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Multiple rna interactions position mrd1 at the site of the small subunit pseudoknot within the 90s pre ribosome
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
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2013 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 2, 1178-1190 p.Article in journal (Refereed) Published
Abstract [en]

Ribosomal subunit biogenesis in eukaryotes is a complex multistep process. Mrd1 is an essential and conserved small (40S) ribosomal subunit synthesis factor that is required for early cleavages in the 35S pre-ribosomal RNA (rRNA). Yeast Mrd1 contains five RNA-binding domains (RBDs), all of which are necessary for optimal function of the protein. Proteomic data showed that Mrd1 is part of the early pre-ribosomal complexes, and deletion of individual RBDs perturbs the pre-ribosomal structure. In vivo ultraviolet cross-linking showed that Mrd1 binds to the pre-rRNA at two sites within the 18S region, in helix 27 (h27) and helix 28. The major binding site lies in h27, and mutational analyses shows that this interaction requires the RBD1-3 region of Mrd1. RBD2 plays the dominant role in h27 binding, but other RBDs also contribute directly. h27 and helix 28 are located close to the sequences that form the central pseudoknot, a key structural feature of the mature 40S subunit. We speculate that the modular structure of Mrd1 coordinates pseudoknot formation with pre-rRNA processing and subunit assembly.

Place, publisher, year, edition, pages
2013. Vol. 41, no 2, 1178-1190 p.
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-88326DOI: 10.1093/nar/gks1129ISI: 000314121100048OAI: oai:DiVA.org:su-88326DiVA: diva2:611800
Funder
Swedish Research Council
Note

AuthorCount:9;

Available from: 2013-03-18 Created: 2013-03-12 Last updated: 2017-07-28Bibliographically approved
In thesis
1. Eukaryotic Ribosome Biogenesis: Focus on the function of the assembly factor Mrd1p
Open this publication in new window or tab >>Eukaryotic Ribosome Biogenesis: Focus on the function of the assembly factor Mrd1p
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The ribosome, the protein factory of the cell, is essential for all life forms. The ribosome is a large RNA-protein machine. It is built in a complex, multi-step process that involves a large number of accessory trans-acting factors and the synthesis consumes a considerable part of the cellular energy. The ribosomal RNA is transcribed as a large precursor rRNA (pre-rRNA) molecule that undergoes extensive processing during maturation, including chemical modifications, pre-rRNA cleavage events, pre-rRNA folding and assembly with ribosomal proteins. More than 200 non-ribosomal proteins and small nucleolar RNAs ensure a successful maturation of the two ribosomal subunits during a pathway that starts with coupled synthesis and processing of the pre-rRNA within the nucleolus. Processing continues through the nucleus and ends with the final maturation in the cytoplasm.

We have studied one of the eukaryotic ribosomal biogenesis proteins, Mrd1 to learn about its essential function in the pre-ribosome maturation process in the yeast, Saccharomyces cerevisiae. Mrd1 contains multiple RNA-binding domains and the protein and its modular design is conserved throughout eukarya. Evolution of Mrd1 is most likely coupled to a common eukaryotic way of producing ribosomes. Together with a large set of other factors, Mrd1 associates early with the nascent pre-rRNA and forms a 90S pre-ribosome that can be seen in Chromatin Miller spreads of active rRNA genes as large terminal knob structures on the growing pre-rRNA. In the absence of Mrd1, essential steps in pre-ribosome maturation cannot occur and small ribosomal subunits are not produced. We have demonstrated that Mrd1 interacts with the pre-rRNA in vivo at two specific sites within the 18S rRNA sequence, both located close in space to where the essential and universally conserved central pseudoknot of the small ribosomal subunit is formed. Furthermore, we have shown that Mrd1 influences the release of the U3 snoRNA from the pre-ribosome. U3 snoRNA is essential for synthesis of the small ribosomal subunit and is involved in pseudoknot formation. Our results show that Mrd1 is present within the pre-ribosome at a crucial location and that it is required for essential maturation steps. Based on our results, we hypothesize that Mrd1 modulates the pre-rRNA folding and assembly to assist pre-ribosome structures necessary for pseudoknot formation and early cleavages. This essential function is conserved in all eukaryotes. 

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University, 2012. 83 p.
Keyword
Ribosome biogenesis, nucleolus, pre-rRNA processing, RNA-binding proteins, Mrd1
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-75829 (URN)978-91-7447-509-8 (ISBN)
Public defence
2012-06-07, De Geersalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (English)
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Supervisors
Note

At the time of doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript. 

Available from: 2012-05-10 Created: 2012-04-30 Last updated: 2017-07-28Bibliographically approved

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Segerstolpe, ÅsaBjörk, PetraAndersson, Charlotta S.Högbom, MartinWieslander, Lars
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