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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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Transient transfection of the ACTN1 minigene and the IR minigene. (A) Schematic representation of the ACTN1 minigene. Exons are indicated as black boxes with alternatively spliced exons indicated as gray boxes. Introns are indicated with a central narrow line. The arrows show the primer sites. (B) Results of the transient transfection experiment. COS7 cells were transiently transfected with the ACTN1 minigene with/without the CUGBP, CUGBP2 R3δ isoform, or Etr-1 expression vectors. Alternatively spliced products were analyzed by RT-PCR. (C) Densitometric analysis of the transfection products. Quantification of the alternatively spliced products was performed by densitometry. The percentage of SM exon inclusion with respect to total product is shown in graphical representation. The error bars indicate the standard error. (D) Schematic representation of the IR minigene. Exons are indicated as black boxes and alternatively spliced exons are indicated as gray boxes. Introns are shown with a central narrow line. The arrows show the primer sites. (E) Results of the transient transfection experiment. HeLa cells were transiently transfected with the IR minigene with/without the CUGBP, R3δ isoform, or Etr-1 expression vectors. Alternatively spliced products were analyzed by RT-PCR. (F) Densitometric analysis of the transfection products. Quantification of the alternatively spliced products was performed by densitometry. The percentage of exon 11 inclusion with respect to total product is shown in graphical representation. The error bars indicate the standard error.
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Figure 3: Transient transfection of the ACTN1 minigene and the IR minigene. (A) Schematic representation of the ACTN1 minigene. Exons are indicated as black boxes with alternatively spliced exons indicated as gray boxes. Introns are indicated with a central narrow line. The arrows show the primer sites. (B) Results of the transient transfection experiment. COS7 cells were transiently transfected with the ACTN1 minigene with/without the CUGBP, CUGBP2 R3δ isoform, or Etr-1 expression vectors. Alternatively spliced products were analyzed by RT-PCR. (C) Densitometric analysis of the transfection products. Quantification of the alternatively spliced products was performed by densitometry. The percentage of SM exon inclusion with respect to total product is shown in graphical representation. The error bars indicate the standard error. (D) Schematic representation of the IR minigene. Exons are indicated as black boxes and alternatively spliced exons are indicated as gray boxes. Introns are shown with a central narrow line. The arrows show the primer sites. (E) Results of the transient transfection experiment. HeLa cells were transiently transfected with the IR minigene with/without the CUGBP, R3δ isoform, or Etr-1 expression vectors. Alternatively spliced products were analyzed by RT-PCR. (F) Densitometric analysis of the transfection products. Quantification of the alternatively spliced products was performed by densitometry. The percentage of exon 11 inclusion with respect to total product is shown in graphical representation. The error bars indicate the standard error.

Mentions: As described previously, CUGBP2 promotes the inclusion of the mutually exclusive SM exon instead of the NM exon in ACTN1 [11,12]. The specific function of the CUGBP2 isoform R3δ was examined by transient transfection using the mouse ACTN1 minigene (Figure 3A). Transfection of COS7 cells with the ACTN1 minigene alone resulted in the detection of a transcript including the NM exon as a major product and another transcript with the SM exon as a minor product (Figure 3B). Control COS7 cells lacking the minigene did not show these transcripts, confirming that the two transcripts containing the NM or SM exon were products of the transfected minigene. Co-transfection with a myc-tagged CUGBP2 expression vector resulted in a decrease in the NM exon product concomitant with an increase in the SM exon product (Figure 3B & Additional file 1: Figure S1). Use of the Etr-1 expression vector as a control showed that Etr-1 promoted the use of the NM exon. These results are consistent with previous reports [11].


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)

Transient transfection of the ACTN1 minigene and the IR minigene. (A) Schematic representation of the ACTN1 minigene. Exons are indicated as black boxes with alternatively spliced exons indicated as gray boxes. Introns are indicated with a central narrow line. The arrows show the primer sites. (B) Results of the transient transfection experiment. COS7 cells were transiently transfected with the ACTN1 minigene with/without the CUGBP, CUGBP2 R3δ isoform, or Etr-1 expression vectors. Alternatively spliced products were analyzed by RT-PCR. (C) Densitometric analysis of the transfection products. Quantification of the alternatively spliced products was performed by densitometry. The percentage of SM exon inclusion with respect to total product is shown in graphical representation. The error bars indicate the standard error. (D) Schematic representation of the IR minigene. Exons are indicated as black boxes and alternatively spliced exons are indicated as gray boxes. Introns are shown with a central narrow line. The arrows show the primer sites. (E) Results of the transient transfection experiment. HeLa cells were transiently transfected with the IR minigene with/without the CUGBP, R3δ isoform, or Etr-1 expression vectors. Alternatively spliced products were analyzed by RT-PCR. (F) Densitometric analysis of the transfection products. Quantification of the alternatively spliced products was performed by densitometry. The percentage of exon 11 inclusion with respect to total product is shown in graphical representation. The error bars indicate the standard error.
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Figure 3: Transient transfection of the ACTN1 minigene and the IR minigene. (A) Schematic representation of the ACTN1 minigene. Exons are indicated as black boxes with alternatively spliced exons indicated as gray boxes. Introns are indicated with a central narrow line. The arrows show the primer sites. (B) Results of the transient transfection experiment. COS7 cells were transiently transfected with the ACTN1 minigene with/without the CUGBP, CUGBP2 R3δ isoform, or Etr-1 expression vectors. Alternatively spliced products were analyzed by RT-PCR. (C) Densitometric analysis of the transfection products. Quantification of the alternatively spliced products was performed by densitometry. The percentage of SM exon inclusion with respect to total product is shown in graphical representation. The error bars indicate the standard error. (D) Schematic representation of the IR minigene. Exons are indicated as black boxes and alternatively spliced exons are indicated as gray boxes. Introns are shown with a central narrow line. The arrows show the primer sites. (E) Results of the transient transfection experiment. HeLa cells were transiently transfected with the IR minigene with/without the CUGBP, R3δ isoform, or Etr-1 expression vectors. Alternatively spliced products were analyzed by RT-PCR. (F) Densitometric analysis of the transfection products. Quantification of the alternatively spliced products was performed by densitometry. The percentage of exon 11 inclusion with respect to total product is shown in graphical representation. The error bars indicate the standard error.
Mentions: As described previously, CUGBP2 promotes the inclusion of the mutually exclusive SM exon instead of the NM exon in ACTN1 [11,12]. The specific function of the CUGBP2 isoform R3δ was examined by transient transfection using the mouse ACTN1 minigene (Figure 3A). Transfection of COS7 cells with the ACTN1 minigene alone resulted in the detection of a transcript including the NM exon as a major product and another transcript with the SM exon as a minor product (Figure 3B). Control COS7 cells lacking the minigene did not show these transcripts, confirming that the two transcripts containing the NM or SM exon were products of the transfected minigene. Co-transfection with a myc-tagged CUGBP2 expression vector resulted in a decrease in the NM exon product concomitant with an increase in the SM exon product (Figure 3B & Additional file 1: Figure S1). Use of the Etr-1 expression vector as a control showed that Etr-1 promoted the use of the NM exon. These results are consistent with previous reports [11].

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