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Eukaryotic Ribosome Biogenesis: Focus on the function of the assembly factor Mrd1p
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
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 [en]
Ribosome biogenesis, nucleolus, pre-rRNA processing, RNA-binding proteins, Mrd1
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-75829ISBN: 978-91-7447-509-8 (print)OAI: oai:DiVA.org:su-75829DiVA: diva2:524227
Public defence
2012-06-07, De Geersalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (English)
Opponent
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
List of papers
1. Mrd1p binds to pre-rRNA early during transcription independent of U3 snoRNA and is required for compaction of the pre-rRNA into small subunit processomes
Open this publication in new window or tab >>Mrd1p binds to pre-rRNA early during transcription independent of U3 snoRNA and is required for compaction of the pre-rRNA into small subunit processomes
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2008 (English)In: Nucleic Acids Research, Vol. 36, no 13, 4364-4380 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-17830 (URN)doi:10.1093/nar/gkn384 (DOI)000257964500021 ()
Available from: 2009-01-21 Created: 2009-01-21 Last updated: 2012-05-03Bibliographically approved
2. Mrd1p Is Required for Release of Base-Paired U3 snoRNA within the Preribosomal Complex
Open this publication in new window or tab >>Mrd1p Is Required for Release of Base-Paired U3 snoRNA within the Preribosomal Complex
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2009 (English)In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 29, no 21, 5763-5774 p.Article in journal (Refereed) Published
Abstract [en]

In eukaryotes, ribosomes are made from precursor rRNA (pre-rRNA) and ribosomal proteins in a maturation process that requires a large number of snoRNPs and processing factors. A fundamental problem is how the coordinated and productive folding of the pre-rRNA and assembly of successive pre-rRNA-protein complexes is achieved cotranscriptionally. The conserved protein Mrd1p, which contains five RNA binding domains (RBDs), is essential for processing events leading to small ribosomal subunit synthesis. We show that full function of Mrd1p requires all five RBDs and that the RBDs are functionally distinct and needed during different steps in processing. Mrd1p mutations trap U3 snoRNA in pre-rRNP complexes both in base-paired and non-base-paired interactions. A single essential RBD, RBD5, is involved in both types of interactions, but its conserved RNP1 motif is not needed for releasing the base-paired interactions. RBD5 is also required for the late pre-rRNP compaction preceding A2 cleavage. Our results suggest that Mrd1p modulates successive conformational rearrangements within the pre-rRNP that influence snoRNA-pre-rRNA contacts and couple U3 snoRNA-pre-rRNA remodeling and late steps in pre-rRNP compaction that are essential for cleavage at A0 to A2. Mrd1p therefore coordinates key events in biosynthesis of small ribosome subunits.

Place, publisher, year, edition, pages
American Society for Microbiology, 2009
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-35367 (URN)10.1128/MCB.00428-09 (DOI)000270716800012 ()
Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2012-05-03Bibliographically approved
3. Multiple rna interactions position mrd1 at the site of the small subunit pseudoknot within the 90s pre ribosome
Open this publication in new window or tab >>Multiple rna interactions position mrd1 at the site of the small subunit pseudoknot within the 90s pre ribosome
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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.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-88326 (URN)10.1093/nar/gks1129 (DOI)000314121100048 ()
Funder
Swedish Research Council
Note

AuthorCount:9;

Available from: 2013-03-18 Created: 2013-03-12 Last updated: 2017-07-28Bibliographically approved
4. Evolutionary Conservation of the Ribosomal Biogenesis Factor Rbm19/Mrd1: Implications for Function
Open this publication in new window or tab >>Evolutionary Conservation of the Ribosomal Biogenesis Factor Rbm19/Mrd1: Implications for Function
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 9, e43786Article in journal (Refereed) Published
Abstract [en]

Ribosome biogenesis in eukaryotes requires coordinated folding and assembly of a pre-rRNA into sequential pre-rRNA-protein complexes in which chemical modifications and RNA cleavages occur. These processes require many small nucleolar RNAs (snoRNAs) and proteins. Rbm19/Mrd1 is one such protein that is built from multiple RNA-binding domains (RBDs). We find that Rbm19/Mrd1 with five RBDs is present in all branches of the eukaryotic phylogenetic tree, except in animals and Choanoflagellates, that instead have a version with six RBDs and Microsporidia which have a minimal Rbm19/Mrd1 protein with four RBDs. Rbm19/Mrd1 therefore evolved as a multi-RBD protein very early in eukaryotes. The linkers between the RBDs have conserved properties; they are disordered, except for linker 3, and position the RBDs at conserved relative distances from each other. All but one of the RBDs have conserved properties for RNA-binding and each RBD has a specific consensus sequence and a conserved position in the protein, suggesting a functionally important modular design. The patterns of evolutionary conservation provide information for experimental analyses of the function of Rbm19/Mrd1. In vivo mutational analysis confirmed that a highly conserved loop 5-beta 4-strand in RBD6 is essential for function.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-82430 (URN)10.1371/journal.pone.0043786 (DOI)000308738500022 ()
Note

AuthorCount:5;

Available from: 2012-11-14 Created: 2012-11-14 Last updated: 2017-12-07Bibliographically approved

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