Structural and Functional Studies of Spliceosome Assembly and Activation

点击翻译按钮获取中文摘要

Pre-messenger RNAs (pre-mRNAs) in eukaryotes are characterized by a split-gene structure where coding exon sequences are separated by non-coding intron sequences. The process by which introns are excised and exons are joined together is known as pre-mRNA splicing and is catalyzed by an RNA-protein complex, the spliceosome, that is highly conserved from yeast to humans. While the faithful execution of constitutive splicing events is required for the maturation of functional mRNAs, alternative splicing events represent a fundamental level of gene regulation in terms of differentiation and development in higher eukaryotic cells and in the life cycle of viruses.******The spliceosome consists of the U1, U2, and U4/U5/U6 snRNPs (small nuclear ribonucleoprotein particles) each containing a unique RNA and associated proteins which assemble on pre-mRNA substrates in an ordered fashion. Although the overall assembly pathway is known, the molecular mechanisms which govern spliceosome assembly and activation remain poorly understood. We are utilizing a variety of biochemical, chemical, and biophysical techniques to study the individual steps of spliceosome assembly as well as transitions in the mature spliceosome. ******The current focus of our studies is based on the characterization of two critical protein components of the spliceosome SF3b14 (p14) and PRP8, both of which recognize key reactive sequences within the pre-mRNA and are critical for spliceosome assembly and regulating the catalytic activity within the spliceosome. These proteins directly contact the pre-mRNA substrate and spliceosomal RNAs recruiting or forming the core of the human spliceosome. We have solved the high-resolution X-ray structures of both p14 and a key domain of PRP8 (the RNase H or RH domain) and investigated both of these factors using biochemistry, yeast genetics, and cell biology. Our structures in turn suggest models for the function of these proteins that can be tested by further experimentation. Our proposed studies involve four sets of experiments: (1) biochemical/structural investigation of p14•RNA interactions (2) characterization in cells and cellular extract of the role of p14 in splicing; (3) assessment of a local conformational switch in the PRP8-RH domain that unmasks a critical metal-binding site; (4) investigation of large-scale conformational change in PRP8 by structure-based mutagenesis.******These studies will provide novel insights into fundamental aspects of an essential gene regulatory pathway — pre-mRNA splicing — including mechanisms of spliceosome assembly and the nature and requirements of spliceosomal rearrangements during the chemical steps of splicing. They will also expand our knowledge of protein and RNA structure and RNA•protein interaction within the cell which are critical in diverse processes ranging from transcription through RNA processing itself to translation.