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Investigating the protein 'assistants' of a modern eukaryotic ribozyme

Reference	BB/F020074/1
Principal Investigator / Supervisor	Dr Johanna Avis
Co-Investigators / Co-Supervisors	Dr Josip Lovric
Institution	The University of Manchester
Department	School of Medical Sciences
Funding type	Research
Value (£)	402,938
Status	Completed
Type	Research Grant
Start date	01/08/2008
End date	31/07/2011
Duration	36 months

Abstract

The human genome project has revealed that the majority of transcribed RNA molecules are non-coding RNAs (ncRNAs) that are emerging as key regulators of gene expression and genome stability. Importantly, ncRNAs associate with proteins, as ribonucleoprotein particles (RNPs), and a complex interplay of protein-RNA interactions thus confers full function. Given their number, diversity and complexity, research into RNPs is vital yet relatively embryonic. Here, we study a eukaryotic RNP, RNase MRP, of which the major function is pre-rRNA cleavage. Remarkably, the enzyme shares high similarity in RNA structure and protein content to eukaryotic RNase P (cleaves pre-tRNA). Bacterial RNase P was the first identified cellular ribozyme. In comparison, the RNAs of its eukaryotic counterpart and the related RNase MRP are poor catalysts, hence the main hypothesis here is that a greater number of proteins are required to assist them. Using recombinant subunits, we have mapped 1:1 interactions and identified subunits that are central to a common RNase P/MRP architecture. Our fundamental interest now lies in determining the assembly and organisation of proteins on MRP RNA forming the holoenzyme and how proteins 'assist' ribozyme activity. To address these interests, we will: 1) assemble recombinant yeast RNase MRP and sub-complexes thereof; 2) purify native RNase MRP from yeast for comparative analysis with recombinant enzyme; 3) assess MRP RNA cleavage activity in the context of different assembly states and thus determine, (a) if the RNA subunit alone exhibits activity, (b) the minimum number of protein subunits required for effective activity and (c) the contribution of additional protein subunits to RNA cleavage efficiency; 4) identify protein-RNA contacts in RNase MRP to gain more insight into molecular organisation and subunit function. The work represents a timely opportunity to truly begin to dissect the structure and function of an ncRNA and its associated proteins.

Summary

The central dogma of life is that genes (DNA) are transcribed into RNA molecules, which then encode proteins, the molecules that ultimately provide cellular structure and function. This is 'gene expression'. The human genome project has revealed that just 2% of our DNA expresses proteins. Despite this statistic, at least 40% of the DNA is nonetheless transcribed into RNA. It transpires that production of this 'non-coding' RNA (ncRNA) is not futile; rather ncRNAs can serve important functions in regulation of gene expression and genome stability across all life kingdoms. For example, the discovery of short interfering RNAs (siRNAs) has revealed a mechanism for gene silencing (inhibition of expression) that few envisaged only a decade ago and which is proving a valuable research tool. However, ncRNAs have a breadth of function that extends beyond that of siRNAs, regulating diverse cellular activities. A usual feature of ncRNAs is that their function requires association with proteins, forming complexes called ribonucleoprotein particles (RNPs). The proteins protect and stablise the RNA and also contribute to their function and regulation. In order to fully understand ncRNA function, therefore, it is crucial to study the RNA in the context of its complex with proteins. Here, we study a ncRNA that functions in production of ribosomal RNA (rRNA), part of the essential protein synthesising machine; the ribosome. In all higher organisms, a precursor form of rRNA, pre-rRNA, has to be cut into smaller rRNA molecules, modified (altered in chemical structure), folded correctly and finally used to make ribosomes. The key participants in this ribosome biogenesis process are >100 ncRNAs, classified as 'small nucleolar' RNAs (snoRNAs). The snoRNA we are studying is called MRP RNA and it associates, in higher organisms, with approximately 10 proteins to form a functional RNP called ribonuclease (RNase) MRP. RNase MRP carries out one of the pre-rRNA cuts, doing so at a precise site. Of added interest, is that it is the MRP RNA component that actually performs this pre-rRNA cleavage; it acts directly as the catalyst. The discovery that RNA, as well as protein, can act as an enzyme has caused considerable interest and excitement, leading to new fields of research into such 'ribozymes', both natural and synthetic. Catalytic RNA has implications for the origins of life (coding and catalytic properties would be useful in a 'first' biological molecule) and as a modern research and therapeutic tool. Our study can effectively be broken down into three aspects: 1) how do the protein subunits of RNase MRP associate with MRP RNA to form an RNP? 2) What insight can this give us into conversion of ncRNAs into RNPs in general? 3) How does association of MRP RNA with a protein complex assist the cleavage of pre-rRNA? This aspect should reveal modes by which proteins can add functionality to an ncRNA, and in particular here, to a ribozyme. More long-term benefits associated with a study of RNase MRP relate, firstly, to its ribozyme properties. Ribozymes have been exploited to develop research and therapeutic tools, modifying them to target desired RNAs (e.g., an RNA that encodes a disease-causing protein). Understanding how RNase MRP recognises and cleaves RNA targets (pre-rRNA is not its only target) is a prerequisite for such biotechnological applications. Secondly, the importance of efficient and accurate RNA cleavage by RNase MRP in humans is exemplified by at least one form of dwarfism that is attributable to MRP RNA mutations. This disease, and the function of about 40% of our DNA, can only truly be understood by studies such as this that aim to unravel how an ncRNA works. The diverse experimental programme, supported by the interests and skills of the investigators, will provide invaluable training for a postdoctoral scientist in a range of highly desirable skills in post-genomic science.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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