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The molecular biology of RNA 3' end processing

English title The molecular biology of RNA 3' end processing
Applicant Keller Walter
Number 133145
Funding scheme Project funding (Div. I-III)
Research institution Abteilung Zellbiologie Biozentrum Universität Basel
Institution of higher education University of Basel - BS
Main discipline Molecular Biology
Start/End 01.10.2010 - 30.09.2012
Approved amount 289'532.00
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Keywords (17)

pre-mRNA 3' end processing; poly(A) site; alternative polyadenylation; PAR-CLIP; RNA-binding proteins; micro RNA; noncoding RNA; UV crosslinking; cleavage and polyadenylation factor; electron microscopy; RNA processing; mRNA polyadenylation; poly(A) polymerase; 3' end formation; RNA splicing; transcription,; regulation of alternative RNA processing

Lay Summary (English)

Lead
Lay summary
The recent observations that alternative pre-mRNA 3' end processing results in a global decreased susceptibility of mRNAs to micro RNA-dependent inhibition in dividing cells including cancer cells caused renewed interest in 3' end processing. The mechanism by which a change in the use of proximal versus distal polyadenylation sites in resting compared to dividing cells occurs is not known. Our previous work contributed to the elucidation of the dynamics of 3' end processing factors within the 3' end processing complex itself and within the transcriptomes of selected mammalian cell systems and is of relevance to the unsolved puzzles concerning the systematic changes in 3' end processing in relation to the cellular state. In collaboration with the group of Professor Mihaela Zavolan at the Biozentrum, we recently initiated a study of binding sites of various 3' end processing factors in a number of cell types and under various conditions. We could show that the binding sites that we identified reflect the known sequence specificities of the 3' end processing factors and their relative location within individual transcripts is consistent with earlier reports.The second project concerns the structure of the cleavage and polyadenylation factor CPF from yeast cells. In collaboration with the group of Professor Andreas Engel at the Biozentrum, we have determined the molecular mass of this complex and the stoichiometry of its subunits by scanning transmission electron microscopy. Professor Holger Stark (Max-Planck-Institute for Biophysical Chemistry, Göttingen) has provided us with his newly developed protein crosslinking-gradient centrifugation procedure "GraFix" to purify CPF preparations suitable for electron microscopy and he has established the three-dimensional structure of the complex at a resolution of 25Å in negatively stained preparations and by single-particle cryo-electron microscopy. CPF has a complex asymmetric architecture in which an outer protein wall surrounds a large inner cavity. Moreover, three GFP-tagged subunits could be localized within the structure and the X-ray structure of poly(A) polymerase could be fitted to the complex. Professor Stark is currently analyzing more images by cryo-electron microscopy to improve the resolution of the structure. We hope that our experiments will contribute to the understanding of the mechanism of 3' end processing and of how this fundamental step of mRNA metabolism interfaces with other cellular processes such as transcription, alternative splicing and polyadenylation as well as with the susceptibility to cotranscriptional and post-transcriptional regulation.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length
Martin Georges, Gruber Andreas, Keller Walter, Zavolan Mihaela (2012), Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length, in Cell Reports, 1(6), 753-763.
PAPD5, a noncanonical poly(A) polymerase with an unusual RNA-binding motif.
Rammelt Christiane, Bilen Biter, Zavolan Mihaela, Keller Walter (2011), PAPD5, a noncanonical poly(A) polymerase with an unusual RNA-binding motif., in RNA (New York, N.Y.), 17(9), 1737-46.

Associated projects

Number Title Start Funding scheme
143977 The molecular biology of RNA 3' end processing 01.10.2012 Project funding (Div. I-III)
102132 Biochemical and genetic characterization of components involved in messenger RNA processing and in RNA editing 01.10.2003 Project funding (Div. I-III)

Abstract

Recent observations that alternative pre-mRNA 3' end processing results in a global, decreased susceptibility of mRNAs to miRNA-dependent inhibition in dividing cells including cancer cells caused renewed interest in the 3' end processing of mRNAs. The mechanism by which a systematic change in the use of proximal versus distal polyadenylation sites in resting and dividing cells occurs is not known. The previous work within the scope of our grant contributes to the elucidation of the dynamics of 3’ end processing factors within the 3' end processing complex itself and also within the transcriptomes of selected mammalian cell systems. This is of obvious relevance to the currently unsolved puzzles concerning systematic changes in the 3’ end processing and polyadenylation in relation to the cellular state. In collaboration with the Zavolan group at the Biozentrum, we recently initiated a study of binding sites of various 3’ end processing factors in a number of cell types and under various conditions. Our preliminary results are very promising in that the binding sites that we identified reflect the known sequence specificities of the factors that we studied and their relative location agrees with prior findings. We therefore request that funding for our project is continued in order to complete these studies which will further the understanding of how 3’ end processing interfaces with other cellular processes such as transcription, alternative splicing and polyadenylation as well as with the susceptibility to post-transcriptional regulation. A second part of the project is aimed at the elucidation of the structure of the cleavage and polyadenylation factor CPF, the key protein complex involved in the 3’ end processing of yeast pre-mRNAs and snoRNAs. In collaboration with Professor Holger Stark (MPI Göttingen) we plan to achieve a high-resolution structure by cryo-electron microscopy.
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