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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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Structure transitions of the CUGBP2 and R3δ isoforms. (A) Initial structure of the CUGBP2 RRM3. The structure was predicted using CUGBP1 RRM3 (2rq4a) because the amino acid sequences of RRM3 of CUGBP1 and 2 are almost identical. (B) MD simulation of the CUGBP2 RRM3. MD simulation was performed for 5 ns. (C) Initial structure of the R3δ isoform RRM3. The RRM3 and the linker residues of the R3δ isoform were analyzed by comparative modeling by SFAS in PBDj. (D) MD simulation of the R3δ isoform. The result of comparative modeling was used for MD simulation for 5 ns to analyze the folding and stability of the predicted structure.
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Figure 4: Structure transitions of the CUGBP2 and R3δ isoforms. (A) Initial structure of the CUGBP2 RRM3. The structure was predicted using CUGBP1 RRM3 (2rq4a) because the amino acid sequences of RRM3 of CUGBP1 and 2 are almost identical. (B) MD simulation of the CUGBP2 RRM3. MD simulation was performed for 5 ns. (C) Initial structure of the R3δ isoform RRM3. The RRM3 and the linker residues of the R3δ isoform were analyzed by comparative modeling by SFAS in PBDj. (D) MD simulation of the R3δ isoform. The result of comparative modeling was used for MD simulation for 5 ns to analyze the folding and stability of the predicted structure.

Mentions: There-dimensional structures of the RRM3 isoforms were examined by MD simulation and NMR spectroscopy. The structural analysis of the RRM3 of CUGBP1 has been reported previously [22]. Several unaligned residues were found between the RRM3s of CUGBP1, and 2. The results of homology modeling showed that the third domain of CUGBP2 formed an RRM structure (Figure 4A). No significant difference could be observed between the MD simulations of RRM3 of CUGBP1 and RRM3 of CUGBP2. (data not shown). Further, CUGBP2 RRM3 maintained an RRM structure for 5 ns in the MD simulation (Figure 4B). The 1H-15N HSQC spectrum of the RRM3 of CUGBP2 in the absence of RNA showed that, unlike RRM3 of CUGBP1, the resonances of the N-terminal residues in the linker domain were concentrated in the center of the spectrum, suggesting a random coil structure. This result was probably due to the inclusion of a long N-terminal sequence in the present constructs, which forms a flexible linker region connecting RRM2 with RRM3 (Figure 5A).


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)

Structure transitions of the CUGBP2 and R3δ isoforms. (A) Initial structure of the CUGBP2 RRM3. The structure was predicted using CUGBP1 RRM3 (2rq4a) because the amino acid sequences of RRM3 of CUGBP1 and 2 are almost identical. (B) MD simulation of the CUGBP2 RRM3. MD simulation was performed for 5 ns. (C) Initial structure of the R3δ isoform RRM3. The RRM3 and the linker residues of the R3δ isoform were analyzed by comparative modeling by SFAS in PBDj. (D) MD simulation of the R3δ isoform. The result of comparative modeling was used for MD simulation for 5 ns to analyze the folding and stability of the predicted structure.
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Related In: Results  -  Collection

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Figure 4: Structure transitions of the CUGBP2 and R3δ isoforms. (A) Initial structure of the CUGBP2 RRM3. The structure was predicted using CUGBP1 RRM3 (2rq4a) because the amino acid sequences of RRM3 of CUGBP1 and 2 are almost identical. (B) MD simulation of the CUGBP2 RRM3. MD simulation was performed for 5 ns. (C) Initial structure of the R3δ isoform RRM3. The RRM3 and the linker residues of the R3δ isoform were analyzed by comparative modeling by SFAS in PBDj. (D) MD simulation of the R3δ isoform. The result of comparative modeling was used for MD simulation for 5 ns to analyze the folding and stability of the predicted structure.
Mentions: There-dimensional structures of the RRM3 isoforms were examined by MD simulation and NMR spectroscopy. The structural analysis of the RRM3 of CUGBP1 has been reported previously [22]. Several unaligned residues were found between the RRM3s of CUGBP1, and 2. The results of homology modeling showed that the third domain of CUGBP2 formed an RRM structure (Figure 4A). No significant difference could be observed between the MD simulations of RRM3 of CUGBP1 and RRM3 of CUGBP2. (data not shown). Further, CUGBP2 RRM3 maintained an RRM structure for 5 ns in the MD simulation (Figure 4B). The 1H-15N HSQC spectrum of the RRM3 of CUGBP2 in the absence of RNA showed that, unlike RRM3 of CUGBP1, the resonances of the N-terminal residues in the linker domain were concentrated in the center of the spectrum, suggesting a random coil structure. This result was probably due to the inclusion of a long N-terminal sequence in the present constructs, which forms a flexible linker region connecting RRM2 with RRM3 (Figure 5A).

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