-helical structure and thus the classic configuration of an RRM domain (shown as a colour change from dark blue to cyan by the Yasara software). Exclusion of the valine was associated with an α-helix. Substitution of the V for an E resulted in two slightly different configurations, depending on the prediction program used: both programs, however, predicted a change to, but a retention of, an α-helical structure compared to either the V354del or the V354. These modelling results suggest that conformational changes to the RBM10v1 protein could be responsible for altering the protein’s ability to interact with RNA.Figure 2


Insight into the role of alternative splicing within the RBM10v1 exon 10 tandem donor site.

Tessier SJ, Loiselle JJ, McBain A, Pullen C, Koenderink BW, Roy JG, Sutherland LC - BMC Res Notes (2015)

Conformation of RBM10v1 RRM2. The RBM10 RRM2 conformation was modeled using SwissProtKB (A) or Phyre2(B). (i) Isoforms V354. (ii) Isoforms V354E. (iii) Isoforms V354del. Arrow indicates the position of the +/− valine. Yasara structure colors indicate beta-sheets (red), alpha-helixes (dark blue), turns (green) and random coils (cyan).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4336493&req=5

Fig2: Conformation of RBM10v1 RRM2. The RBM10 RRM2 conformation was modeled using SwissProtKB (A) or Phyre2(B). (i) Isoforms V354. (ii) Isoforms V354E. (iii) Isoforms V354del. Arrow indicates the position of the +/− valine. Yasara structure colors indicate beta-sheets (red), alpha-helixes (dark blue), turns (green) and random coils (cyan).
Mentions: To better understand how a change to this residue might contribute to RBM10 functional alterations we compared the structures of a valine-retaining, a glutamic acid-substituting and a valine-lacking amino acid within RRM2. We uploaded these altered RBM10v1 RRM2 sequences into SwissProtKB/Swiss-Prot (www.expasy.org), a program that predicts a two-dimensional configuration and ranks it against similar configurations of previously crystalized structures. A crystal structure for a minus-valine RBM10 RRM2 (designated 2m2d) [17,18] was the reference structure for the V354, V354E and V354del RBM10v1 RRM2 predictions. We also uploaded the RRM2 sequences into Phyre2 (the Protein Homology/analogY Recognition Engine v2.0, www.sbg.bio.ic.ac.uk). To visualize a rotatable three-dimensional structure, the structure predictions for V354, V354E and V354del from both SwissProtKB and Phyre2 were uploaded into the Yasara modeling program (Yet Another Scientific Artificial Reality Application, www.yasara.org). A comparison of all the predictions (Figure 2) revealed that the addition of valine did, as anticipated, disrupt the α-helical structure and thus the classic configuration of an RRM domain (shown as a colour change from dark blue to cyan by the Yasara software). Exclusion of the valine was associated with an α-helix. Substitution of the V for an E resulted in two slightly different configurations, depending on the prediction program used: both programs, however, predicted a change to, but a retention of, an α-helical structure compared to either the V354del or the V354. These modelling results suggest that conformational changes to the RBM10v1 protein could be responsible for altering the protein’s ability to interact with RNA.Figure 2

Bottom Line: We then show 2-fold elevated expression of the transcripts encoding the minus valine RBM10v1 isoform in GLC20 cells, compared to those encoding the plus valine isoform.This expression correlates with preferential expression of the lung cancer-associated NUMB exon 11 inclusion variant.A model is presented.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Laurentian University, 935 Ramsey Lake Road, Sudbury, ON, P3E 2C6, Canada. sarah.tessier@live.ca.

ABSTRACT

Background: RBM10 is an RNA binding protein involved in the regulation of transcription, alternative splicing and message stabilization. Mutations in RBM10, which maps to the X chromosome, are associated with TARP syndrome, lung and pancreatic cancers. Two predominant isoforms of RBM10 exist, RBM10v1 and RBM10v2. Both variants have alternate isoforms that differ by one valine residue, at amino acid 354 (RBM10v1) or 277 (RBM10v2). It was recently observed that a novel point mutation at amino acid 354 of RBM10v1, replacing valine with glutamic acid, correlated with preferential expression of an exon 11 inclusion variant of the proliferation regulatory protein NUMB, which is upregulated in lung cancer.

Findings: We demonstrate, using the GLC20 male-derived small cell lung cancer cell line - confirmed to have only one X chromosome - that the two (+/-) valine isoforms of RBM10v1 and RBM10v2 result from alternative splicing. Protein modeling of the RNA Recognition Motif (RRM) within which the alteration occurs, shows that the presence of valine inhibits the formation of one of the two α-helices associated with RRM tertiary structure, whereas the absence of valine supports the α-helical configuration. We then show 2-fold elevated expression of the transcripts encoding the minus valine RBM10v1 isoform in GLC20 cells, compared to those encoding the plus valine isoform. This expression correlates with preferential expression of the lung cancer-associated NUMB exon 11 inclusion variant.

Conclusions: Our observations suggest that the ability of RBM10v1 to regulate alternative splicing depends, at least in part, on a structural alteration within the second RRM domain, which influences whether RBM10v1 functions to support or repress splicing. A model is presented.

Show MeSH
Related in: MedlinePlus