Location function and structural properties of the Nhm1 nuclear decapping enzyme in fission yeast

The information contained in the genes of living cells has to be converted into cellular components that form structures and enable biochemical reactions to take place. This process is called gene expression and it is vital to all life. Gene expression comprises two main steps, called transcription and translation. In transcription, the information in the DNA sequences of the genes is converted into equivalent sequences in so-called messenger RNA (mRNA) molecules. In translation, the mRNA molecules are 'read' by a large molecular structure called the ribosome, which uses the information to dictate the synthesis of proteins. Each cell has to synthesize and break down its mRNA molecules, because this continuous turnover allows the cell to adapt its patterns of gene expression more rapidly to changes in the demand for particular proteins which are needed under certain growth conditions. Our work will improve understanding of an important enzyme that is involved in the cellular metabolism of such RNA molecules. We intend to use methods of structural biology, biochemistry and microscopy to work out where in the cell this enzyme works, how it chooses the RNA molecules it cleaves on the basis of their length, and how its structure enables it to work efficiently as an enzyme.

The m7G cap at the 5'end of mRNA plays an important role in many eukaryotic processes, including translation, polyadenylation, splicing, mRNA export and mRNA decay. The removal of the 5'cap (decapping) is therefore also of potential significance to all of these processes. The 'scavenger'-type pyrophosphatase in S.cerevisiae, Dcs1, decaps short capped RNA species (length less than 10 nucleotides most efficiently) as well as m7GDP, the product of Dcp1/Dcp2-catalysed decapping of longer mRNAs. This overall group of short m7G-oligoribonucleotides is known to exert inhibitory effects on a number of the processes mentioned above. Moreover, we have obtained preliminary evidence indicating that m7G-misincorporation into nucleic acids is linked to accelerated cell senescence. Schizosaccharomyces pombe has a pyrophosphatase of this type called Nhm1, which is unusual in that it is capable of decapping much longer RNA substrates than can be cleaved by its counterparts in budding yeast and mammalian cells and it is found to be preominantly nuclear under mid-log growth conditions. These properties suggest that Nhm1 may operate on nuclear RNA species as well as cytoplasmic substrates, and thus may be involved in the metabolism of a range of types of RNA. We propose to: Determine the subcellular localisation of Nhm1 in fission yeast as a function of growth phase and stress conditions. The resulting information will be vital to our understanding of this enzyme's cellular function(s). Examine the molecular mechanism underpinning the ability of enzymes of this type to discriminate different lengths of RNA substrate thus elucidating, for the first time, how a homodimeric enzyme can effectively measure the length of an RNA polymer. Investigate the role of N-terminal domain mobility in the Nhm1 dimer - how does it influence catalysis and length discrimination? Does it need to flip-flop between states in order to promote catalysis?