Functional characterisation of RNA helicases in the remodelling of pre-ribosomal subunits
by Lukas Brüning
Date of Examination:2017-12-08
Date of issue:2018-07-13
Advisor:Prof. Dr. Markus Bohnsack
Referee:Prof. Dr. Ralf Ficner
Referee:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Jörg Stülke
Referee:Prof. Dr. Michael Thumm
Referee:Prof. Dr. Gerhard Braus
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Abstract
English
DEAD/H-box RNA helicases are ubiquitously expressed enzymes, which are characterised by a conserved helicase core of two RecA-like domains containing sequence motifs required for substrate binding and hydrolysis of NTP. Since this helicase core predominantly interacts with the sugar phosphate backbone of its target RNA, additional N- and/or C-terminal ancillary domains are thought to confer substrate specificity and/or mediate cofactor interactions. On the molecular level, some RNA helicases were shown to have unwinding activity, but others can also anneal RNA duplexes or mediate the release of proteins from protein-RNA complexes. Through their key functions in structural remodelling of RNAs and RNP complexes, RNA helicases are implicated in all aspects of RNA metabolism including transcription, translation, pre-mRNA splicing, RNA turnover and ribosome biogenesis. The biogenesis of ribosomes is a complex and energy-consuming process, which involves the synthesis and processing of four ribosomal RNAs (rRNAs) and their assembly with approximately 80 ribosomal proteins. Ribosome synthesis involves in excess of 200 biogenesis cofactors, including snoRNPs that introduce rRNA modifications co-transcriptionally as well as enzymes such as nucleases, NTPases and RNA helicases, which act in a strict hierarchical order to mediate the correct assembly and maturation of 40S and 60S subunits. In the yeast Saccharomyces cerevisiae, 21 RNA helicases are proposed to act during the synthesis of ribosomes, where they likely contribute to different steps along the maturation pathway. Some RNA helicases are required for the release of specific snoRNPs from pre-ribosomal complexes, whereas other RNA helicases are suggested to be involved in structural rearrangements of rRNA and remodelling of pre-ribosomal complexes. However, many of the RNA helicases implicated in ribosome biogenesis remain uncharacterised and the lack of information about their binding sites on pre-ribosomal complexes has impeded further functional characterisation of these proteins. In this work, we focused on three essential RNA helicases, Has1, Spb4 and Mak5, which are implicated in both SSU and LSU biogenesis (Has1) or in the later stages of LSU biogenesis (Spb4 and Mak5). Using a crosslinking technique with subsequent analysis of cDNA (CRAC) we revealed their putative binding sites on pre-ribosomal complexes. The observed CRAC sites for Has1 were consistent with its previously reported functions in the release the U14 snoRNA from pre-40S particles and regulating the release of a subset of trans-acting ribosome biogenesis factors from pre-60S complexes, but the identification of two Has1 crosslinking sites in the 18S rRNA suggests that Has1 may have an additional function in the biogenesis of small ribosomal subunits. DMS structure probing experiments demonstrated that the crosslinking sites identified for Spb4 and Mak5 by CRAC are bona-fide protein binding sites. In the case of Spb4, the structure probing data further suggest that one of the identified crosslinking sites likely represents a binding platform for the helicase whereas the other crosslinking site might represent a region of the pre-rRNA that is remodelled by Spb4. Interestingly, this rRNA region is in close proximity to the known binding site of the ribosome biogenesis factors Nog2 and Arx1, suggesting that Spb4 may play a direct role in recruitment or release of these proteins from pre-60S complexes. Excitingly, for Mak5, our data have revealed a function of the helicase in restructuring a region of the pre-ribosome to enable recruitment of the ribosomal protein Rpl10 to cytoplasmic ribosomal particles. Together, these findings extend the range of functions linked to RNA helicases in ribosome biogenesis and add to the understanding of important events during assembly of the large ribosomal subunit.
Keywords: Ribosome Biogenesis; RNA helicases; RNA biology