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Journal Article

Direct probing of RNA structure and RNA-protein interactions in purifies HeLa cells and yeast spliceosomal U4/U6.U5 tri-snRNP particles

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Fabrizio,  P.
Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society;

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Luehrmann,  R.
Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society;

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Citation

Mougin, A., Gottschalk, A., Fabrizio, P., Luehrmann, R., & Branlant, C. (2002). Direct probing of RNA structure and RNA-protein interactions in purifies HeLa cells and yeast spliceosomal U4/U6.U5 tri-snRNP particles. Journal of Biological Chemistry, 317(5), 631-649. Retrieved from http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WK7-45MGMXB-C-1&_cdi=6899&_user=38661&_pii=S0022283602954513&_origin=search&_coverDate=04%2F12%2F2002&_sk=996829994&view=c&wchp=dGLzVtb-zSkzV&md5=9f6568759a3ba0d9c6001bca1ccb2592&ie=/sdarticle.pdf.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0012-F4DD-9
Abstract
The U4/U6.U5 tri-snRNP is a key component of spliceosomes. By using chemical reagents and RNases, we performed the first extensive experimental analysis of the structure and accessibility of U4 and U6 snRNAs in tri-snRNPs. These were purified from HeLa cell nuclear extract and Saccharomyces cerevisiae cellular extract. U5 accessibility was also investigated. For both species, data demonstrate the formation of the U4/U6 Y-shaped structure. In the human tri-snRNP and U4/U6 snRNP, U6 forms the long range interaction, that was previously proposed to be responsible for dissociation of the deproteinized U4/U6 duplex. In both yeast and human tri-snRNPs, U5 is more protected than U4 and U6, suggesting that the U5 snRNP-specific protein complex and other components of the tri-snRNP wrapped the 5' stem-loop of U5. Loop I of U5 is partially accessible, and chemical modifications of loop I were identical in yeast and human tri-snRNPs. This reflects a strong conservation of the interactions of proteins with the functional loop I. Only some parts of the U4/U6 Y-shaped motif (the 5' stem-loop of U4 and helix II) are protected. Due to difference of protein composition of yeast and human tri-snRNP, the U6 segment linking the 5' stem-loop to the Y-shaped structure and the U4 central single-stranded segment are more accessible in the yeast than in the human tri-snRNP, especially, the phylogenetically conserved ACAGAG sequence of U6. Data are discussed taking into account knowledge on RNA and protein components of yeast and human snRNPs and their involvement in splicesome assembly.