Structure and Function of the Human RBM25PWI Domain and Its Flanking Basic Region
|School||University of Science and Technology of China|
|Course||Biochemistry and Molecular Biology|
|Keywords||RBM25 Luc7A PWI domain the flanking basic region alternativesplicing Bcl-x pre-mRNA RNA-binding domain SR protein U1snRNP splicing site|
Human RBM25is a novel splicing factor that can regulate Bcl-x pre-mRNA alternative splicing. Bcl-x pre-mRNA contains three exons:the exon1and3are constitutive exon, while the exon2is an alternative exon. Increased RBM25expression correlates with increased Bcl-xS isoform expression. It was reported that RBM25can bind specifically to the exon splicing enchancer CGGGCA that located in the exon2. In addition, RBM25can interact with Luc7A both in vivo and in vitro, which is a U1snRNP-associated factor. Interestingly, there are no direct interactions between RBM25with the U1snRNP specific proteins (Sm, U1A, U1C, and U1-70K). The mechanism of the activation of Bcl-xS5’splicing site is:RBM25specifically binds to the CGGGCA sequence and facilitates the recruitment of Ul snRNP to the Bcl-xS5’splicing site via the interactions with U1sn RNP--ssociated factor Luc7A. RBM25contains a PWI domain, which is a newly identified RNA-binding domain. It was reported that the PWI domain has no preference for binding to single-and double-stranded nucleic acids, but litter is known about its function except that the SRm160PWI domain can facilitate the stimulation of3’-end formation by SRm160. PWI domain has a flanking basic region that contains about ten residues, the flanking basic region acts as an essential partner of the PWI domain in the binding of nucleic acids, the PWI domain without the flanking basic region is insufficient for binding nucleic acids. There are only two solved structures of the PWI domains in the Protein Data Bank before our work:the Prp3PWI domain (PDB entry1X4Q) and the SRm160PWI domain (PDB entry1MP1). Both structures reveal that the PWI domain is mainly composed of a conserved four-helix bundle, but the flanking basic regions were absent in these two structures.To investigate whether the RBM25PWI domain plays a role in the regulation of Bcl-x pre-mRNA alternative splicing by RBM25, and whether the flanking basic region has a secondary structure, and how the flanking basic region facilitates the binding of the PWI domain for nucleic acids, and how the PWI domain interacts with nucleic acids, we study the structure and function of the RBM25PWI domain and its flanking basic region. In our work, we solved the crystal structure of the RBM25PWI domain and its flanking basic region with the resolution at2.9A. We report for the first time the structure of the flanking basic region of PWI domain, which forms two a-helices and associates with the core helix H4of the PWI domain, these inteactions directly promote the accumulation of the basic residues in the PWI domain and in the flanking basic region, accordingly, an enlarged nucleic acid-binding surface is formed. We found the important contact residues both in the PWI domain and in the flanking basic region for nucleic acid-binding, which reveal that the nucleic acid-binding surface of the RBM25PWI differs entirely from the SRm160PWI domain. These difference indicate that the PWI domain adopts highly plastic binding surface for nucleic acid-binding. The results of RT-PCR assays indicate that the PWI domain plays an essential role in the regulation of Bcl-x pre-mRNA alternative splicing by RBM25. Interestingly, we found that some RNA molecules can act as a bridge between the PWI domain and Luc7AC (C-terminal region of Luc7A), but whether these complexs play a functional role in the regulation of Bcl-x pre-mRNA alternative splicing by RBM25is still unknown, some further research will be needed to investigate the function.The dissertation is comprised of three chapters:in the first chapter, we will introduce the backgroud about our research; in the second chapter, we will introduce the materials and methods; in the last chapter, we will introduce the results and discussion.