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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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Solution structure of the hTra2-β RRM in the RNA-free form. (A) Superposition of the 20 conformers of the hTra2-β RRM (Arg111–Thr201) for the best fit of the backbone atoms of residues Leu120–Phe193. The N-terminal extension (Arg111–Cys119) and the C-terminal extension (Ser194–Thr201) are colored cyan and magenta, respectively. (B) Ribbon representation of the hTra2-β RRM. The β-strands and α-helices are colored green and pink, respectively. The side chains of Phe123, Val150, Phe161, Phe163 and Tyr165 are shown in green; and the side chains of Arg157, 159, 187, 188 and 190 are shown in blue. (C) Electrostatic potential surface of the hTra2-β RRM. Positively and negatively charged regions are colored blue and red, respectively. All of the structural representations were prepared with the software MOLMOL (35).
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Figure 2: Solution structure of the hTra2-β RRM in the RNA-free form. (A) Superposition of the 20 conformers of the hTra2-β RRM (Arg111–Thr201) for the best fit of the backbone atoms of residues Leu120–Phe193. The N-terminal extension (Arg111–Cys119) and the C-terminal extension (Ser194–Thr201) are colored cyan and magenta, respectively. (B) Ribbon representation of the hTra2-β RRM. The β-strands and α-helices are colored green and pink, respectively. The side chains of Phe123, Val150, Phe161, Phe163 and Tyr165 are shown in green; and the side chains of Arg157, 159, 187, 188 and 190 are shown in blue. (C) Electrostatic potential surface of the hTra2-β RRM. Positively and negatively charged regions are colored blue and red, respectively. All of the structural representations were prepared with the software MOLMOL (35).

Mentions: We determined the solution structure of the region spanning Arg111–Thr201 in the hTra2-β protein, which contained the RRM domain [the hTra2-β RRM- (Arg111–Thr201)], by NMR experiments. In total, 1257 NOE distance restraints derived from 3D 15N-edited [1H, 1H]-NOESY and 13C-edited [1H, 1H]-NOESY spectra were assigned and used in the structure calculation, together with 37 dihedral angle restraints (Table 1). The 40 structures obtained from the CYANA calculation were further refined with the AMBER program, resulting in the 20 energy-refined conformers that represent the solution structure of the hTra2-β RRM (Figure 2A). The region spanning Leu120–Phe193 is the core RRM body that adopts the canonical RRM fold, consisting of a four-stranded anti-parallel β-sheet and two α-helices with a βαββαβ topology (β1: Leu120–Phe123, α1: Glu131–Phe138, β2: Asp146–Val150, β3: Phe161–Phe166, α2: Val169–Ala179 and β4: Arg190–Phe193, respectively) (Figures 1B and 2B). In addition, the hTra2-β RRM has an additional β-hairpin, β3′–β3″ (residues: Glu183–Arg188), between helix α2 and strand β4 (Figures 1B and 2B), and a 10-residue loop structure between the β2 and β3 strands (the β2–β3 loop).Table 1.


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)

Solution structure of the hTra2-β RRM in the RNA-free form. (A) Superposition of the 20 conformers of the hTra2-β RRM (Arg111–Thr201) for the best fit of the backbone atoms of residues Leu120–Phe193. The N-terminal extension (Arg111–Cys119) and the C-terminal extension (Ser194–Thr201) are colored cyan and magenta, respectively. (B) Ribbon representation of the hTra2-β RRM. The β-strands and α-helices are colored green and pink, respectively. The side chains of Phe123, Val150, Phe161, Phe163 and Tyr165 are shown in green; and the side chains of Arg157, 159, 187, 188 and 190 are shown in blue. (C) Electrostatic potential surface of the hTra2-β RRM. Positively and negatively charged regions are colored blue and red, respectively. All of the structural representations were prepared with the software MOLMOL (35).
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Figure 2: Solution structure of the hTra2-β RRM in the RNA-free form. (A) Superposition of the 20 conformers of the hTra2-β RRM (Arg111–Thr201) for the best fit of the backbone atoms of residues Leu120–Phe193. The N-terminal extension (Arg111–Cys119) and the C-terminal extension (Ser194–Thr201) are colored cyan and magenta, respectively. (B) Ribbon representation of the hTra2-β RRM. The β-strands and α-helices are colored green and pink, respectively. The side chains of Phe123, Val150, Phe161, Phe163 and Tyr165 are shown in green; and the side chains of Arg157, 159, 187, 188 and 190 are shown in blue. (C) Electrostatic potential surface of the hTra2-β RRM. Positively and negatively charged regions are colored blue and red, respectively. All of the structural representations were prepared with the software MOLMOL (35).
Mentions: We determined the solution structure of the region spanning Arg111–Thr201 in the hTra2-β protein, which contained the RRM domain [the hTra2-β RRM- (Arg111–Thr201)], by NMR experiments. In total, 1257 NOE distance restraints derived from 3D 15N-edited [1H, 1H]-NOESY and 13C-edited [1H, 1H]-NOESY spectra were assigned and used in the structure calculation, together with 37 dihedral angle restraints (Table 1). The 40 structures obtained from the CYANA calculation were further refined with the AMBER program, resulting in the 20 energy-refined conformers that represent the solution structure of the hTra2-β RRM (Figure 2A). The region spanning Leu120–Phe193 is the core RRM body that adopts the canonical RRM fold, consisting of a four-stranded anti-parallel β-sheet and two α-helices with a βαββαβ topology (β1: Leu120–Phe123, α1: Glu131–Phe138, β2: Asp146–Val150, β3: Phe161–Phe166, α2: Val169–Ala179 and β4: Arg190–Phe193, respectively) (Figures 1B and 2B). In addition, the hTra2-β RRM has an additional β-hairpin, β3′–β3″ (residues: Glu183–Arg188), between helix α2 and strand β4 (Figures 1B and 2B), and a 10-residue loop structure between the β2 and β3 strands (the β2–β3 loop).Table 1.

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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