The University of Sheffield
Department of Molecular Biology and Biotechnology

Yeast Genetics


Dr P J Mitchell
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The lab’s research addresses the nature of RNA surveillance processes in eukaryotic cells. These are quality control systems that recognise and degrade incorrectly synthesised or damaged RNA molecules. If left unchecked, such faulty RNAs could lead to the production of truncated and potentially toxic protein products or block essential cellular processes. A major component of the cellular armoury for the degradation of faulty RNA molecules is a conserved eukaryotic nuclease complex called the exosome (Mitchell et al., 1997; Mitchell and Tollervey, 2003). We have recently focussed on the RNA surveillance function of Rrp47/Lrp1, an exosome-associated protein that was initially shown to play an important role in correct 3’ end formation of stable RNAs (Mitchell et al., 2003). Rrp47 represents a novel class of nucleic acid-binding proteins that shows specificity for structured nucleic acids but binds RNA and DNA with comparable affinity (Stead et al., 2007). The protein comprises an N-terminal helical bundle and a poorly structured C-terminal region. The N-terminal domain is sufficient for normal cell growth, while residues within the C-terminal region are required for stable interaction with RNA or DNA in vitro (Figure 1) and for interaction with components of the transcriptional machinery or assembled RNP particles that may help target the exosome to its substrates. We have also initiated studies to address the processes by which RNA is degraded or repaired after exposure of cells to mutagenic agents.

 

The lab uses molecular biological, genetic and biochemical techniques on the model organism Saccharomyces cerevisiae (baker’s yeast) to address the molecular mechanisms of RNA surveillance in eukaryotes.
  1. Mitchell et al. (1997) The exosome: a conserved eukaryotic RNA processing complex containing multiple 3’->5’ exoribonucleases. Cell 91, 457-466.
  2. Mitchell, P. and Tollervey, D. (2003) An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3’->5’ degradation. Mol. Cell 11, 1405-1413.
  3. Mitchell et al. (2003) Rrp47p is an exosome-associated protein required for the 3’ processing of stable RNAs. Mol. Cell. Biol. 23, 6982-6992.

 

RNA binding analysis Figure 1: The C-terminal region of Rrp47 is required for stable RNA binding in vitro.
Full-length and C-terminally truncated Rrp47 mutants (I162X, G181X) were expressed and purified as recombinant proteins and tested for RNA binding activity in vitro. (A) slot blot analysis of RNA present in the bound (B) and free (F) fractions upon incubation with increasing concentrations of Rrp47. (B) Quantitative analysis of the binding data. Data points shown are the average of three independent assays.

 

Selected Publications

The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein. Stead JA, Costello JL, Livingstone MJ, Mitchell P. Nucleic Acids Res. 2007;35(16):5556-67.