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Spatial control of mRNA stability in yeast
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Tracy Nissan)ORCID iD: 0000-0003-3956-7197
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The degradation of mRNA is an important modulator of gene expression and the ultimate fate of messenger mRNA. Important steps in the degradation of mRNA include initial shortening of its poly(A) tail followed by the subsequent removal of the m7G cap. These two processes are linked temporally as well as spatially. In addition to physical interactions between proteins involved in these two processes, deadenylation and decapping enzymes and accessory factors are found in P bodies. P bodies are aggregates of protein and mRNA that are induced upon stress in all eukaryotes examined. In this thesis, I examine the spatial localization of decapping factors and explore the role of P bodies in mRNA turnover in the yeast Saccharomyces cerevisiae.  

This thesis is based on three underlying principles. First, mRNA decapping factors are membrane associated. More so, we show that decapping factors can be co-localized with the endoplasmic reticulum and Golgi apparatus. Second, although P bodies were proposed as sites of mRNA decay, we found that they stabilize mRNA. We examined the role of P bodies in mRNA turnover using a mutant defective in their assembly, edc3∆ lsm4∆C.  This strain is mutated in two decapping activators.  It combines a deletion of the gene encoding the Edc3 protein and lacks the prion-like domain of Lsm4. Using the edc3∆ lsm4∆C mutant, we demonstrate that mRNA stability is significantly reduced in the absence of P bodies for longer-lived mRNA. The effect of mRNA destabilization was due to increased deadenylation and decapping dependence. Finally, the decapping factor usually found in the cytoplasm, but accumulates in the nucleus in the P body deficient strain (edc3∆ lsm4∆C). This implies a possible role in modulating transcription.

A model for the functioning of P bodies that is consistent with our work is that P bodies serve a role as a cytoplasmic sink for degradation factors. By regulating the access of the cytosol to proteins involved in mRNA turnover, P bodies can modulate mRNA stability. This suggests a role for P bodies under stress and their potential importance in stress adaptation.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2017. , 50 p.
Keyword [en]
mRNA stability, P bodies, mRNA turnover, gene regulation, decapping factors
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-133894ISBN: 978-91-7601-721-0 (print)OAI: oai:DiVA.org:umu-133894DiVA: diva2:1089684
Public defence
2017-05-19, E04, Biomedicinehuset, Norrlands Universitetetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 621-2010-4602
Available from: 2017-04-28 Created: 2017-04-20 Last updated: 2017-04-27Bibliographically approved
List of papers
1. Membrane-association of mRNA decapping factors is independent of stress in budding yeast
Open this publication in new window or tab >>Membrane-association of mRNA decapping factors is independent of stress in budding yeast
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, 25477Article in journal (Refereed) Published
Abstract [en]

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane otation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation. 

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology; cellforskning
Identifiers
urn:nbn:se:umu:diva-120043 (URN)10.1038/srep25477 (DOI)000375544400001 ()27146487 (PubMedID)
Funder
Carl Tryggers foundation , 11:330Swedish Research Council, 621-2010-4602
Available from: 2016-05-05 Created: 2016-05-05 Last updated: 2017-04-27Bibliographically approved
2. The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae
Open this publication in new window or tab >>The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae
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2016 (English)In: Biology Open, ISSN 2046-6390, Vol. 5, no 10, 1388-1399 p.Article in journal (Refereed) Published
Abstract [en]

The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs or alternatively may imply a role for P bodies in mRNA stabilization.

Place, publisher, year, edition, pages
The company of biologists ltd, 2016
Keyword
P bodies, Deadenylation, Exosome, mRNA decapping, mRNA decay, mRNA stability
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-126827 (URN)10.1242/bio.020487 (DOI)000388300900005 ()27543059 (PubMedID)
Funder
Carl Tryggers foundation , 15:345Swedish Research Council, 621-2010-4602
Available from: 2016-10-15 Created: 2016-10-15 Last updated: 2017-04-27Bibliographically approved
3. An mRNA decapping mutant deficient in P body assembly limits mRNA stabilization in response to osmotic stress
Open this publication in new window or tab >>An mRNA decapping mutant deficient in P body assembly limits mRNA stabilization in response to osmotic stress
2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, 44395Article in journal (Refereed) Published
Abstract [en]

Yeast is exposed to changing environmental conditions and must adapt its genetic program to provide a homeostatic intracellular environment. An important stress for yeast in the wild is high osmolarity. A key response to this stress is increased mRNA stability primarily by the inhibition of deadenylation. We previously demonstrated that mutations in decapping activators (edc3∆ lsm4∆C), which result in defects in P body assembly, can destabilize mRNA under unstressed conditions. We wished to examine whether mRNA would be destabilized in the edc3∆ lsm4∆C mutant as compared to the wild-type in response to osmotic stress, when P bodies are intense and numerous. Our results show that the edc3∆ lsm4∆C mutant limits the mRNA stability in response to osmotic stress, while the magnitude of stabilization was similar as compared to the wild-type. The reduced mRNA stability in the edc3∆ lsm4∆C mutant was correlated with a shorter PGK1 poly(A) tail. Similarly, the MFA2 mRNA was more rapidly deadenylated as well as significantly stabilized in the ccr4∆ deadenylation mutant in the edc3∆ lsm4∆C background. These results suggest a role for these decapping factors in stabilizing mRNA and may implicate P bodies as sites of reduced mRNA degradation.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-132468 (URN)10.1038/srep44395 (DOI)000396229600001 ()
Funder
Magnus Bergvall Foundation, 2016-01694Carl Tryggers foundation , 15:347Swedish Research Council, 621-2010-4602
Available from: 2017-03-14 Created: 2017-03-14 Last updated: 2017-05-12Bibliographically approved

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