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Structural and Biochemical Examination of the Yeast U4/U6 snRNP.

Final Report Summary - STRUCTUREU4U6SNRNP (Structural and Biochemical Examination of the Yeast U4/U6 snRNP)

The spliceosome is a large RNA-protein assembly that catalyses the removal of introns from mRNA precursors (pre-mRNA) and the splicing of coding exons to produce mature mRNAs. Four small nuclear ribonucleoprotein particles (U1, U2, U5 and U4/U6 snRNPs) assemble onto pre-mRNA substrates together with non-snRNP proteins to form the spliceosome. After binding of the U1 snRNP and the U2 snRNP to the 5' splice site and the branch point sequence within the intron, a pre-assembled U4/U6.U5 tri-snRNP joins and the spliceosome undergoes extensive remodeling to yield the catalytically active spliceosome. In the tri-snRNP, the U4 and U6 snRNAs are extensively base-paired but upon activation this duplex is unwound by an RNA helicase, Brr2p. The ultimate aim of this project is to determine the structure of the U4/U6 snRNP by X-ray crystallography and reveal details of spliceosome activation / mechanism. The aim of this work is to initially employ electrophoretic gel mobility shift assays and gel filtration chromatography for characterisation of assembly, isothermal titration calorimetry to identify / quantify protein-protein interactions, chemical / enzymatic RNA footprinting to identify protein-RNA contacts and protein-induced conformational changes in the RNA, and pulse-chase quantitative mass spectrometry to further substantiate and characterize the pathway of assembly of the U4/U6 snRNP and the U4/U6.U5 tri-snRNP biochemically and thermodynamically. These experiments will also aid crystallisation. Unwinding directionality and helicase-induced protein displacement by the U5 snRNP protein Brr2p is proposed to be examined in the context of the fully assembled U4/U6 snRNP and tri-snRNP complexes. This will reveal details of spliceosome activation / mechanism. The proposed project will exploit the researcher's extensive knowledge and experience with RNA-protein complex biochemistry, complex engineering skills, and crystallographic approaches but will also allow him to develop new knowledge and skills.

The primary goal of this project is to assemble and attain the structure of the U4/U6 di-snRNP by xray crystallography. To accomplish this, it has firstly been necessary to produce all of the components (18 proteins and 2 RNAs) in quantities sufficient for crystal screening. Secondly, it is deemed wise to biochemically characterize extensively the assembly behaviour of these components in-vitro. As of this final report, we have accomplished both of these goals. For the most part, our findings have revealed that it is possible to assemble a very well-behaved complex and that all components assemble into this complex with high affinity. However, we have found that there appears to be a high degree of either comformational or compositional heterogeneity present upon binding of one of the key components (the LSm complex). This results in complex formation that is not likely to be suitable for crystallization trials. As we entered the second half of funding for the project the primary focus had unexpectedly changed towards experiments designed to better understand LSm complex behaviour and on seeking ways in which this behaviour might be improved. The hope was that, once the LSm behaviour issues are resolved, we could once again rejoin the path towards obtaining crystals and ultimately obtain the structure of the entire U4/U6 di-snRNP. Because of the issues encountered, all subsequent planned investigations (isothermal titration calorimetry, chemical / enzymatic RNA footprinting, pulse-chase quantitative mass spectrometry, and Brr2p unwinding / protein displacement experiments) that depend firstly upon obtaining well-behaved in-vitro reconstituted U4/U6 di-snRNP were necessarily deferred until such a complex can be produced. With our focus on LSm behaviour, we found that we were able to obtain material that was far more homogeneous as determined through use of native mass spectrometry. This material formed very well-defined discrete complexes by electrophoretic mobility shift assays when titrated against a truncated U4/U6 snRNA construct and thus the issues with LSm complex behaviour appeared to be solved. However, complex behaviour with full length U4/U6 snRNAs surprisingly remained poor. It was soon discovered that this is likely due to the presence of secondary high affinity binding sites on the U4/U6 snRNA duplex that lie outside of the predicted LSm binding site at the 3' end of U6 snRNA. Work continues to understand this observation and to determine the largest U4/U6 snRNA construct that allows formation of a discrete LSm containing complex suitable for crystallisation. In the meantime, we have pursued several truncated complexes that have not yet yielded crystals despite appearing to be well-behaved. Recently, as a direct result of this work, we have engaged in a collaboration to better understand the conformational changes that occur within the U4/U6 snRNA upon binding of protein components through fluorescence resonance energy transfer experiments. We have also, sought to produce material more suitable for electron microscopy studies on the U4/U6 di-snRNP and the U5.U4/U6 tri-snRNP complexes purified from the native source. This places the project in position for a number of forthcoming potential publications as a direct result of the Marie Curie fellowship funding.

Ultimately, we want to understand the splicing mechanism in atomic resolution detail. This very important and fundamental cellular process is currently only poorly understood and the results of this project will contribute to our understanding overall. It is anticipated that our understanding of U4/U6 di-snRNP and tri-snRNP assembly will be greatly enhanced as a direct result of this work as we continue to build upon the foundation begun in this study but this is clearly a long-term goal and will likely require many additional years to yield the detailed information on the splicing process sought. The wider implications of such information are currently difficult to ascertain although there is hope for the eventual utilisation of such knowledge in the prevention and treatment of disorders originating in defective splicing or in various cancers.