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Regulation of pre-mRNA splicing and mRNA degradation in Saccharomyces cerevisiae
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Marcus Johansson)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Messenger RNAs are transcribed and co-transcriptionally processed in the nucleus, and transported to the cytoplasm. In the cytoplasm, mRNAs serve as the template for protein synthesis and are eventually degraded. The removal of intron sequences from a precursor mRNA is termed splicing and is carried out by the dynamic spliceosome. In this thesis, I describe the regulated splicing of two transcripts in Saccharomyces cerevisiae. I also describe a study where the mechanisms that control the expression of magnesium transporters are elucidated.

The pre-mRNA retention and splicing (RES) complex is a spliceosome-associated protein complex that promotes the splicing and nuclear retention of a subset of pre-mRNAs. The RES complex consists of three subunits, Bud13p, Snu17p and Pml1p. We show that the lack of RES factors causes a decrease in the formation of N4-acetylcytidine (ac4C) in tRNAs. This phenotype is caused by inefficient splicing of the pre-mRNA of the TAN1 gene, which is required for the formation of ac4C in tRNAs. The RES mutants also show growth defects that are exacerbated at elevated temperatures. We show that the temperature sensitive phenotype of the bud13Δ and snu17Δ cells is caused by the inefficient splicing of the MED20 pre-mRNA. The MED20 gene encodes a subunit of the Mediator complex. Unspliced pre-mRNAs that enter the cytoplasm are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway, which targets transcripts that contain premature translation termination codons. Consistent with the nuclear retention function of the RES complex, we find that NMD inactivation in the RES mutants leads to the accumulation of both TAN1 and MED20 pre-mRNAs. We also show that the cis-acting elements that promote RES-dependent splicing are different between the TAN1 and MED20 pre-mRNAs.

The NMD pathway also targets transcripts with upstream ORFs (uORFs) for degradation. The ALR1 gene encodes the major magnesium importer in yeast, and its expression is controlled by the NMD pathway via a uORF in the 5’ untranslated region. We show that the ribosome reaches the downstream main ORF by a translation reinitiation mechanism. The NMD pathway was shown to control cellular Mg2+ levels by regulating the expression of the ALR1 gene. We further show that the NMD pathway targets the transcripts of the vacuolar Mg2+ exporter Mnr2p and the mitochondrial Mg2+ exporter Mme1p for degradation.

In summary, we conclude that the RES complex has a role in the splicing regulation of a subset of transcripts. We also suggest a regulatory role for the NMD pathway in maintaining the cellular Mg2+ concentration by controlling the expression of Mg2+ transporters.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2017. , p. 53
Keywords [en]
NMD, RES complex, pre-mRNA splicing, magnesium homeostasis
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-138142ISBN: 978-91-7601-749-4 (print)OAI: oai:DiVA.org:umu-138142DiVA, id: diva2:1131394
Public defence
2017-09-22, KBE303-Stora hörsalen, KBC-huset, 09:00 (English)
Opponent
Supervisors
Available from: 2017-08-15 Created: 2017-08-14 Last updated: 2018-06-09Bibliographically approved
List of papers
1. The pre-mRNA retention and splicing complex controls tRNA maturation by promoting TAN1 expression
Open this publication in new window or tab >>The pre-mRNA retention and splicing complex controls tRNA maturation by promoting TAN1 expression
2013 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 11, p. 5669-5678Article in journal (Refereed) Published
Abstract [en]

The conserved pre-mRNA retention and splicing (RES) complex, which in yeast consists of Bud13p, Snu17p and Pml1p, is thought to promote nuclear retention of unspliced pre-mRNAs and enhance splicing of a subset of transcripts. Here, we find that the absence of Bud13p or Snu17p causes greatly reduced levels of the modified nucleoside N-4-acetylcytidine (ac(4)C) in tRNA and that a lack of Pml1p reduces ac(4)C levels at elevated temperatures. The ac(4)C nucleoside is normally found at position 12 in the tRNA species specific for serine and leucine. We show that the tRNA modification defect in RES-deficient cells is attributable to inefficient splicing of TAN1 pre-mRNA and the effects of reduced Tan1p levels on formation of ac(4)C. Analyses of cis-acting elements in TAN1 pre-mRNA showed that the intron sequence between the 5' splice site and branchpoint is necessary and sufficient to mediate RES dependency. We also show that in RES-deficient cells, the TAN1 pre-mRNA is targeted for degradation by the cytoplasmic nonsense-mediated mRNA decay pathway, indicating that poor nuclear retention may contribute to the tRNA modification defect. Our results demonstrate that TAN1 pre-mRNA processing has an unprecedented requirement for RES factors and that the complex controls the formation of ac(4)C in tRNA.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-76798 (URN)10.1093/nar/gkt269 (DOI)000320116200019 ()23605039 (PubMedID)
Available from: 2013-07-16 Created: 2013-07-15 Last updated: 2018-06-08Bibliographically approved
2. The pre-mRNA retention and splicing complex controls expression of the Mediator subunit Med20
Open this publication in new window or tab >>The pre-mRNA retention and splicing complex controls expression of the Mediator subunit Med20
2017 (English)In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 14, no 10, p. 1411-1417Article in journal (Refereed) Published
Abstract [en]

The heterotrimeric pre-mRNA retention and splicing (RES) complex, consisting of Bud13p, Snu17p and Pml1p, promotes splicing and nuclear retention of a subset of intron-containing pre-mRNAs. Yeast cells deleted for individual RES genes show growth defects that are exacerbated at elevated temperatures. Although the growth phenotypes correlate to the splicing defects in the individual mutants, the underlying mechanism is unknown. Here, we show that the temperature sensitive (Ts) growth phenotype of bud13Δ and snu17Δ cells is a consequence of inefficient splicing of MED20 pre-mRNA, which codes for a subunit of the Mediator complex; a co-regulator of RNA polymerase II transcription. The MED20 pre-mRNA splicing defect is less pronounced in pml1Δ cells, explaining why they grow better than the other 2 RES mutants at elevated temperatures. Inactivation of the cytoplasmic nonsense-mediated mRNA decay (NMD) pathway in the RES mutants leads to accumulation of MED20 pre-mRNA, indicating that inefficient nuclear retention contributes to the growth defect. Further, the Ts phenotype of bud13Δ and snu17Δ cells is partially suppressed by the inactivation of NMD, showing that the growth defects are augmented by the presence of a functional NMD pathway. Collectively, our results demonstrate an important role of the RES complex in maintaining the Med20p levels.

Place, publisher, year, edition, pages
Taylor & Francis, 2017
Keywords
Med20, NMD, RES complex, mediator, splicing
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-135241 (URN)10.1080/15476286.2017.1294310 (DOI)000418050000015 ()28277935 (PubMedID)
Available from: 2017-05-23 Created: 2017-05-23 Last updated: 2018-06-09Bibliographically approved
3. The nonsense-mediated mRNA decay pathway controls the expression of magnesium transporters in Saccharomyces cerevisiae
Open this publication in new window or tab >>The nonsense-mediated mRNA decay pathway controls the expression of magnesium transporters in Saccharomyces cerevisiae
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-138148 (URN)
Available from: 2017-08-14 Created: 2017-08-14 Last updated: 2018-06-09

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Zhou, Yang

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