NYU School of Medicine - Belasco Laboratory

Skirball Institute of Biomolecular Medicine
BELASCO LAB

Skirball Institute of Biomolecular Medicine

BELASCO LAB

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.

Belasco Lab links

mRNA degradation

5'-end dependence
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 mRNA degradation in E. coli 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. This modification creates better substrates for the endonuclease RNase E, whose cleavage activity is greatly enhanced when the RNA 5' end is monophosphorylated. We have identified the pyrophosphate-removing hydrolase responsible for that 5'-terminal event, the first such bacterial enzyme ever characterized. That the action of the pyrophosphohydrolase is impeded when the 5' end is 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 E. coli not only catalyzes a process functionally reminiscent of eukaryotic mRNA decapping but also bears an evolutionary relationship to the eukaryotic decapping enzyme Dcp2.

RNase E autoregulation
To ensure a steady supply of RNase E, E. coli and related bacteria have evolved a homeostatic mechanism for tightly regulating the synthesis of that important enzyme by modulating the decay rate of rne (RNase E) mRNA in response to changes in cellular RNase E activity. We have determined the secondary structure of the rne 5' untranslated region and identified the elements within it that function in cis to mediate feedback regulation by RNase E. In vitro studies with purified components indicate that these 5' UTR elements, like a 5' monophosphate, act directly to expedite RNA degradation by binding to RNase E and guiding it to nearby cleavage sites.

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 imperfectly 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. 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 suggest that microRNAs utilize two distinct post-transcriptional mechanisms to downregulate gene expression.

Small interfering RNAs (siRNAs), the mediators of RNA interference, are closely related to microRNAs. Although siRNAs were originally thought to inhibit the function of fully complementary messages solely by guiding endonucleolytic cleavage, our recent data indicate that they can also repress translation of those messages. In addition, we have found that the specificity of RNA interference by siRNAs is cell-type-dependent due to disparities in the tissue distribution and activity of the four Ago proteins that deliver siRNAs to their mRNA targets in human cells.

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.	Belasco Lab links Home Research Summary PI Bio Publications Lab Personnel Contact Us
mRNA degradation 5'-end dependence 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 mRNA degradation in E. coli 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. This modification creates better substrates for the endonuclease RNase E, whose cleavage activity is greatly enhanced when the RNA 5' end is monophosphorylated. We have identified the pyrophosphate-removing hydrolase responsible for that 5'-terminal event, the first such bacterial enzyme ever characterized. That the action of the pyrophosphohydrolase is impeded when the 5' end is 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 E. coli not only catalyzes a process functionally reminiscent of eukaryotic mRNA decapping but also bears an evolutionary relationship to the eukaryotic decapping enzyme Dcp2.
RNase E autoregulation To ensure a steady supply of RNase E, E. coli and related bacteria have evolved a homeostatic mechanism for tightly regulating the synthesis of that important enzyme by modulating the decay rate of rne (RNase E) mRNA in response to changes in cellular RNase E activity. We have determined the secondary structure of the rne 5' untranslated region and identified the elements within it that function in cis to mediate feedback regulation by RNase E. In vitro studies with purified components indicate that these 5' UTR elements, like a 5' monophosphate, act directly to expedite RNA degradation by binding to RNase E and guiding it to nearby cleavage sites.
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 imperfectly 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. 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 suggest that microRNAs utilize two distinct post-transcriptional mechanisms to downregulate gene expression. Small interfering RNAs (siRNAs), the mediators of RNA interference, are closely related to microRNAs. Although siRNAs were originally thought to inhibit the function of fully complementary messages solely by guiding endonucleolytic cleavage, our recent data indicate that they can also repress translation of those messages. In addition, we have found that the specificity of RNA interference by siRNAs is cell-type-dependent due to disparities in the tissue distribution and activity of the four Ago proteins that deliver siRNAs to their mRNA targets in human cells.