Professor, Department of Microbiology
RNA has played a central role in biology since the origin of life on Earth. Research in the Belasco laboratory is aimed at elucidating the diverse molecular mechanisms employed by this vitally important family of macromolecules to control gene expression post-transcriptionally. Our strategy for achieving this goal is multifaceted, combining molecular biological, biochemical, biophysical, and genetic methods. We are particularly interested in understanding how gene expression in bacterial and mammalian cells is regulated by mRNA degradation.
5′-terminal deprotection of mRNA
In bacteria, the lifetimes of individual mRNAs can differ by up to two orders of magnitude, with profound consequences for gene expression. For many years it had been assumed that bacterial mRNA degradation always begins with endonucleolytic cleavage at internal sites. However, our findings have overturned that view by showing that mRNA decay is often triggered by a prior non-nucleolytic event that marks transcripts for rapid turnover: the stepwise conversion of the 5′ terminus from a triphosphate to a monophosphate. In Escherichia coli and related organisms, 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 and other bacterial species that lack RNase E, it enables 5′-exonucleolytic degradation by RNase J. We have discovered and characterized a family of RNA pyrophosphohydrolases (RppH) crucial for phosphate removal from the 5′ terminus. The inability of RppH to bind monophosphorylated 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. Current efforts are aimed at identifying the E. coli RNA triphosphatase(s) whose action stimulates subsequent ß phosphate removal by RppH and elucidating the mechanism by which a metabolic enzyme important for cell wall synthesis enhances the catalytic activity of RppH.
Linear scanning by RNase E
The diverse lifetimes of bacterial mRNAs seem difficult to reconcile with the relaxed cleavage-site specificity of RNase E, which can cut most single-stranded regions of RNA. Mounting evidence indicates that, in E. coli, rates of mRNA decay are determined not by the number or intrinsic quality of internal cleavage sites but rather by the ease with which RNase E can gain access to them. We have recently discovered that RNase E locates cleavage sites in monophosphorylated RNA by a novel monorail-like mechanism involving linear diffusion from the 5′ terminus along RNA segments that are single-stranded. Consequently, the rate of cleavage at those internal sites is governed not only by the ability of RNase E to initially bind the 5′ end but also by any obstacles that this key regulatory endonuclease may encounter as it scans downstream. We are now investigating the mechanism of scanning, the features of RNase E that enable it to diffuse linearly on RNA, and the characteristics of obstacles that determine their efficacy.
RNase E autoregulation
RNase E is an essential regulatory enzyme whose overproduction or underproduction can impair cell growth. To ensure a steady supply of this protein, E. coli has evolved a homeostatic mechanism for tightly regulating its synthesis by modulating the decay rate of rne (RNase E) mRNA in response to changes in cellular RNase E activity. Our studies have shown that feedback regulation by RNase E is mediated by the 361-nucleotide rne 5′ untranslated region (UTR), which can confer this property onto heterologous messages to which it is fused. The rne 5′ UTR is composed of several structural domains, two of which play critical roles in feedback regulation. However, these two domains are not themselves cleavage sites. We now wish to determine the mechanism by which they expedite RNase E cleavage elsewhere within the transcript and thereby control rne gene expression.
Professor, Department of Microbiology
PhD from Harvard University
Fellowship, Stanford University, Genetics
Fellowship, Harvard University, Chemistry
Molecular cell. 2019 Apr 18; 74(2):284-295.e5
Molecular cell. 2017 07 06; 67(1):44-54.e6
Journal of biological chemistry. 2016 Mar 04; 291(10):5038-48
Journal of biological chemistry. 2015 Apr 10; 290(15):9478-86
Nucleic acids research. 2015 Jan; 43(1):309-23
Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2013 May 28; 110(22):8864-9
Molecular cell. 2011 Sep 16; 43(6):940-9
Nature. 2008 Jan 17; 451(7176):355-8