Understanding Pre-mRNA Dynamics in Single Spliceosome Complexes.

Spliceosome;Pre-mRNA Splicing;Single Molecule FRET;Conformational Dynamics;Biological Chemistry;Chemistry;Molecular;Cellular and Developmental Biology;Science (General);Science;Chemistry

Krishnan, RamyaFierke, Carol A. ;

University of Michigan

关键词: Spliceosome;    Pre-mRNA Splicing;    Single Molecule FRET;    Conformational Dynamics;    Biological Chemistry;    Chemistry;    Molecular;    Cellular and Developmental Biology;    Science (General);    Science;    Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/102364/ramyak_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 large RNA-protein complex that catalyzes pre-mRNA splicing by removing sequences called introns and joining of the remaining sequences, called exons, to produce a mature mRNA. Despite over a quarter century of research on splicing, little is known about the compositional and conformational rearrangements, timing, and coordination of this process. Particularly, the pre-mRNA, which provides the template for spliceosome assembly and the reactive sites for splicing chemistry, has been largely ignored. This thesis utilizes single molecule fluorescence resonance energy transfer (smFRET) approaches on a short budding yeast pre-mRNA to address this challenge. Specifically, we have placed fluorescent dyes near the conserved splice sites that are recognized by the spliceosome and have monitored dynamic changes in the distance between these sites, in real-time throughout the splicing cycle.We find that, contrary to conventional depictions, the splice sites are highly dynamic and explore reversible transitions. By stalling the progress of splicing at various steps, we have been able to associate unique dynamic information of the splice sites with specific steps of splicing using novel analysis methods that have broad applicability. We find that the splice sites explore reversible splice site proximity in a non-monotonic fashion throughout the process. Our results show that even at very early steps of splicing assembly, the splice sites are brought close together via a novel ATP-independent role of a helicase. Furthermore, employing a combination of smFRET and affinity purification (termed SiMPull-FRET), we have been able to describe the conformational dynamics of single isolated spliceosomes and find them to follow a biased Brownian ratcheting mechanism leading up to the first chemical step of splicing. Our results hint at the possibility that, much like the ribosome, the spliceosome and its substrates often toggle between active and inactive conformations that are subsequently locked into the preferred state by a specific cofactor. The work presented in this thesis provides a structural and dynamic view of the pre-mRNA in the spliceosome, finds associated roles for protein factors, and pioneers single molecule techniques to answer focused questions about the mechanisms of RNA:protein complex assembly and catalysis in general.