Crystal structure and functional characterization of the human RBM25 PWI domain and its flanking basic region
| Autor: | Zhenhua Shao, Fan Yang, Jihui Wu, Fudong Li, Dandan Qian, Minhao Wu, Yunyu Shi, Deshun Gong, Mian Wu |
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| Rok vydání: | 2013 |
| Předmět: |
Models
Molecular ssRNA single-stranded RNA RNA-binding protein Biochemistry Luc7AC C-terminal region of Luc7A Protein structure GST glutathione transferase Protein Isoforms GFP green fluorescent protein Genetics dsDNA double-stranded DNA flanking basic region RT reverse transcription Nuclear Proteins RNA-Binding Proteins NF nucleic-acid-free IPTG isopropyl β-D-thiogalactopyranoside RNA splicing SR serine- and arginine-rich Research Article Binding domain SeMet selenomethionine RBM25 RNA-binding motif protein 25 rmsd root mean square deviation RNA Splicing Molecular Sequence Data bcl-X Protein Luc7A Computational biology Biology ss splicing site alternative splicing HEK human embryonic kidney Splicing factor FPA fluorescence polarization assay ssDNA single-stranded DNA Humans Amino Acid Sequence Binding site Molecular Biology Binding Sites NP40 Nonidet P40 Alternative splicing snRNP small nuclear ribonucleoprotein RNA Bcl-x pre-mRNA PWI domain Cell Biology RRM RNA-recognition motif WT wild-type Protein Structure Tertiary HEK293 Cells RNA-binding protein motif 25 (RBM25) dsRNA double-stranded RNA SAD single-wavelength anomalous dispersion FAM 6-carboxyfluorescein |
| Zdroj: | Biochemical Journal |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj20121382 |
| Popis: | Human RBM25 (RNA-binding motif protein 25) is a novel splicing factor that contains a PWI domain, a newly identified RNA/DNA-binding domain, and regulates Bcl-x pre-mRNA alternative splicing. The flanking basic region has been suggested to serve as a co-operative partner of the PWI domain in the binding of nucleic acids, but the structure of this basic region is unknown. In the present paper, we report the crystal structure of the RBM25 PWI domain and its flanking basic region. The PWI domain is revealed to comprise a conserved four-helix bundle, and the flanking basic region forms two α-helices and associates with helix H4 of the PWI domain. These interactions promote directly the formation of an enlarged nucleic-acid-binding platform. Structure-guided mutagenesis reveals a positively charged nucleic-acid-binding surface in the RBM25 PWI domain that is entirely different from that in the SRm160 PWI domain. Furthermore, we show that the promotion of the pro-apoptotic Bcl-xS isoform expression by RBM25 is facilitated by the PWI domain in vivo. Thus the present study suggests that the PWI domain plays an important role in the regulation of Bcl-x pre-mRNA alternative splicing. |
| Databáze: | OpenAIRE |
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