The case for membrane-associated RNA-binding proteins in Saccharomyces cerevisiae

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Dokumentart: PhDThesis
Date: 2021-08-21
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Biochemie
Advisor: Jansen, Ralf-Peter (Prof. Dr.)
Day of Oral Examination: 2019-08-21
DDC Classifikation: 500 - Natural sciences and mathematics
Keywords: Nucleinsäuren
Other Keywords:
mRNA localization
Protein translation
Endoplasmic reticulum
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The endoplasmic reticulum (ER) is the site of protein synthesis for secretory and membrane proteins. I aimed to identify RNA-binding proteins (RBPs) in yeast that associate with ER and to assess their role in mRNA localization and/or translation at ER. I identified several RBPs that partially colocalize with ER such as Khd1p, Mrn1p, Whi3p, Pbp2p, Slf1p, Ngr1p, and Puf1p. Only the deletion of Khd1p affected the localization of mRNAs such as MID2 and SLG1 to ER. Additionally, MID2 can localize to ER in a translation-independent manner. In contrast to the popular SRP model, RNA seq analysis in combination with qPCR and microscopic data revealed that mRNAs coding for nuclear-related proteins can also localize to ER. By deleting ER-associated RBPs such as Mrn1p, Whi3p, Pbp2p, Slf1p, and Ngr1p, I see an upregulation of mRNAs coding for nuclear-encoded MRPs and at the same time a loss in the protein levels of Mrpl39p, Mrpl44p, and Img2p. Furthermore, there is an absence of transcription for mitochondrial-encoded COX genes and the fivefold deletion strain cannot utilize nonfermentable carbon source such as glycerol. I propose that that Mrn1p, Whi3p, Pbp2p, Slf1p, and Ngr1p are involved in controlling the expression of nuclear-encoded MRPs. For ASH1 E3 LE recognition by She2p/She3p, the she2p tetramer uses two identical sets of amino acids (N36, R43, R52, K60, and R63) to contact RNA, while its protruding helix seems to be important for interaction with She3p. Mutations in the contact sites in She3p revealed that they were important for mRNA localization in vivo.

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