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Biochemical and genetic characterization of components involved in messenger RNA processing and in RNA editing

English title Biochemical and genetic characterization of components involved in messenger RNA processing and in RNA editing
Applicant Keller Walter
Number 102132
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 01.10.2003 - 30.09.2010
Approved amount 1'301'596.00
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Keywords (6)

RNA polymerase II; RNA processing; mRNA polyadenylation; poly(A) polymerase; 3' end formation;

Lay Summary (English)

Lead
Lay summary
Our work is focused on the biochemical and genetic characterization of the protein machinery involved in the processing of messenger RNA precursors (pre-mRNAs) in the nucleus of eukaryotic cells. Pre-mRNAs are synthesized by RNA polymerase II and undergo three processing steps that include the addition of a 7-methyl guanosine "cap" at the 5' end, the removal of intron sequences by RNA splicing and the addition of a poly(A) tail at their 3' ends. The cap and the poly(A) tails have multiple functions. In the nucleus they interact with a complex of cap binding proteins and with a nuclear form of poly(A) binding protein and thereby signal to the RNA export machinery that they are ready to be transported to the cytoplasm. In the cytoplasm they interact with a different set of proteins and form circular mRNAs by interactions between the proteins bound to the cap and the cytoplasmic version of the poly(A) binding protein. Circularization serves to protect the mRNAs against premature degradation and increases the efficiency of their translation into proteins.The capping, splicing and 3' end processing reactions are coupled to each other and to the transcription process as well. We are studying the composition of the multiprotein complexes and the reaction mechanism of the steps involved in 3' end formation: endonucleolytic cleavage of the pre-mRNA followed by polyadenylation. The overall reaction involves a number of trans-acting protein factors that interact with sequence signals in the pre-mRNA substrates and which each other, forming a network of RNA-protein and protein-protein interactions. Moreover, we are investigating the coupling of 3' end processing to splicing and to the termination step of transcription. The repertoire of the proteins making up the different 3' end processing factors is known. However, except for poly(A) polymerase, the enzyme catalyzing the synthesis of the poly(A) tails, and the putative endonuclease involved in the cleavage step of the reaction, the precise functions of the many other subunits remain to be elucidated.We and other research groups have discovered a new type of poly(A) addition complex in yeast cells that function in the quality control of RNAs by recognizing defective molecules and targetting them for degradation by the nuclear exosome. We are further characterizing this unconventional polyadenylation system biochemically and by genetic approaches and we have expanded this project by including mammalian relatives of the yeast proteins.We are using mammalian tissue culture cells and the yeast Saccharomyces cerevisiae as starting material for our experiments. In addition to conventional biochemical fractionation methods and the standard molecular biology tools we are collaborating with experts in microarray analyses and bioinformatics and with structural biology colleagues to investigate proteins and protein complexes by X-ray crystallography, nuclear magnetic resonance and cryo-electron microscopy. The results of these efforts should contribute to a better understanding of the mechanism of eukaryotic gene expression and its regulation.
Direct link to Lay Summary Last update: 21.02.2013

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Associated projects

Number Title Start Funding scheme
133145 The molecular biology of RNA 3' end processing 01.10.2010 Project funding (Div. I-III)
53897 Biochemical and genetic characterization of components involved in messenger RNA processing and in RNA-editing 01.10.1998 Project funding (Div. I-III)

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