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Structural basis for the dual RNA-recognition modes of human Tra2-β RRM.

Tsuda K, Someya T, Kuwasako K, Takahashi M, He F, Unzai S, Inoue M, Harada T, Watanabe S, Terada T, Kobayashi N, Shirouzu M, Kigawa T, Tanaka A, Sugano S, Güntert P, Yokoyama S, Muto Y - Nucleic Acids Res. (2010)

Bottom Line: We then solved the complex structure of the hTra2-β RRM with the (GAA)(2) sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the β-sheet surface.Further NMR experiments revealed that the hTra2-β RRM recognizes the CAA sequence when it is integrated in the stem-loop structure.This study indicates that the hTra2-β RRM recognizes two types of RNA sequences in different RNA binding modes.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Systems and Structural Biology Center, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.

ABSTRACT
Human Transformer2-β (hTra2-β) is an important member of the serine/arginine-rich protein family, and contains one RNA recognition motif (RRM). It controls the alternative splicing of several pre-mRNAs, including those of the calcitonin/calcitonin gene-related peptide (CGRP), the survival motor neuron 1 (SMN1) protein and the tau protein. Accordingly, the RRM of hTra2-β specifically binds to two types of RNA sequences [the CAA and (GAA)(2) sequences]. We determined the solution structure of the hTra2-β RRM (spanning residues Asn110-Thr201), which not only has a canonical RRM fold, but also an unusual alignment of the aromatic amino acids on the β-sheet surface. We then solved the complex structure of the hTra2-β RRM with the (GAA)(2) sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the β-sheet surface. Further NMR experiments revealed that the hTra2-β RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-β RRM recognizes two types of RNA sequences in different RNA binding modes.

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Comparison of ribbon representations of the hTra2-β RRM in complex with [5′-(GAAGAA)-3′] RNA (A); and the first RRM domain of poly pyrimidine-tract binding protein (PTBP RRM1) (PDBID: 2AD9) (B). In both structures, the N-terminal extension is colored cyan (the side-chain of the Pro residue in the N-terminal extension is depicted in blue), and the C-terminal extension is colored magenta (the side chain of Pro199 of the hTra2-β RRM, and the Leu residue of the PTBP RRM1 in the C-terminal extension are depicted in red). The side chain of the aromatic ring residue located at the end of the β4 strand and the side chains interacting with the residue in the C-terminal extension on the β-sheet surface are depicted in green. The RNA molecule is shown in gold.
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Figure 6: Comparison of ribbon representations of the hTra2-β RRM in complex with [5′-(GAAGAA)-3′] RNA (A); and the first RRM domain of poly pyrimidine-tract binding protein (PTBP RRM1) (PDBID: 2AD9) (B). In both structures, the N-terminal extension is colored cyan (the side-chain of the Pro residue in the N-terminal extension is depicted in blue), and the C-terminal extension is colored magenta (the side chain of Pro199 of the hTra2-β RRM, and the Leu residue of the PTBP RRM1 in the C-terminal extension are depicted in red). The side chain of the aromatic ring residue located at the end of the β4 strand and the side chains interacting with the residue in the C-terminal extension on the β-sheet surface are depicted in green. The RNA molecule is shown in gold.

Mentions: When an RRM binds to the target RNA molecule, other regions besides the core RRM body are involved in the RNA recognition in several cases, such as the C-terminal extension for Fox-1 (40) and U1A (41), the N-terminal extension for the CUG-BP1 RRM3 (32), and both the N- and C-terminal extensions for the hnRNP A1 RRM1 (42) and the RRM1 of poly pyrimidine-tract binding protein (the PTBP RRM1) (43). Structural similarity was especially observed in both the N- and C-terminal extensions between the PTBP RRM1 and the hTra2-β RRM, when they were bound to their target RNAs (Figure 6B). In the PTBP RRM1, even without an RNA molecule, the C-terminal extension is fixed on and traverses the β-sheet surface of the core RRM body, because the hydrophobic aliphatic amino acid residue (Leu) in the C-terminal extension interacts with the hydrophobic residues on the β-sheet surface (Figures 1B and 8B). When the target RNA molecule is located on the β-sheet surface, several carboxyl oxygens and/or amide protons of the C-terminal extension are involved in the recognition of the poly-pyrimidine tracts. On the other hand, in the case of the hTra2-β RRM, there are no hydrophobic aliphatic amino acid residues on the C-terminal extension, corresponding to the above-mentioned Leu residue of the PTBP RRM1, for the interaction with the amino acid residues on the β-sheet surface. Therefore, without the RNA molecules, the C-terminal extension is rather flexible and does not closely contact the β-sheet surface. However, in the presence of the RNA molecule, Pro199 in the C-terminal extension forms a van der Waals contact with the aromatic ring of Tyr165 on the β-sheet surface (Figure 6A), and the C-terminal extension adopts a similar conformation as seen in the PTBP RRM1 for the recognition of the base moieties of the RNA molecule (Figure 6A and B).Figure 6.


Structural basis for the dual RNA-recognition modes of human Tra2-β RRM.

Tsuda K, Someya T, Kuwasako K, Takahashi M, He F, Unzai S, Inoue M, Harada T, Watanabe S, Terada T, Kobayashi N, Shirouzu M, Kigawa T, Tanaka A, Sugano S, Güntert P, Yokoyama S, Muto Y - Nucleic Acids Res. (2010)

Comparison of ribbon representations of the hTra2-β RRM in complex with [5′-(GAAGAA)-3′] RNA (A); and the first RRM domain of poly pyrimidine-tract binding protein (PTBP RRM1) (PDBID: 2AD9) (B). In both structures, the N-terminal extension is colored cyan (the side-chain of the Pro residue in the N-terminal extension is depicted in blue), and the C-terminal extension is colored magenta (the side chain of Pro199 of the hTra2-β RRM, and the Leu residue of the PTBP RRM1 in the C-terminal extension are depicted in red). The side chain of the aromatic ring residue located at the end of the β4 strand and the side chains interacting with the residue in the C-terminal extension on the β-sheet surface are depicted in green. The RNA molecule is shown in gold.
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Figure 6: Comparison of ribbon representations of the hTra2-β RRM in complex with [5′-(GAAGAA)-3′] RNA (A); and the first RRM domain of poly pyrimidine-tract binding protein (PTBP RRM1) (PDBID: 2AD9) (B). In both structures, the N-terminal extension is colored cyan (the side-chain of the Pro residue in the N-terminal extension is depicted in blue), and the C-terminal extension is colored magenta (the side chain of Pro199 of the hTra2-β RRM, and the Leu residue of the PTBP RRM1 in the C-terminal extension are depicted in red). The side chain of the aromatic ring residue located at the end of the β4 strand and the side chains interacting with the residue in the C-terminal extension on the β-sheet surface are depicted in green. The RNA molecule is shown in gold.
Mentions: When an RRM binds to the target RNA molecule, other regions besides the core RRM body are involved in the RNA recognition in several cases, such as the C-terminal extension for Fox-1 (40) and U1A (41), the N-terminal extension for the CUG-BP1 RRM3 (32), and both the N- and C-terminal extensions for the hnRNP A1 RRM1 (42) and the RRM1 of poly pyrimidine-tract binding protein (the PTBP RRM1) (43). Structural similarity was especially observed in both the N- and C-terminal extensions between the PTBP RRM1 and the hTra2-β RRM, when they were bound to their target RNAs (Figure 6B). In the PTBP RRM1, even without an RNA molecule, the C-terminal extension is fixed on and traverses the β-sheet surface of the core RRM body, because the hydrophobic aliphatic amino acid residue (Leu) in the C-terminal extension interacts with the hydrophobic residues on the β-sheet surface (Figures 1B and 8B). When the target RNA molecule is located on the β-sheet surface, several carboxyl oxygens and/or amide protons of the C-terminal extension are involved in the recognition of the poly-pyrimidine tracts. On the other hand, in the case of the hTra2-β RRM, there are no hydrophobic aliphatic amino acid residues on the C-terminal extension, corresponding to the above-mentioned Leu residue of the PTBP RRM1, for the interaction with the amino acid residues on the β-sheet surface. Therefore, without the RNA molecules, the C-terminal extension is rather flexible and does not closely contact the β-sheet surface. However, in the presence of the RNA molecule, Pro199 in the C-terminal extension forms a van der Waals contact with the aromatic ring of Tyr165 on the β-sheet surface (Figure 6A), and the C-terminal extension adopts a similar conformation as seen in the PTBP RRM1 for the recognition of the base moieties of the RNA molecule (Figure 6A and B).Figure 6.

Bottom Line: We then solved the complex structure of the hTra2-β RRM with the (GAA)(2) sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the β-sheet surface.Further NMR experiments revealed that the hTra2-β RRM recognizes the CAA sequence when it is integrated in the stem-loop structure.This study indicates that the hTra2-β RRM recognizes two types of RNA sequences in different RNA binding modes.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Systems and Structural Biology Center, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.

ABSTRACT
Human Transformer2-β (hTra2-β) is an important member of the serine/arginine-rich protein family, and contains one RNA recognition motif (RRM). It controls the alternative splicing of several pre-mRNAs, including those of the calcitonin/calcitonin gene-related peptide (CGRP), the survival motor neuron 1 (SMN1) protein and the tau protein. Accordingly, the RRM of hTra2-β specifically binds to two types of RNA sequences [the CAA and (GAA)(2) sequences]. We determined the solution structure of the hTra2-β RRM (spanning residues Asn110-Thr201), which not only has a canonical RRM fold, but also an unusual alignment of the aromatic amino acids on the β-sheet surface. We then solved the complex structure of the hTra2-β RRM with the (GAA)(2) sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the β-sheet surface. Further NMR experiments revealed that the hTra2-β RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-β RRM recognizes two types of RNA sequences in different RNA binding modes.

Show MeSH