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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
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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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. Vol. 5, no 10, 1388-1399 p.
Keyword [en]
P bodies, Deadenylation, Exosome, mRNA decapping, mRNA decay, mRNA stability
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-126827DOI: 10.1242/bio.020487ISI: 000388300900005PubMedID: 27543059OAI: oai:DiVA.org:umu-126827DiVA: diva2:1037457
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
In thesis
1. mRNA degradation factors as regulators of the gene expression in Saccharomyces cerevisiae
Open this publication in new window or tab >>mRNA degradation factors as regulators of the gene expression in Saccharomyces cerevisiae
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
mRNA nedbrytningsfaktorer som regulatorer av genexpression i Saccharomyces cerevisiae.
Abstract [en]

Messenger RNA degradation is crucial for the regulation of eukaryotic gene expression. It not only modulates the basal mRNA levels but also functions as a quality control system, thereby controlling the availability of mRNA for protein synthesis. In Saccharomyces cerevisiae, the first and the rate-limiting step in the process of mRNA degradation is the shortening of the poly(A) tail by deadenylation complex. After the poly(A) tail shortens, mRNA can be degraded either through the major 5' to 3' decapping dependent or the 3' to 5' exosome-mediated degradation pathway. In this thesis, we show some of the means by which mRNA decay factors can modulate gene expression.

First, Pat1 is a major cytoplasmic mRNA decay factor that can enter the nucleus and nucleo-cytoplasmically shuttle.  Recent evidence suggested several possible nuclear roles for Pat1. We analyzed them and showed that Pat1 might not function in pre-mRNA decay or pre-mRNA splicing, but it is required for normal rRNA processing and transcriptional elongation. We show that the mRNA levels of the genes related to ribosome biogenesis are dysregulated in the strain lacking Pat1, a possible cause of the defective pre-rRNA processing. In conclusion, we theorize that Pat1 might regulate gene expression both at the level of transcription and mRNA decay.

Second, Edc3 and Lsm4 are mRNA decapping activators and mRNA decay factors that function in the assembly of RNA granules termed P bodies. Mutations in mRNA degradation factors stabilize mRNA genome-wide or stabilize individual mRNAs. We demonstrated that paradoxically, deletion of Edc3 together with the glutamine/asparagine-rich domain of Lsm4 led to a decrease in mRNA stability. We believe that the decapping activator Edc3 and the glutamine/asparagine-rich domain of Lsm4 functions together, to modify mRNA decay pathway by altering cellular mRNA decay protein abundance or changing the mRNP composition or by regulating P bodies, to enhance mRNA stability.

Finally, mRNA decay was recently suggested to occur on translating ribosomes or within P bodies. We showed that mRNA degradation factors associate with large structures in sucrose density gradients and this association is resistant to salt and sensitive to detergent. In flotation assay, mRNA decay factors had buoyancy consistent with membrane association, and this association is independent of stress, translation, P body formation or RNA. We believe that such localization of mRNA degradation to membranes may have important implications in gene expression.

In conclusion, this thesis adds to the increasing evidence of the importance of the mRNA degradation factors in the gene expression.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2016. 66 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1844
Keyword
mRNA decapping, mRNA degradation, Pat1, Edc3, Lsm4, Lsm1-7, P bodies, Dcp2, Transcription, Ribosome biogenesis, Exosome.
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-127358 (URN)978-91-7601-560-5 (ISBN)
Public defence
2016-11-02, Hörsal E04, Unod R01, Umeå University, Umeå, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilCarl Tryggers foundation
Available from: 2016-11-11 Created: 2016-11-09 Last updated: 2016-11-24Bibliographically approved
2. Spatial control of mRNA stability in yeast
Open this publication in new window or tab >>Spatial control of mRNA stability in yeast
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
mRNA stability, P bodies, mRNA turnover, gene regulation, decapping factors
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-133894 (URN)978-91-7601-721-0 (ISBN)
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

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