Faculty Bibliography

The downregulation of gene expression by miRNAs and siRNAs is a complex process involving both translational repression and accelerated mRNA turnover, each of which appears to occur by multiple mechanisms. Moreover, under certain conditions, miRNAs are also capable of activating translation. A variety of cellular proteins have been implicated in these regulatory mechanisms, yet their exact roles remain largely unresolved

The long-standing assumption that messenger RNA (mRNA) degradation in Escherichia coli begins with endonucleolytic cleavage has been challenged by the recent discovery that RNA decay can 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 at internal sites is greatly enhanced when the RNA 5' end is monophosphorylated. Moreover, it suggests an explanation for the influence of 5' termini on the endonucleolytic cleavage of primary transcripts, which are triphosphorylated. However, no enzyme capable of removing pyrophosphate from RNA 5' ends has been identified in any bacterial species. Here we show that the E. coli protein RppH (formerly NudH/YgdP) is the RNA pyrophosphohydrolase that initiates mRNA decay by this 5'-end-dependent pathway. In vitro, RppH efficiently removes pyrophosphate from the 5' end of triphosphorylated RNA, irrespective of the identity of the 5'-terminal nucleotide. In vivo, it accelerates the degradation of hundreds of E. coli transcripts by converting their triphosphorylated 5' ends to a more labile monophosphorylated state that can stimulate subsequent ribonuclease cleavage. 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. Together, these findings suggest a possible basis for the effect of RppH and its orthologues on the invasiveness of bacterial pathogens. Interestingly, this master regulator of 5'-end-dependent mRNA degradation in E. coli not only catalyses a process functionally reminiscent of eukaryotic mRNA decapping but also bears an evolutionary relationship to the eukaryotic decapping enzyme Dcp2

MicroRNAs (miRNAs) utilize multiple posttranscriptional mechanisms to downregulate gene expression in metazoan organisms. These include translation repression and accelerated mRNA decay, the latter being triggered either by deadenylation or, less frequently, by endonucleolytic cleavage, as governed by the degree of complementarity of the targeted message. This chapter describes methods for examining the effect of miRNAs on the translation and turnover of complementary mRNAs in cultured mammalian cells. Among these are procedures for quantifying their influence on the cytoplasmic concentration and translation efficiency of luciferase reporter mRNAs, for monitoring their impact on the deadenylation and decay of beta-globin reporter mRNAs, and for detecting miRNA-directed internal mRNA cleavage

Recent studies have revealed that 5'-end-dependent RNA degradation in prokaryotes is triggered by pyrophosphate removal from the 5'-terminus to generate a monophosphorylated intermediate that is readily degraded. This chapter describes how to examine the 5'-phosphorylation state of any specific bacterial RNA by PABLO analysis. The method is based on the ability of monophosphorylated, but not triphosphorylated, RNA 5'-ends to undergo splinted ligation to a DNA oligonucleotide when juxtaposed by base pairing to a bridging oligonucleotide. PABLO analysis not only makes it possible to quantify the proportion of a particular RNA that is monophosphorylated in bacterial cells but also provides a more reliable method than primer extension for high-resolution mapping of RNA 5'-termini

The common belief that endonucleolytic cleavage is the initial, rate-determining step of mRNA decay in Escherichia coli fails to explain the influence of 5' termini on the half-lives of primary transcripts. We have re-examined the initial events of RNA degradation in that organism by devising an assay to probe the 5' phosphorylation state of RNA and by employing a self-cleaving hammerhead ribozyme to investigate the degradative consequences of an unphosphorylated 5' end. These studies have identified a previously unrecognized prior step in decay that triggers subsequent internal cleavage by the endonuclease RNase E and thereby governs RNA longevity: the rate-determining conversion of a triphosphorylated to a monophosphorylated 5' terminus. Our findings redefine the role of RNase E in RNA degradation and explain how unpaired 5'-terminal nucleotides can facilitate access to internal cleavage sites within primary transcripts. Moreover, these results reveal a striking parallel between the mechanisms of mRNA decay in prokaryotic and eukaryotic organisms

MicroRNAs (miRNAs) are ubiquitous regulators of eukaryotic gene expression. In addition to repressing translation, miRNAs can down-regulate the concentration of mRNAs that contain elements to which they are imperfectly complementary. Using miR-125b and let-7 as representative miRNAs, we show that in mammalian cells this reduction in message abundance is a consequence of accelerated deadenylation, which leads to rapid mRNA decay. The ability of miRNAs to expedite poly(A) removal does not result from decreased translation; nor does translational repression by miRNAs require a poly(A) tail, a 3' histone stem-loop being an effective substitute. These findings suggest that miRNAs use two distinct posttranscriptional mechanisms to down-regulate gene expression

The lifetimes of bacterial mRNAs are strongly affected by their association with ribosomes. Events occurring at any stage during translation, including ribosome binding, polypeptide elongation, or translation termination, can influence the susceptibility of mRNA to ribonuclease attack. Ribosomes usually act as protective barriers that impede mRNA cleavage, but in some instances they can instead trigger the decay of the mRNA to which they are bound or send a signal that leads to widespread mRNA destabilization within a cell. The influence of translation on mRNA decay provides a quality-control mechanism for minimizing the use of poorly or improperly translated mRNAs as templates for the production of abnormal proteins that might be toxic to bacteria

Vertebrate genomes each encode hundreds of micro-RNAs (miRNAs), yet for few of these miRNAs is there empirical evidence as to which mRNA(s) they regulate. Here we report the identification of human lin-28 mRNA as a regulatory target of human miR-125b and its homolog miR-125a. Studies of miR-125b function in mouse P19 embryonal carcinoma cells induced to develop into neurons suggest a role for this regulatory miRNA in mammalian neuronal differentiation, since its increased concentration in these cells contributes to lin-28 downregulation. Within the lin-28 3' untranslated region (UTR) are two conserved miRNA responsive elements (miREs) that mediate repression by miR-125b and miR-125a. Simultaneous deletion of both miREs renders the lin-28 3' UTR almost completely insensitive to these miRNAs, indicating that these two miREs are the principal elements in the lin-28 3' UTR that respond to miR-125. At the 3' end of each element is an adenosine residue that makes a significant contribution to function irrespective of its complementarity to the 5'-terminal nucleotide of miR-125. By contrast to most earlier reports of gene repression by other miRNAs that are imperfectly complementary to their targets, lin-28 downregulation by miR-125 involves reductions in both translational efficiency and mRNA abundance. The decrease in the mRNA concentration is achieved by a posttranscriptional mechanism that is independent of the inhibitory effect on translation

RNase E is an endonuclease that plays a central role in RNA processing and degradation in Escherichia coli. Like its E. coli homolog RNase G, RNase E shows a marked preference for cleaving RNAs that bear a monophosphate, rather than a triphosphate or hydroxyl, at the 5' end. To investigate the mechanism by which 5'-terminal phosphorylation can influence distant cleavage events, we have developed fluorogenic RNA substrates that allow the activity of RNase E and RNase G to be quantified much more accurately and easily than before. Kinetic analysis of the cleavage of these substrates by RNase E and RNase G has revealed that 5' monophosphorylation accelerates the reaction not by improving substrate binding, but rather by enhancing the catalytic potency of these ribonucleases. Furthermore, the presence of a 5' monophosphate can increase the specificity of cleavage site selection within an RNA. Although monomeric forms of RNase E and RNase G can cut RNA, the ability of these enzymes to discriminate between RNA substrates on the basis of their 5' phosphorylation state requires the formation of protein multimers. Among the molecular mechanisms that could account for these properties are those in which 5'-end binding by one enzyme subunit induces a protein structural change that accelerates RNA cleavage by another subunit