Faculty Bibliography

The involvement of the recently characterized 5' exonuclease activity of RNase J1 and endonuclease activity of RNase Y in the turnover of DeltaermC mRNA in Bacillus subtilis was investigated. Evidence is presented that both of these activities determine the half-life of DeltaermC mRNA.

In Escherichia coli, RNA degradation often begins with conversion of the 5'-terminal triphosphate to a monophosphate, creating a better substrate for internal cleavage by RNase E. Remarkably, no homolog of this key endonuclease is present in many bacterial species, such as Bacillus subtilis and various pathogens. Here, we report that the degradation of primary transcripts in B. subtilis can nevertheless be triggered by an analogous process to generate a short-lived, monophosphorylated intermediate. Like its E. coli counterpart, the B. subtilis RNA pyrophosphohydrolase that catalyzes this event is a Nudix protein that prefers unpaired 5' ends. However, in B. subtilis, this modification exposes transcripts to rapid 5' exonucleolytic degradation by RNase J, which is absent in E. coli but present in most bacteria lacking RNase E. This pathway, which closely resembles the mechanism by which deadenylated mRNA is degraded in eukaryotic cells, explains the stabilizing influence of 5'-terminal stem-loops in such bacteria

New structures of RNase J reported by Dorleans et al. and Newman et al. in this issue of Structure suggest how an enzyme whose identical subunits each contain a single buried active site can function as both a 5' exonuclease and an endonuclease

Despite its universal importance for controlling gene expression, mRNA degradation was initially thought to occur by disparate mechanisms in eukaryotes and bacteria. This conclusion was based on differences in the structures used by these organisms to protect mRNA termini and in the RNases and modifying enzymes originally implicated in mRNA decay. Subsequent discoveries have identified several striking parallels between the cellular factors and molecular events that govern mRNA degradation in these two kingdoms of life. Nevertheless, some key distinctions remain, the most fundamental of which may be related to the different mechanisms by which eukaryotes and bacteria control translation initiation

Regulated transport and local translation of mRNA in neurons are critical for modulating synaptic strength, maintaining proper neural circuitry, and establishing long term memory. Neuronal RNA granules are ribonucleoprotein particles that serve to transport mRNA along microtubules and control local protein synthesis in response to synaptic activity. Studies suggest that neuronal RNA granules share similar structures and functions with somatic P-bodies. We recently reported that the Huntington disease protein huntingtin (Htt) associates with Argonaute (Ago) and localizes to cytoplasmic P-bodies, which serve as sites of mRNA storage, degradation, and small RNA-mediated gene silencing. Here we report that wild-type Htt associates with Ago2 and components of neuronal granules and co-traffics with mRNA in dendrites. Htt was found to co-localize with RNA containing the 3'-untranslated region sequence of known dendritically targeted mRNAs. Knockdown of Htt in neurons caused altered localization of mRNA. When tethered to a reporter construct, Htt down-regulated reporter gene expression in a manner dependent on Ago2, suggesting that Htt may function to repress translation of mRNAs during transport in neuronal granules

MicroRNAs repress gene expression post-transcriptionally by inhibiting translation and by expediting deadenylation so as to trigger rapid mRNA decay. Their regulatory influence is mediated by the protein components of the RNA-induced silencing complex (RISC), which deliver miRNAs and siRNAs to their mRNA targets. Here we present evidence that CCR4-NOT is the deadenylase that removes poly(A) from messages destabilized by miRNAs in human cells. Overproducing a mutationally inactivated form of either of the catalytic subunits of this deadenylase (CCR4 or CAF1/POP2) significantly impedes the deadenylation and decay of mRNA targeted by a partially complementary miRNA. The same deadenylase initiates the degradation of 'off-target' mRNAs that are bound by an imperfectly complementary siRNA introduced by transfection. The greater inhibitory effect of inactive CAF1 or POP2 (versus inactive CCR4) suggests a predominant role for this catalytic subunit of CCR4-NOT in mi/siRNA-mediated deadenylation. These effects of mi/siRNAs and CCR4-NOT can be fully reproduced by directly tethering RISC to mRNA without the guidance of a small RNA, indicating that the ability of RISC to accelerate deadenylation is independent of RNA base pairing. Despite its importance for mi/siRNA-mediated deadenylation, CCR4-NOT appears not to associate significantly with RISC, as judged by the failure of CAF1 and POP2 to co-immunoprecipitate detectably with either the Ago or TNRC6 subunit of RISC, a finding at odds with deadenylase recruitment as the mechanism by which RISC accelerates poly(A) removal

RNase E autoregulates its production in Escherichia coli by governing the decay rate of rne (RNase E) mRNA. It does so by a mechanism that is dependent in part on hp2, a cis-acting stem-loop within the rne 5' untranslated region. In principle, hp2 could function either as a cleavage site for RNase E or as a binding site for that protein or an ancillary factor. Here we show that the effector region at the top of hp2 is cleaved poorly by RNase E yet binds the catalytic domain of that ribonuclease with a sequence specificity reflecting its efficacy in feedback regulation. These findings suggest that hp2 controls RNase E synthesis by binding to RNase E and expediting cleavage elsewhere within the rne transcript

Until recently, the mechanism of mRNA decay in bacteria was thought to be different from that of eukaryotes. This paradigm changed with the discovery that RppH (ORF176/NudH/YgdP), an Escherichia coli enzyme that belongs to the Nudix superfamily, is an RNA pyrophosphohydrolase that initiates mRNA decay by cleaving pyrophosphate from the 5'-triphosphate. Here we report the 1.9 A resolution structure of the Nudix hydrolase BdRppH from Bdellovibrio bacteriovorus, a bacterium that feeds on other Gram-negative bacteria. Based on the structure of the enzyme alone and in complex with GTP-Mg(2+), we propose a mode of RNA binding similar to that of the nuclear decapping enzyme from Xenopus laevis, X29. In additional experiments, we show that BdRppH can indeed function in vitro and in vivo as an RNA pyrophosphohydrolase. These findings set the basis for the identification of possible decapping enzymes in other bacteria

In this issue of Molecular Cell, Bouvier et al. (2008) show that bacterial sRNAs can repress mRNA translation not only by binding to the Shine-Dalgarno element but also by base pairing anywhere within the first few codons of the protein-coding region

In animals, both siRNAs and miRNAs are thought to diminish protein synthesis from transcripts that are perfectly complementary by directing endonucleolytic cleavage where they anneal, thereby triggering rapid degradation of the entire message [1-4]. By contrast, partially complementary messages are downregulated by a combination of translational repression and accelerated decay caused by rapid poly(A) tail removal [3, 5-12]. Here we present evidence that translational repression can also make a substantial contribution to the downregulation of fully complementary messages by RNA interference. Unlike mRNA destabilization, this inhibitory effect on translation is greater for perfectly complementary elements located in the 3' untranslated region rather than in the protein-coding region. In addition to known disparities in their endonucleolytic activity [13, 14], the four Ago proteins with which siRNAs associate in humans differ significantly in their capacity to direct translational repression. As a result, the relative effect of siRNA on targets that are fully versus partially complementary is influenced by the comparative abundance of the three nonnucleolytic Ago proteins, causing this on-target/off-target ratio to vary in a cell-type-dependent manner because of the dissimilar tissue distribution of these proteins. These findings have important implications for the efficacy and specificity of RNA interference