Identification and analysis of factors that regulate the activity of the yeast exosome complex of exoribonucleases

Lead Research Organisation: University of Sheffield
Department Name: Molecular Biology and Biotechnology

Abstract

Understanding biological processes at the molecular level remains one of the greatest challenges of the modern age. In addition to understanding what makes us tick, fundamental biological research can potentially aid the treatment or prevention of human diseases, lead to increased agricultural production/diversity, support environment management and trigger the development of new biology-based technologies. Central to all cellular biological processes is the flow of genetic information through what is known as the gene expression pathway. In this pathway, the linear DNA sequence of our genes is copied into a chemically similar but less stable molecule, called RNA. These short-lived gene copies, known as messenger RNA (mRNA), are then transported to highly specialised structures called ribosomes. Here, the information encoded within the mRNA is converted into functional protein molecules, which then carry out the specific processes required in the cell. A mistake at any point within this pathway can have very serious consequences for the cell. For example, a copying error inserted into the mRNA sequence can result in an incorrectly folded or shortened protein that is unable to perform its function correctly. Mistakes in gene expression occur rather often, even in normal, healthy cells. Therefore, it is essential that cells are able to recognise faulty mRNA molecules and destroy them efficiently. To monitor the production of their mRNA, cells have evolved a number of quality control systems that are known collectively as mRNA surveillance. These processes ensure that incorrectly produced mRNA molecules are selected out and rapidly degraded, thereby preventing the production of defective proteins. A major piece of the cell's armoury for degrading RNA is the exosome. The exosome consists of 10 different enzymes associated with one another in a complex. Packaged together, these enzymes can work more efficiently than they could on their own. The complex also allows coordinated regulation; all the enzymes can be switched on or swiched off at the same time. Previous studies have identified the components of the exosome complex and demonstrated its function in RNA degradation. However, little is known about how the complex is regulated. This research project will identify proteins that function either to promote the activity of the exosome or to inhibit its activity. The function of the identified proteins in exosome-dependent cellular processes such as mRNA surveillance will then be studied. Further studies on mRNA surveillance will increase our understanding of how gene expression is controlled in the cell. This knowledge will be of potential benefit in the design and development of new biotechnological strategies for protein production. Moreover, mRNA surveillance phenomena are directly linked to the basis of some prevalent human genetic disorders, such as breast cancer. The exosome is also a biological target of a successful anti-cancer drug. Hence, a knowledge of how the activity of the exosome complex is regulated may provide an important resource of information in developing future strategies in the treatment or prevention of disease.

Technical Summary

The exosome is a multienzyme complex of 3prime -5prime exoribonucleases that has been conserved throughout eukaryotic evolution. The exosome complex plays a key role in the 3prime end maturation of diverse stable RNAs, including 5.8S ribosomal RNA (rRNA), the small nuclear RNAs (snRNAs) required for pre-mRNA splicing and the small nucleolar RNAs (snoRNAs) that function in ribosome biogenesis. The exosome also functions in mRNA turnover pathways and plays a central role in RNA quality control pathways that degrade improperly processed or assembled RNAs. The diverse nature of the different exosome substrates and their very distinct fates (productive RNA synthesis or complete RNA degradation) suggests that the activity of the complex must be tightly regulated. How the activity of the exosome is regulated is, as yet, largely unknown. In vitro enzymatic studies on purified exosomes suggest two distinct regulatory mechanisms for this complex. Exosome complexes purified from cell lysates exhibit a rather weak exonucleolytic activity in vitro, given the robust nature of exosome-mediated processes in vivo. Biochemical fractionation studies have shown that a small subpopulation of exosome complexes exhibits very high specific activity. Since exosome complexes are reported to interact functionally with a number of different proteins, the high specific activity of this fraction of exosome complexes is predicted to be due to the presence of one or more additional, as yet uncharacterised, protein factors. The activity of exosome complexes can also be downregulated. Exosome complexes can bind to GTP and are specifically inhibited in the presence of this NTP. The research outlined in this proposal will employ nanoelectrospray MALDI/MS mass spectrometry and an established, robust in vitro exonuclease assay to identify proteins that copurify with the exosome complex and regulate its activity. Subsequent analyses on the identified proteins will characterise their biochemical function in vitro and analyse their role in exosome-mediated processes in vivo. It is estimated that proteins present in as little as approximately 1 percent of all exosome complexes will be readily detected by MALDI/MS in this study. Exosome complexes will be purified by affinity chromatography, using strains expressing an epitope-tagged form of the exosome component Rrp4p that contains a TEV protease recognition site. In vitro exonuclease assays will be performed on the affinity purified complexes using short, synthetic 5prime labelled RNA substrates and the reaction products will be resolved by PAGE. Exosome complexes with increased specific activity will be resolved by a combination of ion exchange chromatography and activity profiling. Exosome complexes and associated proteins will be resolved by PAGE and candidate regulatory proteins will be identified by MALDI/MS. Covalent exosome-GTP complexes will be generated by irradiation with ultraviolet light. Crosslinked proteins will be resolved from the exosome components by 2D-PAGE and identified by MALDI/MS, as above. Interactions between the exosome complex and the identified proteins will be confirmed by reverse tagging experiments, using specific antiserum raised against Rrp4p. The functions of the identified proteins will be analysed both in vitro and in vivo. The proteins will be expressed in recombinant form and tested for their putative function in vitro (stimulation of exosome exonuclease activity, GTPase activity or GTP binding). To analyse the protein's role in exosome function in vivo, RNA will be isolated from mutant strains before and after its genetic depletion or overexpression and analysed by Northern blot hybridisation. To analyse effects on mRNA surveillance pathways, experiments will be performed using available reporter constructs expressing defective mRNA transcripts or in characterised mutants that show conditional defects in mRNA synthesis or assembly.
 
Description Understanding biological processes at the molecular level remains one of the greatest challenges of the modern age.
Central to all cellular biological processes is the flow of genetic information through what is known as the gene expression
pathway. In this pathway, the genetic information that is stored in stretches of the cell's DNA called "genes" is copied into
short-lived RNA intermediates and then decoded to generate the proteins required by the cell at any given time.
A distinct set of key cellular RNA molecules are required to facilitate this flow of genetic information. These RNAs are
made from longer precursor molecules by the cell through the activity of enzymes called ribonucleases. In addition to the
productive synthesis of functional RNAs, some of these ribonucleases are also involved in the elimination of certain RNAs
from the cell. In some instances, this is due to RNA "quality control" processes that detect and eliminate incorrectly made
or assembled RNAs, or degrade those that lack crucial modifications normally incorporated after synthesis. In addition,
cells produce a huge raft of short-lived regulatory RNA molecules that are maintained at a low level through active
degradation.
The exosome is a key component of the cell's armoury of nucleases that both processes precursor RNA molecules to
mature species and degrades unwanted RNAs. Recently, detailed structures have been determined for the exosome and
we know which components of this complex have ribonuclease activity, i.e. the enzymatic activity to break down RNA. A
key outstanding question is how the complex is regulated to act both in a productive and in a destructive manner. The
focus of this research project lay in understanding how additional proteins may interact with and regulate the activity of
the exosome complex.
We have shown that a protein called Rrp47 can bind directly and simultaneously to both RNA and to Rrp6, one of the
exosome components that has ribonuclease activity. In the presence of Rrp47, Rrp6 degrades RNA more efficiently and
in a manner that is dependent upon the RNA binding activity of Rrp47. Using both biochemical and genetic experiments,
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we have determined which portions of the Rrp47 protein are required for specific functions, either by analysing purified
proteins in a test tube or by studying the RNAs in cells expressing variant forms of each protein. These data show that
one end of the Rrp47 protein binds directly to Rrp6, while the other end interacts with a number of proteins that are either
components of the molecular machinery involved in RNA production or RNA-binding proteins that are found associated
with correctly processed RNAs. The ability of Rrp47 to interact with these RNA-bound proteins correlates with proper
maturation of the RNA. Taken together, these results suggest three distinct mechanisms by which Rrp47 contributes to
the activity of the exosome. Firstly, Rrp47 interacts with both the exosome and the machinery that makes RNA, promoting
the recruitment of the exosome complex to the site of RNA production. Secondly, Rrp47 binds RNA and can present the
end of the RNA to Rrp6, thereby facilitating the degradation of the RNA. Thirdly, Rrp47 can sense the assembly of RNAbinding
proteins onto the correctly made RNA and ensure correct final trimming.
Exploitation Route This work was successfully continued in a research grant funded by the Wellcome Trust.
Sectors Agriculture, Food and Drink,Education,Energy,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description Wellcome Trust project Grant
Amount £265,261 (GBP)
Funding ID 088306/Z/09/Z 
Organisation The Wellcome Trust Ltd 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 11/2009 
End 08/2013
 
Title anti-Rrp47 antibody 
Description rabbit polyclonal raised against a recombinant His(6)-tagged Rrp47 
Type Of Material Antibody 
Year Produced 2013 
Provided To Others? Yes  
Impact The antiserum was used by us in the initial publication in 2007 and by another lab in a subsequent publication in 2013