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Biochemical characterization and three-dimensional structure analysis of the yeast cleavage and polyadenylation factor CPF

Paguet, Bertrand. Biochemical characterization and three-dimensional structure analysis of the yeast cleavage and polyadenylation factor CPF. 2008, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_8403

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Abstract

In eukaryotes, protein-encoding genes are transcribed in the nucleus by RNA
polymerase II (RNAP II). Before the gene transcript (pre-mRNA) is transported to the
cytoplasm and can function in translation, it has to undergo three specific maturation
steps: capping, splicing and 3’ end processing. It is well established now that premRNA
processing events occur cotranscritpionally, while RNAP II is still
transcribing the gene.
Pre-mRNA 3’ end formation is an essential step in gene expression. With the
exception of mRNAs coding for replication-dependent histone proteins, all eukaryotic
pre-mRNAs are processed at their 3’ end by a coupled two-step reaction that involves
a specific endonucleolytic cleavage at the poly(A) site and subsequent poly(A) tail
addition to the upstream cleavage fragment catalyzed by poly(A) polymerase (Pap1p).
The complete maturation of the pre-mRNA 3’ end is directed by the presence of cisacting
sequence elements on the pre-mRNA that recruit protein factors. Surprisingly,
3’ end processing is accomplished by a complex protein machinery, which is highly
conserved from yeast to mammals. Indeed, most of the polypeptides involved in 3’
end formation in mammals have homologues in yeast. In S. cerevisiae, the cleavage
and polyadenylation factor (CPF), cleavage factor IA (CF IA), cleavage factor IB (CF
IB or Nab4p/Hrp1p) and the poly(A) binding protein (Pab1p) are required for specific
and accurate 3’ end processing activities.
The main pre-mRNA 3’ end processing factor, CPF, is a multiprotein complex
that consists of 15 polypeptides and is required for both pre-mRNA 3’ end processing
reactions. Most of its subunits are essential (only two are not essential) and all the
components are involved in RNA recognition or protein-protein interactions within
CPF or with other protein complexes, such as CF IA or RNAP II. Biochemical studies
of all CPF subunits have confirmed that they belong to the complex and allowed
characterization of their function in the context of pre-mRNA 3’ end formation.
Affinity purification of CPF identified a novel protein that co-purified with the
previously known components and was therefore proposed to be a new subunit of the
complex. This putative new component is non-essential and was named Cpf11p. In
this work, we showed that Cpf11p is not stably associated with CPF, as TAP tag
purification of Cpf11p did not result in the purification of CPF components (Chapter 3). Furthermore, in vitro cleavage and polyadenylation assays performed with
Cpf11p-depleted extracts did not show any defect, indicating that Cpf11p is not
involved in pre-mRNA 3’ end formation. We also found that expression of Cpf11p, in
contrast to the expression of CPF subunits, is controlled in a sugar-dependent manner.
Taken together, our results strongly suggest that Cpf11p has no function in premRNA
3’ end processing.
CPF plays a central role in pre-mRNA 3’ end processing. Its requirement for
cleavage and polyadenylation reactions is mediated by cooperative interactions with
CF IA and recognition of cis-acting polyadenylation signals on the primary transcript.
Despite every polypeptide involved in pre-mRNA 3’ end processing was
characterized, the mechanism by which the pre-mRNA is cleaved and polyadenylated
is not known. One aim of this thesis is to provide insight into the three-dimensional
structure of CPF. This would allow a better understanding in the arrangement of the
subunits in the complex and provide information on the molecular mechanism of premRNA
3’ end processing. We have developed an efficient purification procedure that
yields highly pure and active CPF. Here we report for the first time the 3D structure
of the complex at a resolution of 25 Å, determined by single-particle electron
microscopy on natively purified CPF using angular reconstitution and random conical
tilt (Chapter 4). The 3D model reveals a rough globular shape of the complex and a
strikingly large central cavity. We discuss the possibility that the inner cavity
represents a reaction chamber in which pre-mRNA 3’ end processing reactions could
take place. Furthermore, we have determined the mass of CPF particles by scanning
transmission electron microscopy (STEM) at approximately one megaDalton.
The work reported in this thesis should contribute to a better understanding of
the mechanism by which pre-mRNAs are processed at their 3’ end by presenting the
first 3D model of the CPF complex, and by refining the protein composition of CPF.
In addition, a part of this work is dedicated to investigation of in vivo interconnections
between transcription and 3’ end processing of pre-mRNAs (Chapter 2).
Advisors:Keller, Walter
Committee Members:Engel, Andreas
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Cell Biology (Keller)
UniBasel Contributors:Keller, Walter
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:8403
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:174
Language:English
Identification Number:
Last Modified:22 Jan 2018 15:50
Deposited On:03 Jun 2009 12:00

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