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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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Alternative splicing of RBM10. (A) FISH analysis of GLC20 cells, with painted X and Y chromosomes, demonstrating the presence of only one X chromosome. (B) RBM10v1 and RBM10v2 RNA expression in various cell lines, including GLC20. Representative raw RT-PCR data using RBM10 exon 4 spanning primers. Bi: RBM10F with RBM10RS primers. Bii: RBM10F with RBM10v1/v2R primers. M: 100 bp DNA ladder (FroggaBio Inc., Toronto, Canada). NTC: no template control. (C) Protein expression by Western blot. Ci shows RBM10 expression in whole cell lysates from three cell lines, including GLC20. The numbers 1, 2 and 3 after JKM1 and GLC20 delineate cells from three biological replicates. Cii includes control HeLa protein and in vitro translated RBM10v1 and RBM10v2 protein, to confirm the location of RBM10v2 is the cell line extracts. (D) Cartoon of full-length RBM10v1 mRNA, not drawn to scale. Boxes represent exons. Left and right black arrows represent primer placement for sequencing. Approximate positioning of consensus functional motifs is indicated by text and differential shading. (E) Alignment of the two GLC20 RBM10v1 isoform sequences. Circled area indicates the region that differs between the two RBM10v1 isoforms. (F) Nucleotide and amino acid sequences of the RBM10v1 exon10/intron 10/exon 11 donor and acceptor sites for (i) RBM10v1(V354), and (ii) RBM10v1(V354del).
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Fig1: Alternative splicing of RBM10. (A) FISH analysis of GLC20 cells, with painted X and Y chromosomes, demonstrating the presence of only one X chromosome. (B) RBM10v1 and RBM10v2 RNA expression in various cell lines, including GLC20. Representative raw RT-PCR data using RBM10 exon 4 spanning primers. Bi: RBM10F with RBM10RS primers. Bii: RBM10F with RBM10v1/v2R primers. M: 100 bp DNA ladder (FroggaBio Inc., Toronto, Canada). NTC: no template control. (C) Protein expression by Western blot. Ci shows RBM10 expression in whole cell lysates from three cell lines, including GLC20. The numbers 1, 2 and 3 after JKM1 and GLC20 delineate cells from three biological replicates. Cii includes control HeLa protein and in vitro translated RBM10v1 and RBM10v2 protein, to confirm the location of RBM10v2 is the cell line extracts. (D) Cartoon of full-length RBM10v1 mRNA, not drawn to scale. Boxes represent exons. Left and right black arrows represent primer placement for sequencing. Approximate positioning of consensus functional motifs is indicated by text and differential shading. (E) Alignment of the two GLC20 RBM10v1 isoform sequences. Circled area indicates the region that differs between the two RBM10v1 isoforms. (F) Nucleotide and amino acid sequences of the RBM10v1 exon10/intron 10/exon 11 donor and acceptor sites for (i) RBM10v1(V354), and (ii) RBM10v1(V354del).

Mentions: The GLC20 cell line was established from a small cell lung cancer (SCLC) of male origin [12]. It is RBM5- and was determined, by FISH, to contain only one X chromosome (Figure 1A). Alternative splicing of the X-linked RBM10 gene [13], to generate both RBM10v1 and RBM10v2, was confirmed in the GLC20 cells, using RT-PCR (Figure 1B). Most of the cancer, or transformed, cell lines that we have tested express more RBM10v1 than RBM10v2, at both the mRNA (Figure 1B) and protein (Figure 1C and data not shown) levels. This observation is particularly true for GLC20 cells, which express such a small amount of RBM10v2 protein that it is technically challenging to detect.Figure 1


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)

Alternative splicing of RBM10. (A) FISH analysis of GLC20 cells, with painted X and Y chromosomes, demonstrating the presence of only one X chromosome. (B) RBM10v1 and RBM10v2 RNA expression in various cell lines, including GLC20. Representative raw RT-PCR data using RBM10 exon 4 spanning primers. Bi: RBM10F with RBM10RS primers. Bii: RBM10F with RBM10v1/v2R primers. M: 100 bp DNA ladder (FroggaBio Inc., Toronto, Canada). NTC: no template control. (C) Protein expression by Western blot. Ci shows RBM10 expression in whole cell lysates from three cell lines, including GLC20. The numbers 1, 2 and 3 after JKM1 and GLC20 delineate cells from three biological replicates. Cii includes control HeLa protein and in vitro translated RBM10v1 and RBM10v2 protein, to confirm the location of RBM10v2 is the cell line extracts. (D) Cartoon of full-length RBM10v1 mRNA, not drawn to scale. Boxes represent exons. Left and right black arrows represent primer placement for sequencing. Approximate positioning of consensus functional motifs is indicated by text and differential shading. (E) Alignment of the two GLC20 RBM10v1 isoform sequences. Circled area indicates the region that differs between the two RBM10v1 isoforms. (F) Nucleotide and amino acid sequences of the RBM10v1 exon10/intron 10/exon 11 donor and acceptor sites for (i) RBM10v1(V354), and (ii) RBM10v1(V354del).
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Related In: Results  -  Collection

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Fig1: Alternative splicing of RBM10. (A) FISH analysis of GLC20 cells, with painted X and Y chromosomes, demonstrating the presence of only one X chromosome. (B) RBM10v1 and RBM10v2 RNA expression in various cell lines, including GLC20. Representative raw RT-PCR data using RBM10 exon 4 spanning primers. Bi: RBM10F with RBM10RS primers. Bii: RBM10F with RBM10v1/v2R primers. M: 100 bp DNA ladder (FroggaBio Inc., Toronto, Canada). NTC: no template control. (C) Protein expression by Western blot. Ci shows RBM10 expression in whole cell lysates from three cell lines, including GLC20. The numbers 1, 2 and 3 after JKM1 and GLC20 delineate cells from three biological replicates. Cii includes control HeLa protein and in vitro translated RBM10v1 and RBM10v2 protein, to confirm the location of RBM10v2 is the cell line extracts. (D) Cartoon of full-length RBM10v1 mRNA, not drawn to scale. Boxes represent exons. Left and right black arrows represent primer placement for sequencing. Approximate positioning of consensus functional motifs is indicated by text and differential shading. (E) Alignment of the two GLC20 RBM10v1 isoform sequences. Circled area indicates the region that differs between the two RBM10v1 isoforms. (F) Nucleotide and amino acid sequences of the RBM10v1 exon10/intron 10/exon 11 donor and acceptor sites for (i) RBM10v1(V354), and (ii) RBM10v1(V354del).
Mentions: The GLC20 cell line was established from a small cell lung cancer (SCLC) of male origin [12]. It is RBM5- and was determined, by FISH, to contain only one X chromosome (Figure 1A). Alternative splicing of the X-linked RBM10 gene [13], to generate both RBM10v1 and RBM10v2, was confirmed in the GLC20 cells, using RT-PCR (Figure 1B). Most of the cancer, or transformed, cell lines that we have tested express more RBM10v1 than RBM10v2, at both the mRNA (Figure 1B) and protein (Figure 1C and data not shown) levels. This observation is particularly true for GLC20 cells, which express such a small amount of RBM10v2 protein that it is technically challenging to detect.Figure 1

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