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Nucleolar Ribosome Assembly
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
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 [en]
Ribosome biogenesis, RNA, Nucleolus
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-145639ISBN: 978-91-7649-921-4 (print)ISBN: 978-91-7649-922-1 (electronic)OAI: oai:DiVA.org:su-145639DiVA: diva2:1131496
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
List of papers
1. 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
Show others...
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: 2017-08-14Bibliographically approved
2. Linker 2 of the eukaryotic pre-ribosomal processing factor Mrd1p is an essential interdomain functionally coupled to upstream RNA Binding Domain 2 (RBD2)
Open this publication in new window or tab >>Linker 2 of the eukaryotic pre-ribosomal processing factor Mrd1p is an essential interdomain functionally coupled to upstream RNA Binding Domain 2 (RBD2)
2017 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 4, e0175506Article in journal (Refereed) Published
Abstract [en]

Ribosome synthesis is an essential process in all cells. In Sacharomyces cerevisiae, the precursor rRNA, 35S pre-rRNA, is folded and assembled into a 90S pre-ribosomal complex. The 40S ribosomal subunit is processed from the pre-ribosomal complex. This requires concerted action of small nucleolar RNAs, such as U3 snoRNA, and a large number of transacting factors. Mrd1p, one of the essential small ribosomal subunit synthesis factors is required for cleavage of the 35S pre-rRNA to generate 18S rRNA of the small ribosomal subunit. Mrd1p is evolutionary conserved in all eukaryotes and in yeast it contains five RNA Binding Domains (RBDs) separated by linker regions. One of these linkers, Linker 2 between RBD2 and RBD3, is conserved in length, predicted to be structured and contains conserved clusters of amino acid residues. In this report, we have analysed Linker 2 mutations and demonstrate that it is essential for Mrd1p function during pre-ribosomal processing. Extensive changes of amino acid residues as well as specific changes of conserved clusters of amino acid residues were found to be incompatible with synthesis of pre-40S ribosomes and cell growth. In addition, gross changes in primary sequence of Linker 2 resulted in Mrd1p instability, leading to degradation of the N-terminal part of the protein. Our data indicates that Linker 2 is functionally coupled to RBD2 and argues for that these domains constitute a functional module in Mrd1p. We conclude that Linker 2 has an essential role for Mrd1p beyond just providing a defined length between RBD2 and RBD3.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-143600 (URN)10.1371/journal.pone.0175506 (DOI)000399375800063 ()28388671 (PubMedID)
Available from: 2017-05-31 Created: 2017-05-31 Last updated: 2017-08-14Bibliographically approved
3. Mrd1 is involved in restructuring the U3 snoRNP and central regions in the 90S pre-ribosome
Open this publication in new window or tab >>Mrd1 is involved in restructuring the U3 snoRNP and central regions in the 90S pre-ribosome
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-145622 (URN)
Available from: 2017-08-14 Created: 2017-08-14 Last updated: 2017-08-14Bibliographically approved
4. High-throughput RNA structure probing reveals critical folding events during early 60S ribosome assembly in yeast
Open this publication in new window or tab >>High-throughput RNA structure probing reveals critical folding events during early 60S ribosome assembly in yeast
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-145624 (URN)
Available from: 2017-08-14 Created: 2017-08-14 Last updated: 2017-08-14Bibliographically approved

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