Splicing at Single Molecule Resolution:Pre-MRNA Dynamics throughout Spliceosome Assembly and Catalysis.

Pre-mRNA Splicing;Single Molecule;FRET;Molecular;Cellular and Developmental Biology;Science;Cellular & Molecular Biology

Blanco, Mario ReynaldoMeyhofer, Edgar ;

University of Michigan

关键词: Pre-mRNA Splicing;    Single Molecule;    FRET;    Molecular;    Cellular and Developmental Biology;    Science;    Cellular & Molecular Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/99864/mrblanc_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The spliceosome is a multi-megadalton RNA-protein complex that catalyzes the removal of introns and the ligation of exons during pre-mRNA splicing. In humans, approximately 95% of all pre-mRNAs undergo alternative splicing, which allows for the dynamic expression of various protein isoforms from a single gene through cell- and tissue-specific networks of regulated splicing events. Catalytic activation of the spliceosome involves an intricate set of RNA:RNA and RNA:protein interactions which must be carefully coordinated at various points of assembly. The pre-mRNA substrate is an integral component of the catalytically competent spliceosome. It serves as both a scaffold for assembly and provides the reactive sites for chemistry. Despite 25 years of study, questions about the specific conformational rearrangements and particularly their kinetics leading to catalysis remain unanswered. We have developed single molecule fluorescence resonance energy transfer (smFRET) assays that have begun to dissect pre-mRNA conformational changes during splicing. These assays allow us to track the relative position of conserved splice sites in real-time throughout spliceosome assembly and catalysis. We observed a series of reversible conformational states that were dependent on bona fide splicing signals and the presence of ATP. Furthermore, we dissected the sequence of conformational changes that lead to the first step of catalysis. Assigning pre-mRNA conformations to specific assembly events required the development of novel analysis methods that have broad applicability to the rest of the single molecule field. Finally, we utilized recently developed single molecule immunopurification techniques to help isolate a pre-catalytic spliceosomal complex. We then tracked the dynamics of this complex as it was chased through the first step of catalysis. We determined that, much like the ribosome, trans-acting factors influence catalysis by acting on the intrinsic dynamics of the spliceosome. By establishing smFRET as a viable tool for the investigation of splicing we have opened the field to new experimental possibilities. The results from these studies are already challenging some canonical assumptions, providing evidence for others, as well as providing new avenues of investigation.