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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 143977
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 01.10.2012 - 31.05.2015
Approved amount 421'104.00
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Keywords (10)

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

Lay Summary (English)

Lead
Lay summary

Expression of eukaryotic protein-coding genes proceeds through multiple steps, including transcription, addition of a guanosine cap to the 5’-end of the nascent messenger RNA, splicing, cleavage of the mRNA precursor (pre-mRNA) to define the 3’ end of the transcript and in most cases, addition of a poly(A) tail. Like alternative splicing, 3' end processing of a given pre-mRNA can occur at different sites. This gives rise to mRNAs with different coding regions or 3' untranslated regions (UTRs) with different sets of binding sites for regulatory factors such as microRNAs. Recently, a global tendency towards the use of proximal polyadenylation sites has been reported in dividing compared to resting cells and in cancer cells relative to their normal counterparts, highlighting the importance of polyadenylation for cell physiology.

In collaboration with the group of Professor Mihaela Zavolan at the Biozentrum, we have generated the first combined maps of binding sites of core cleavage and polyadenylation factors and of the poly(A) sites that are used in a human cell type. In addition, we have identified factors whose binding is most informative for the location of the cleavage site. We further investigated the change in poly(A) site selection induced by the knockdown of CF Im68 and CstF-64, two core components that were also reported previously to affect poly(A) site choice, and we found that the knockdown of CF Im68 results in a global shift towards proximal poly(A) sites. Thus, changes in relative abundance of a single 3’ end processing factor can modulate the length of 3’ untranslated regions across the transcriptome and suggested a mechanism behind the previously observed increase in tumor cell invasiveness upon CF Im68 knockdown. We now plan to investigate in detail the mechanism by which reduced levels of CF Im68 results in the use of proximal poly(A) sites. Perhaps the concentration of 3’ end processing factors is limiting in dividing compared to resting cells. We will attempt to measure the mRNA as well as protein levels of several cleavage factors such as CF Im and CstF in resting and in actively dividing cells but also in tumor tissue and tumor cell lines. Transcript levels will be determined by quantitative real time PCR or Northern blotting and protein levels by Western blotting or immunostaining. Preliminary data indicate that immortalized cells such as HEK293 have a high concentration of CF Im68 in the cytoplasm compared to the nucleus. The reason for this is unknown but regulation of nuclear import could be a way to keep the concentration of this factor low in the nucleus. A further line of investigation will be to determine whether modifications such as phosphorylation or methylation influence the nuclear import of these factors.

The second project we pursued in the previous years concerns the cleavage and polyadenylation factor CPF of yeast. In collaboration with the group of Professor Andreas Engel 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 established the three-dimensional structure of the complex at a resolution of 25Å from 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 located within the structure and the X-ray structure of poly(A) polymerase could be fitted to the complex. Professor Stark is now planning to fit the X-ray structure of DDB1 (DNA damage binding protein 1) to the CPF structure. Mammalian CPSF160 and yeast YHH1 are homologues of DDB1 over the entire protein sequence.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Reflections on the RNA world
Zavolan Mihaela, Keller Walter (2015), Reflections on the RNA world, in RNA, 21, 531-533.
Crystal structure of human poly(A) polymerase gamma reveals a conserved catalytic core for canonical poly(A) polymerases
Yang Quin, Nausch Lydia W.M., Martin Georges, Keller Walter, Doublié Sylvie (2014), Crystal structure of human poly(A) polymerase gamma reveals a conserved catalytic core for canonical poly(A) polymerases, in Journal of Molecular Biology, 426, 43-50.
Global 3' UTR shortening has a limited effect on protein abundance in proliferating T cells
Gruber Andreas R., Martin Georges, Müller Philipp, Schmidt Alexander, Gruber Andreas J., Gummienny Rafal, Nitish Mittal, Jayachandran Rajesh, Pieters Jean, Keller Walter, van Nimwegen Erik, Zavolan Mihaela (2014), Global 3' UTR shortening has a limited effect on protein abundance in proliferating T cells, in Nature Communications, 5:5465, 1-10.
Means to en end: mechanisms of alternative polyadenylation of messenger RNA precursors
Gruber Andreas R, Martin Georges, Keller Walter, Zavolan Mihaela (2014), Means to en end: mechanisms of alternative polyadenylation of messenger RNA precursors, in WIREs RNA, 5, 183-196.
Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33
Schönemann Lars, Kühn Uwe, Martin Georges, Schäfer Peter (2014), Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33, in Genes & Development, 28, 2381-2393.
RNA polymerase III promoter screen uncovers a novel noncoding RNA family conserved in Caenorhabditis and other clade V nematodes
Gruber Andreas R. (2014), RNA polymerase III promoter screen uncovers a novel noncoding RNA family conserved in Caenorhabditis and other clade V nematodes, in Gene, 544, 236-240.
Cleavage Factor Im is a key regulator of 3' UTR length
Gruber Andreas R., Martin Georges, Keller Walter, Zavolan Mihaela (2012), Cleavage Factor Im is a key regulator of 3' UTR length, in RNA Biology, 9(12), 1405-1412.
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, 753-763.

Collaboration

Group / person Country
Types of collaboration
Professor Elmar Wahle, University of Halle Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Mihaela Zavolan, Biozentrum, Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Professor Holger Stark MPI Göttingen Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure

Associated projects

Number Title Start Funding scheme
133145 The molecular biology of RNA 3' end processing 01.10.2010 Project funding (Div. I-III)
170216 Regulation of mRNA translation and its relationship with disease processes 01.10.2016 Project funding (Div. I-III)

Abstract

Recent findings 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. In the current grant period we sought to generate the first combined maps of binding sites of core cleavage and polyadenylation factors and of the poly(A) sites that are used in a human cell type and to identify factors whose binding is most informative for the location of the cleavage site. We further investigated the change in poly(A) site selection induced by the knockdown of CF Im68 and CstF-64, two core components that were also reported previously to affect poly(A) site choice, and we found that the knockdown of CF Im68 results in a global shift towards proximal poly(A) sites. We now plan to investigate in detail the mechanism by which reduced CF Im68 results in the use of proximal poly(A) sites.The second project we pursued in the previous years and is now almost completed concerns the cleavage and polyadenylation factor CPF of yeast. In collaboration with the group of Professor Andreas Engel 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 established the three-dimensional structure of the complex at a resolution of 25Å from 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 now planning to fit the X-ray structure of DDB1 (DNA damage binding protein 1) to the CPF structure. Mammalian CPSF160 and yeast YHH1 are homologues of DDB1 over the entire protein sequence.
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