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L7Ae- and LSm-RNA interactomes of Sulfolobus acidocaldarius
The archaeal L7Ae and Sm-like proteins (LSm) are universal RNA-binding proteins. L7Ae stabilizes non-coding RNA species, including ribosomal RNA, by recognizing a structural RNA motif, termed kink-turn (k-turn). Sm family proteins, like bacterial Hfq and eukaryotic Sm/LSm, are involved in multiple R...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2017
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| Zusammenfassung: | The archaeal L7Ae and Sm-like proteins (LSm) are universal RNA-binding proteins. L7Ae stabilizes non-coding RNA species, including ribosomal RNA, by recognizing a structural RNA motif, termed kink-turn (k-turn). Sm family proteins, like bacterial Hfq and eukaryotic Sm/LSm, are involved in multiple RNA-related processes including small RNA (sRNA)-based translational regulation, mRNA decay or splicing. However, the function of the archaeal members is elusive.
Using RNA-immunoprecipitation sequencing (RIP-Seq) methodology, this thesis aimed to identify the global RNA interaction partners (RNA interactome) of L7Ae and the three LSm proteins of the thermoacidophilic archaeon Sulfolobus acidocaldarius. Besides many known non-coding RNAs, the SRP RNA was identified as a novel binding partner of the L7Ae protein. Mobility shift assays demonstrated L7Ae binding to a k-turn motif that was found to be conserved among archaeal SRP RNAs. Interestingly, mRNAs, including the l7ae transcript, were enriched in the RIP-Seq analysis and found to comprise putative k-turns that facilitate L7Ae binding. In vivo studies showed that L7Ae autoregulates the translation of its mRNA by binding to a k-turn motif in the 5' untranslated region. A GFP reporter system was established in Escherichia coli that verified the conservation of L7Ae-mediated feedback regulation in archaea and provides a new tool for the modulation of synthetic gene circuits in bacteria. Mobility shift assays confirmed binding of L7Ae to a k-turn in the transcript of nop5-fibrillarin, suggesting that the synthesis of all C/D box sRNP core proteins (L7Ae, Nop5 and fibrillarin) is regulated by L7Ae. These studies revealed the regulation of mRNA translation as a novel function of the archaeal L7Ae protein.
The LSm RIP-Seq study found mRNAs and sRNAs as LSm1 and LSm2 interactors, including a recently reported sRNA that regulates biofilm formation in S. acidocaldarius. No RNA-binding capacity was observed for LSm3. A computational analysis of the interaction partners identified the U-rich 3' termination signal of RNAs and a motif composed of UAG triplets as potential LSm binding sites, which was verified by mobility shift assays. Knock-out studies revealed that only the lsm3 gene is dispensable, whereas the genes for lsm1 and lsm2 seem to be essential. Mutant strains producing tagged versions of LSm1 and LSm2 displayed a pleiotropic phenotype. In resemblance to the roles of bacterial Hfq and eukaryotic LSm proteins, this study provides hints that archaeal LSm proteins may be involved in mRNA degradation, C/D box sRNA biogenesis and sRNA-regulated processes, like tRNA maturation and translational regulation of mRNAs. |
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| Umfang: | 145 Seiten |
| DOI: | 10.17192/z2018.0118 |