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Research Summary

Post-transcriptional Gene Regulation

Post-transcriptional processes play a crucial role in controlling gene expression in all organisms. Our research is aimed at elucidating the molecular mechanisms by which such control is imposed. We are particularly interested in two important means by which genes are regulated post-transcriptionally: messenger RNA degradation and repression by microRNAs and siRNAs. The goal of our investigations is to identify and characterize the proteins, RNA elements, and molecular mechanisms that govern these key regulatory processes in bacterial and mammalian cells.

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mRNA degradation

In bacteria, the lifetimes of mRNAs can differ by more than an order of magnitude, with profound consequences for gene expression. For many years it had been assumed that bacterial mRNA degradation begins with endonucleolytic cleavage at internal sites. However, our recent findings have challenged that view by showing that mRNA decay can instead be triggered by a prior non-nucleolytic event that marks transcripts for rapid turnover: the rate-determining conversion of the 5' terminus from a triphosphate to a monophosphate. In Escherichia coli, this modification creates better substrates for the endonuclease RNase E, whose cleavage activity is greatly enhanced when the RNA 5' end is monophosphorylated, whereas in Bacillus subtilis it triggers 5'-exonucleolytic degradation by RNase J. We have identified the pyrophosphate-removing hydrolase responsible for that 5'-terminal event, the first such bacterial enzyme ever characterized. The inability of this RNA pyrophosphohydrolase to modify 5' ends that are structurally sequestered by a stem-loop helps to explain the stabilizing influence of 5'-terminal base pairing on mRNA lifetimes in vivo. Interestingly, this master regulator of 5'-end-dependent mRNA degradation in bacteria not only catalyzes a process functionally reminiscent of eukaryotic mRNA decapping but also bears an evolutionary relationship to the eukaryotic decapping enzyme Dcp2.


MicroRNA function

Human cells contain hundreds of different microRNAs, short RNA molecules that function as negative genetic regulators. In animal cells, microRNAs act by annealing to mRNAs to which they are partially complementary. Our studies have shown that microRNAs inhibit gene expression not only by repressing translation but also by directing rapid poly(A) tail removal, thereby hastening mRNA degradation. Analogously, small interfering RNAs (siRNAs), the mediators of RNA interference, inhibit the function of fully complementary mRNAs both by guiding endonucleolytic cleavage of those messages and by repressing their translation. We have further shown that, in human cells, CCR4-NOT is the deadenylase whose action is stimulated by the multiprotein complex (RISC) that accompanies microRNAs to their mRNA targets. Interestingly, that complex appears to facilitate deadenylation not by recruiting CCR4-NOT to mRNA but rather by making the autonomous encounters of the deadenylase with poly(A) more productive. The ability of microRNAs to expedite deadenylation does not result from decreased translation; nor does translational repression by microRNAs require a poly(A) tail. These findings indicate that microRNAs utilize two distinct post-transcriptional mechanisms to downregulate gene expression.