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Alternative splicing produces structural and functional changes in CUGBP2.

Suzuki H, Takeuchi M, Sugiyama A, Alam AK, Vu LT, Sekiyama Y, Dam HC, Ohki SY, Tsukahara T - BMC Biochem. (2012)

Bottom Line: In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure.Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms.The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Nano Materials and Technology, Japan Advanced Institute of Science and Technology, Ishikawa 923-1292, Japan. suzuki-h@jaist.ac.jp

ABSTRACT

Background: CELF/Bruno-like proteins play multiple roles, including the regulation of alternative splicing and translation. These RNA-binding proteins contain two RNA recognition motif (RRM) domains at the N-terminus and another RRM at the C-terminus. CUGBP2 is a member of this family of proteins that possesses several alternatively spliced exons.

Results: The present study investigated the expression of exon 14, which is an alternatively spliced exon and encodes the first half of the third RRM of CUGBP2. The ratio of exon 14 skipping product (R3δ) to its inclusion was reduced in neuronal cells induced from P19 cells and in the brain. Although full length CUGBP2 and the CUGBP2 R3δ isoforms showed a similar effect on the inclusion of the smooth muscle (SM) exon of the ACTN1 gene, these isoforms showed an opposite effect on the skipping of exon 11 in the insulin receptor gene. In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure.

Conclusion: Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms. Alternative splicing of CUGBP2 exon 14 contributes to the regulation of the splicing of the insulin receptor. The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.

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Related in: MedlinePlus

Alternative splicing of the CUGBP2 gene. (A) Schematic representation of the CUGBP2 protein and the CUGBP2 R3δ isoform. The upper panel shows CUGBP2 and its domains. RRMs represent the RNA-binding domains. The NLS (line), NES (broken line), and splicing activation domain (gray line) were determined in a previous report [27]. The lower panel shows the alternatively spliced form of CUGBP2, the R3δ isoform. (B) Schematic representation of exon 14 and its adjacent region in the CUGBP2 gene. Exons are indicated as black boxes with the alternatively spliced exons indicated as gray boxes. Introns are indicated by a central narrow line. Arrows show primer sites. (C) Expression analysis of CUGBP2 in adult mouse tissues. Semi-quantitative RT-PCR was performed using primers to detect the alternatively spliced exon of CUGBP2. The right side indicates the positions of exon 14 skipping or inclusion products. β-Actin was used as a control. (D) Expression analysis of CUGBP2 in P19 neural differentiation. The right side indicates the positions of exon 14 skipping or inclusion products. β-Actin was used as a control. Relative amounts of exon 14 skipping and inclusion products were estimated by densitometry. Changes of total expression levels were normalized using brain samples (C) or Day 0 samples (D). The error bars indicate the standard error. The values under the gel images indicate the percentage of the exon 14 skipping in total CUGBP2 transcripts. (E) Western blot analysis of CUGBP2. Whole cell extracts of P19 cells (2 μg) were used to detect the changes in the amount of full-length CUGBP2 in the upper panel. The middle panel shows the R3δ isoform detected using 7 μg of each extract. GAPDH was used as a control and is shown in the lower panel.
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Figure 1: Alternative splicing of the CUGBP2 gene. (A) Schematic representation of the CUGBP2 protein and the CUGBP2 R3δ isoform. The upper panel shows CUGBP2 and its domains. RRMs represent the RNA-binding domains. The NLS (line), NES (broken line), and splicing activation domain (gray line) were determined in a previous report [27]. The lower panel shows the alternatively spliced form of CUGBP2, the R3δ isoform. (B) Schematic representation of exon 14 and its adjacent region in the CUGBP2 gene. Exons are indicated as black boxes with the alternatively spliced exons indicated as gray boxes. Introns are indicated by a central narrow line. Arrows show primer sites. (C) Expression analysis of CUGBP2 in adult mouse tissues. Semi-quantitative RT-PCR was performed using primers to detect the alternatively spliced exon of CUGBP2. The right side indicates the positions of exon 14 skipping or inclusion products. β-Actin was used as a control. (D) Expression analysis of CUGBP2 in P19 neural differentiation. The right side indicates the positions of exon 14 skipping or inclusion products. β-Actin was used as a control. Relative amounts of exon 14 skipping and inclusion products were estimated by densitometry. Changes of total expression levels were normalized using brain samples (C) or Day 0 samples (D). The error bars indicate the standard error. The values under the gel images indicate the percentage of the exon 14 skipping in total CUGBP2 transcripts. (E) Western blot analysis of CUGBP2. Whole cell extracts of P19 cells (2 μg) were used to detect the changes in the amount of full-length CUGBP2 in the upper panel. The middle panel shows the R3δ isoform detected using 7 μg of each extract. GAPDH was used as a control and is shown in the lower panel.

Mentions: A search of the UCSC genome browser, BLAT, suggested that the R3δ isoform is the product of skipping of exon 14 in 25% of CUGBP2 transcripts. Exon14 is 144 nt and the skipping transcript does not generate a new premature termination codon. To assess in which organ alternative splicing of RRM3 in CUGBP2 takes place, RT-PCR was performed in adult mouse tissues. The products of exon 14 inclusion (CUGBP2) and exon 14 skipping (R3δ), which encode a complete and partial RRM3, respectively, were detected (Figure 1A &1B). CUGBP2 mRNA was highly expressed in the brain, where the main product was the exon 14 inclusion transcript encoding a complete RRM3 (Figure 1C, R3δ percentage of CUGBP2 and R3δ in the brain: 8.1%). The total amount of CUGBP2 transcripts in the kidney or liver was low compared to that in brain, but the percentage of the R3δ isoform was relatively high (Figure 1C, R3δ percentage of CUGBP2 and R3δ in the kidney: 22.1%; in the liver, 19.5%). These results suggest that the R3δ isoform is one of major products when their gene expression of CUGBP2 is low, and the CUGBP2 isoform, but not R3δ, is expressed as the major product when their gene expression is high. To further investigate the alternative splicing pattern of CUGBP2, RT-PCR was performed in P19 cells during neural differentiation. Increased levels of the exon 14 inclusion product were detected during neural differentiation even though the exon 14 skipping product was not essentially changed (Figure 1D). The R3δ percentage on day 7 (neural cell stage) was 18.9%, which was the lowest during P19 neural differentiation (R3δ was 29.3% in undifferentiated P19 cells). The relatively low expression of R3δ with respect to alternative splicing patterns was also found in tissues of the adult mouse. Western blot analysis of CUGBP2 proteins showed that the full length isoform was the main protein in P19 cells and that its level increased at the neural stage. By contrast, the level of R3δ decreased at the neural stage, although it was observed in undifferentiated P19 cells (Figure 1E).


Alternative splicing produces structural and functional changes in CUGBP2.

Suzuki H, Takeuchi M, Sugiyama A, Alam AK, Vu LT, Sekiyama Y, Dam HC, Ohki SY, Tsukahara T - BMC Biochem. (2012)

Alternative splicing of the CUGBP2 gene. (A) Schematic representation of the CUGBP2 protein and the CUGBP2 R3δ isoform. The upper panel shows CUGBP2 and its domains. RRMs represent the RNA-binding domains. The NLS (line), NES (broken line), and splicing activation domain (gray line) were determined in a previous report [27]. The lower panel shows the alternatively spliced form of CUGBP2, the R3δ isoform. (B) Schematic representation of exon 14 and its adjacent region in the CUGBP2 gene. Exons are indicated as black boxes with the alternatively spliced exons indicated as gray boxes. Introns are indicated by a central narrow line. Arrows show primer sites. (C) Expression analysis of CUGBP2 in adult mouse tissues. Semi-quantitative RT-PCR was performed using primers to detect the alternatively spliced exon of CUGBP2. The right side indicates the positions of exon 14 skipping or inclusion products. β-Actin was used as a control. (D) Expression analysis of CUGBP2 in P19 neural differentiation. The right side indicates the positions of exon 14 skipping or inclusion products. β-Actin was used as a control. Relative amounts of exon 14 skipping and inclusion products were estimated by densitometry. Changes of total expression levels were normalized using brain samples (C) or Day 0 samples (D). The error bars indicate the standard error. The values under the gel images indicate the percentage of the exon 14 skipping in total CUGBP2 transcripts. (E) Western blot analysis of CUGBP2. Whole cell extracts of P19 cells (2 μg) were used to detect the changes in the amount of full-length CUGBP2 in the upper panel. The middle panel shows the R3δ isoform detected using 7 μg of each extract. GAPDH was used as a control and is shown in the lower panel.
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Figure 1: Alternative splicing of the CUGBP2 gene. (A) Schematic representation of the CUGBP2 protein and the CUGBP2 R3δ isoform. The upper panel shows CUGBP2 and its domains. RRMs represent the RNA-binding domains. The NLS (line), NES (broken line), and splicing activation domain (gray line) were determined in a previous report [27]. The lower panel shows the alternatively spliced form of CUGBP2, the R3δ isoform. (B) Schematic representation of exon 14 and its adjacent region in the CUGBP2 gene. Exons are indicated as black boxes with the alternatively spliced exons indicated as gray boxes. Introns are indicated by a central narrow line. Arrows show primer sites. (C) Expression analysis of CUGBP2 in adult mouse tissues. Semi-quantitative RT-PCR was performed using primers to detect the alternatively spliced exon of CUGBP2. The right side indicates the positions of exon 14 skipping or inclusion products. β-Actin was used as a control. (D) Expression analysis of CUGBP2 in P19 neural differentiation. The right side indicates the positions of exon 14 skipping or inclusion products. β-Actin was used as a control. Relative amounts of exon 14 skipping and inclusion products were estimated by densitometry. Changes of total expression levels were normalized using brain samples (C) or Day 0 samples (D). The error bars indicate the standard error. The values under the gel images indicate the percentage of the exon 14 skipping in total CUGBP2 transcripts. (E) Western blot analysis of CUGBP2. Whole cell extracts of P19 cells (2 μg) were used to detect the changes in the amount of full-length CUGBP2 in the upper panel. The middle panel shows the R3δ isoform detected using 7 μg of each extract. GAPDH was used as a control and is shown in the lower panel.
Mentions: A search of the UCSC genome browser, BLAT, suggested that the R3δ isoform is the product of skipping of exon 14 in 25% of CUGBP2 transcripts. Exon14 is 144 nt and the skipping transcript does not generate a new premature termination codon. To assess in which organ alternative splicing of RRM3 in CUGBP2 takes place, RT-PCR was performed in adult mouse tissues. The products of exon 14 inclusion (CUGBP2) and exon 14 skipping (R3δ), which encode a complete and partial RRM3, respectively, were detected (Figure 1A &1B). CUGBP2 mRNA was highly expressed in the brain, where the main product was the exon 14 inclusion transcript encoding a complete RRM3 (Figure 1C, R3δ percentage of CUGBP2 and R3δ in the brain: 8.1%). The total amount of CUGBP2 transcripts in the kidney or liver was low compared to that in brain, but the percentage of the R3δ isoform was relatively high (Figure 1C, R3δ percentage of CUGBP2 and R3δ in the kidney: 22.1%; in the liver, 19.5%). These results suggest that the R3δ isoform is one of major products when their gene expression of CUGBP2 is low, and the CUGBP2 isoform, but not R3δ, is expressed as the major product when their gene expression is high. To further investigate the alternative splicing pattern of CUGBP2, RT-PCR was performed in P19 cells during neural differentiation. Increased levels of the exon 14 inclusion product were detected during neural differentiation even though the exon 14 skipping product was not essentially changed (Figure 1D). The R3δ percentage on day 7 (neural cell stage) was 18.9%, which was the lowest during P19 neural differentiation (R3δ was 29.3% in undifferentiated P19 cells). The relatively low expression of R3δ with respect to alternative splicing patterns was also found in tissues of the adult mouse. Western blot analysis of CUGBP2 proteins showed that the full length isoform was the main protein in P19 cells and that its level increased at the neural stage. By contrast, the level of R3δ decreased at the neural stage, although it was observed in undifferentiated P19 cells (Figure 1E).

Bottom Line: In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure.Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms.The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Nano Materials and Technology, Japan Advanced Institute of Science and Technology, Ishikawa 923-1292, Japan. suzuki-h@jaist.ac.jp

ABSTRACT

Background: CELF/Bruno-like proteins play multiple roles, including the regulation of alternative splicing and translation. These RNA-binding proteins contain two RNA recognition motif (RRM) domains at the N-terminus and another RRM at the C-terminus. CUGBP2 is a member of this family of proteins that possesses several alternatively spliced exons.

Results: The present study investigated the expression of exon 14, which is an alternatively spliced exon and encodes the first half of the third RRM of CUGBP2. The ratio of exon 14 skipping product (R3δ) to its inclusion was reduced in neuronal cells induced from P19 cells and in the brain. Although full length CUGBP2 and the CUGBP2 R3δ isoforms showed a similar effect on the inclusion of the smooth muscle (SM) exon of the ACTN1 gene, these isoforms showed an opposite effect on the skipping of exon 11 in the insulin receptor gene. In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure.

Conclusion: Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms. Alternative splicing of CUGBP2 exon 14 contributes to the regulation of the splicing of the insulin receptor. The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.

Show MeSH
Related in: MedlinePlus