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Evolutionary Conservation of the Ribosomal Biogenesis Factor Rbm19/Mrd1: Implications for Function
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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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.

Place, publisher, year, edition, pages
2012. Vol. 7, no 9, e43786
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-82430DOI: 10.1371/journal.pone.0043786ISI: 000308738500022OAI: oai:DiVA.org:su-82430DiVA: diva2:567832
Note

AuthorCount:5;

Available from: 2012-11-14 Created: 2012-11-14 Last updated: 2017-12-07Bibliographically 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)
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
2. Nucleolar Ribosome Assembly
Open this publication in new window or tab >>Nucleolar Ribosome Assembly
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ribosomes are macromolecular machines that are responsible for production of every protein in a living cell. Yet we do not know the details about how these machines are formed. The ribosome consists of four RNA strands and roughly 80 proteins that associate with each other in the nucleolus and form pre-ribosomal complexes. Eukaryotes, in contrast to prokaryotes, need more than 200 non-ribosomal factors to assemble ribosomes. These associate with pre-ribosomal complexes at different stages as they travel from the nucleolus to the cytoplasm and are required for pre-rRNA processing. We do however lack knowledge about the molecular function of most of these factors and what enables pre-rRNA processing. Especially, information is missing about how non-ribosomal factors influence folding of the pre-rRNA and to what extent the pre-ribosomal complexes are restructured during their maturation. 

This thesis aims to obtain a better understanding of the earliest events of ribosome assembly, namely those that take place in the nucleolus. This has been achieved by studying the essential protein Mrd1 by mutational analysis in the yeast Saccharomyces cerevisiae as well as by obtaining structural information of nucleolar pre-ribosomal complexes. Mrd1 has a modular structure consisting of multiple RNA binding domains (RBDs) that we find is conserved throughout eukarya. We show that an evolutionary conserved linker region of Mrd1 is crucial for function of the protein and likely forms an essential module together with adjacent RBDs. By obtaining structural information of pre-ribosomal complexes at different stages, we elucidate what structuring events occur in the nucleolus.  We uncover a direct role of Mrd1 in structuring the pre-rRNA in early pre-ribosomal complexes, which provides an explanation for why pre-rRNA cannot be processed in Mrd1 mutants.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular BiosciDepartment of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2017. 70 p.
Keyword
Ribosome biogenesis, RNA, Nucleolus
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-145639 (URN)978-91-7649-921-4 (ISBN)978-91-7649-922-1 (ISBN)
Public defence
2017-09-20, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

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

Available from: 2017-08-28 Created: 2017-08-14 Last updated: 2017-08-28Bibliographically approved

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