Limits...
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.

Show MeSH
NMR chemical shift perturbations of the hTra2-β RRM upon RNA binding (protein:RNA ratio = 1:1). (A) [5′-(GAAGAA)-3′], (B) [5′-(AAAAAA)-3′], (C) [5′-(UCAAU)-3′] and (D) [5′-(GACUUCAACAAGUC)-3′]. Left panels: Plot of the magnitudes of the chemical shift change on the vertical axis, and the amino acid residue numbers on the horizontal axis. Right panels: Chemical shift perturbations mapped on the tertiary structure of the hTra2-β RRM. The chemical shift perturbation values were obtained from the [1H, 15N]-HSQC spectrum. The absolute values of the chemical shift change Δδ were calculated as follows: Δδ = [(Δδ15N/6.5)2+Δδ1H2]1/2. Amino acid residues with significant chemical shift changes are labeled. Residues with resonances that could not be assigned after the addition of the RNA are indicated by asterisks.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3045587&req=5

Figure 4: NMR chemical shift perturbations of the hTra2-β RRM upon RNA binding (protein:RNA ratio = 1:1). (A) [5′-(GAAGAA)-3′], (B) [5′-(AAAAAA)-3′], (C) [5′-(UCAAU)-3′] and (D) [5′-(GACUUCAACAAGUC)-3′]. Left panels: Plot of the magnitudes of the chemical shift change on the vertical axis, and the amino acid residue numbers on the horizontal axis. Right panels: Chemical shift perturbations mapped on the tertiary structure of the hTra2-β RRM. The chemical shift perturbation values were obtained from the [1H, 15N]-HSQC spectrum. The absolute values of the chemical shift change Δδ were calculated as follows: Δδ = [(Δδ15N/6.5)2+Δδ1H2]1/2. Amino acid residues with significant chemical shift changes are labeled. Residues with resonances that could not be assigned after the addition of the RNA are indicated by asterisks.

Mentions: As described above, the SELEX experiment indicated that the (GAA)2 sequence could bind to the hTra2-β RRM-(Arg111–Thr201). Accordingly, upon the addition of [5′-(GAAGAA)-3′] to the hTra2-β RRM-(Arg111–Thr201), some of the main-chain 1H–15N resonances of the free form gradually disappeared, and correspondingly new resonances of the bound form appeared in the 2D [1H, 15N]-HSQC spectra (Figure 3A). This indicated that the exchange between the RNA-bound and RNA-free forms is in the intermediate to slow regime on the NMR time scale. A more detailed analysis of the resonances that shifted upon the addition of [5′-(GAAGAA)-3′] revealed that the amino acids on the β-sheet surface of the core RRM body of hTra2-β (especially, Gly124 at the end of the β1 strand and Ile189, Arg190 and Val191 on the β4 strand) were obviously shifted (Figure 4A). Intriguingly, the resonances originating from the N-terminal extension (Ala112, Asn113, Asn117, Cys118 and Cys119) and the C-terminal extension (Ser194, Ile195, Thr196, Lys197 and Arg198) were also strongly affected (Figure 4A), indicating that both the N- and C-terminal extensions contributed to the interaction with [5′-(GAAGAA)-3′]. Based on the ITC experiment, we estimated a Kd value of 1.5 µM for the complex with the [5′-(GAAGAA)-3′] sequence (Supplementary Figure S1A). This clearly indicated that the hTra2-β RRM binds to [5′-(GAAGAA)-3′] tightly enough for the complex structure determination. After considering the possibility that the Arg111 residue in the N-terminal extension may also be involved in the RNA binding, we prepared the protein sample for the region spanning Asn110–Thr201 (the hTra2-β RRM) to analyze the RNA binding activity of hTra2-β.Figure 3.


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)

NMR chemical shift perturbations of the hTra2-β RRM upon RNA binding (protein:RNA ratio = 1:1). (A) [5′-(GAAGAA)-3′], (B) [5′-(AAAAAA)-3′], (C) [5′-(UCAAU)-3′] and (D) [5′-(GACUUCAACAAGUC)-3′]. Left panels: Plot of the magnitudes of the chemical shift change on the vertical axis, and the amino acid residue numbers on the horizontal axis. Right panels: Chemical shift perturbations mapped on the tertiary structure of the hTra2-β RRM. The chemical shift perturbation values were obtained from the [1H, 15N]-HSQC spectrum. The absolute values of the chemical shift change Δδ were calculated as follows: Δδ = [(Δδ15N/6.5)2+Δδ1H2]1/2. Amino acid residues with significant chemical shift changes are labeled. Residues with resonances that could not be assigned after the addition of the RNA are indicated by asterisks.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045587&req=5

Figure 4: NMR chemical shift perturbations of the hTra2-β RRM upon RNA binding (protein:RNA ratio = 1:1). (A) [5′-(GAAGAA)-3′], (B) [5′-(AAAAAA)-3′], (C) [5′-(UCAAU)-3′] and (D) [5′-(GACUUCAACAAGUC)-3′]. Left panels: Plot of the magnitudes of the chemical shift change on the vertical axis, and the amino acid residue numbers on the horizontal axis. Right panels: Chemical shift perturbations mapped on the tertiary structure of the hTra2-β RRM. The chemical shift perturbation values were obtained from the [1H, 15N]-HSQC spectrum. The absolute values of the chemical shift change Δδ were calculated as follows: Δδ = [(Δδ15N/6.5)2+Δδ1H2]1/2. Amino acid residues with significant chemical shift changes are labeled. Residues with resonances that could not be assigned after the addition of the RNA are indicated by asterisks.
Mentions: As described above, the SELEX experiment indicated that the (GAA)2 sequence could bind to the hTra2-β RRM-(Arg111–Thr201). Accordingly, upon the addition of [5′-(GAAGAA)-3′] to the hTra2-β RRM-(Arg111–Thr201), some of the main-chain 1H–15N resonances of the free form gradually disappeared, and correspondingly new resonances of the bound form appeared in the 2D [1H, 15N]-HSQC spectra (Figure 3A). This indicated that the exchange between the RNA-bound and RNA-free forms is in the intermediate to slow regime on the NMR time scale. A more detailed analysis of the resonances that shifted upon the addition of [5′-(GAAGAA)-3′] revealed that the amino acids on the β-sheet surface of the core RRM body of hTra2-β (especially, Gly124 at the end of the β1 strand and Ile189, Arg190 and Val191 on the β4 strand) were obviously shifted (Figure 4A). Intriguingly, the resonances originating from the N-terminal extension (Ala112, Asn113, Asn117, Cys118 and Cys119) and the C-terminal extension (Ser194, Ile195, Thr196, Lys197 and Arg198) were also strongly affected (Figure 4A), indicating that both the N- and C-terminal extensions contributed to the interaction with [5′-(GAAGAA)-3′]. Based on the ITC experiment, we estimated a Kd value of 1.5 µM for the complex with the [5′-(GAAGAA)-3′] sequence (Supplementary Figure S1A). This clearly indicated that the hTra2-β RRM binds to [5′-(GAAGAA)-3′] tightly enough for the complex structure determination. After considering the possibility that the Arg111 residue in the N-terminal extension may also be involved in the RNA binding, we prepared the protein sample for the region spanning Asn110–Thr201 (the hTra2-β RRM) to analyze the RNA binding activity of hTra2-β.Figure 3.

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