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Functional characterization of Ysh1p, the yeast endonuclease involved in 3" end processing and in transcription termination of RNA polymerase II transcripts

Garas, Monika. Functional characterization of Ysh1p, the yeast endonuclease involved in 3" end processing and in transcription termination of RNA polymerase II transcripts. 2008, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_8414

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Abstract

Eukaryotic RNA polymerase II (RNAP II) is involved in the synthesis of two major classes
of transcripts: messenger RNAs (mRNAs) and small nuclear and small nucleolar RNAs.
In order to be biologically functional, primary transcripts of RNAP II require extensive
processing and modifications. Biogenesis of mature mRNAs involves capping at the 5’ end,
splicing out of the introns and poly(A) tail addition at the 3’ end. Only correctly processed
mRNAs can be exported to the cytoplasm where they act as templates for protein translation.
Eukaryotic pre-mRNA 3’ end formation is initiated by endonucleolytic cleavage at the
poly(A) site, followed by polyadenylation of the upstream cleavage product. In contrast, small
nuclear RNA (snRNA) and small nucleolar RNA (snoRNA) precursors are cleaved at their
3’ ends, but in their mature form they are not polyadenylated. The seemingly simple reactions
of 3’ end cleavage and polyadenylation are nevertheless performed by surprisingly complex
protein machineries. In yeast, the pre-mRNA 3’ end processing apparatus consists
of cleavage and polyadenylation factor (CPF), cleavage factor IA (CF IA) and cleavage factor
IB (CF IB; reviewed in Zhao et al., 1999). The complexity of the 3’ end processing machinery
is in part due to the necessity of precise RNA sequence recognition and also to the regulation
in a wider transcriptional context. Both the exact mechanism of 3’ end processing, and many
of the factors involved in these reactions exhibit a high level of similarity between metazoans
and yeast. Cleavage and polyadenylation factors are co-transcriptionally recruited to the
carboxy-terminal domain (CTD) of RNAP II and together with the cis-acting 3’ end processing
signals are required for transcription termination on mRNA genes (reviewed in Buratowski,
2005; Proudfoot, 2004).
The original aim of this thesis was the identification and characterization of the yeast
endonuclease involved in pre-mRNA 3’ end processing. Whereas it has long been known that
the poly(A) tails of mRNA are synthesized by poly(A) polymerase, the endonucleolytic activity
involved in 3’ end cleavage remained enigmatic for many years. Therefore, in the beginning of
this work we assigned putative endonucleolytic activity to the yeast CPF subunit Ysh1p/Brr5p
and to its archaean homologue, M. jannaschii MJ1236, based on highly conserved metallo-
β-lactamase and β-CASP domains present in these factors.
Very little has been known about Ysh1/Brr5 protein and its role within the 3’ end
processing machinery. We found that the conserved metallo-β-lactamase motif present
in Ysh1p/Brr5p is essential for yeast viability in vivo, as any mutation within its conserved
β-lactamase signature HXHXDH is detrimental to the cell. Although this fact underscored the
functional importance of the metallo-β-lactamase motif in Ysh1p/Brr5p, it hampered further
attempts to analyze the effects of such mutations. Moreover, biochemical assignment
of a potential enzymatic activity to this factor in vitro was virtually impossible, as recombinant
Ysh1p/Brr5p alone neither bound to RNA nor exhibited any nucleolytic activity. Consistently,
specific cross-linking of the yeast 3’ end processing factors to the poly(A) site did not identify
Ysh1p/Brr5p as the factor present at the cleavage site. Therefore, to better understand the role of Ysh1p/Brr5p in pre-mRNA 3’ end formation, we generated a series of conditional
mutants of YSH1. Analysis of several temperature- and cold- sensitive ysh1 alleles revealed
several important features of Ysh1p/Brr5p in different aspects of RNA processing and their
coupling to RNAP II transcription termination and splicing. Firstly, we showed that
Ysh1p/Brr5p is generally required for 3’ end cleavage and polyadenylation as well as for
poly(A) site selection of ACT1 pre-mRNA. Interestingly, RNAP II transcription termination
defects on a plasmid-borne CYC1 gene were observed in ysh1 mutant strains. Northern blot
analysis of steady-state RNA extracted from ysh1-12 mutant cells detected read-through
transcripts on several endogenous mRNA genes, confirming the general requirement of
Ysh1p/Brr5p for transcription termination. Secondly, a significant proportion of RNAP II
molecules failed to terminate transcription properly on SNR3 snoRNA gene locus in ysh1-12
mutant and extended transcripts produced from several snoRNA genes accumulated in this
strain, pointing towards the involvement of Ysh1p/Brr5p in snoRNA 3’ end formation.
Furthermore, we showed that Ysh1p/Brr5p is involved in the regulation of NRD1 mRNA
levels. Interestingly, mutations in ysh1-12 strain resulted in splicing defects on mRNA and
snoRNA genes, thus suggesting a function for Ysh1p/Brr5p in coupling of pre-mRNA 3’ end
formation and splicing reactions in S. cerevisiae.
In addition, we analyzed functions of Syc1p, a new yeast 3’ end processing subunit,
which exhibits a high level of homology to the C-terminus of Ysh1p/Brr5p. Syc1p has possible
regulatory functions in pre-mRNA 3’ end formation and possibly links the processing
machinery to other nuclear events.
Last not least, we carried out in vitro analyses of the recombinant M. jannaschii
protein MJ1236, which is homologous to the β-lactamase and β-CASP domains
of Ysh1p/Brr5p. Intriguingly, MJ1236 possesses also a KH-RNA binding domain, thus further
suggesting a potential function of this factor in RNA metabolism. Heterogeneous expression
and assaying of MJ1236 revealed its endonucleolytic activity on CYC1, ADH1 and GAL7 RNA
substrates in vitro. This finding strongly implied the same type of hydrolyzing activity for its
S. cerevisiae homologue Ysh1p/Brr5p.
Only recently the pre-mRNA 3’ end endonucleolytic activity has been assigned to
CPSF73, subunit of the mammalian 3’ end processing machinery, as based on its crystal
structure and in vitro activity (Mandel et al., 2006). Because of its high level of homology to
CPSF73, Ysh1p/Brr5p is now generally believed to be the 3’ end processing endonuclease in
S. cerevisiae.
This thesis is a record of a fascinating yet sometimes frustrating quest towards
identification of the yeast pre-mRNA 3’ end processing endonuclease and understanding its
functions in a wider transcriptional context.
Advisors:Keller, Walter
Committee Members:Filipowicz, Witold
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:8414
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:171
Language:English
Identification Number:
Last Modified:22 Jan 2018 15:50
Deposited On:03 Jun 2009 12:08

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