Faculty Bibliography

In Escherichia coli, the endonuclease RNase E can access internal cleavage sites in mRNA either directly or by a 5' end-dependent mechanism in which cleavage is facilitated by prior RppH-catalysed conversion of the 5'-terminal triphosphate to a monophosphate, to which RNase E can bind. The characteristics of transcripts that determine which of these two pathways is primarily responsible for their decay are poorly understood. Here we report the influence of ribosome binding and translocation on each pathway, using yeiP and trxB as model transcripts. Ribosome binding to the translation initiation site impedes degradation by both mechanisms. However, because the effect on the rate of 5' end-independent decay is greater, poor ribosome binding favours degradation by that pathway. Arresting translation elongation with chloramphenicol quickly inhibits RNase E cleavage downstream of the initiation codon but has little or no immediate effect on cleavage upstream of the ribosome binding site. RNase E binding to a monophosphorylated 5' end appears to increase the likelihood of cleavage at sites within the 5' untranslated region. These findings indicate that ribosome binding and translocation can have a major impact on 5' end-dependent mRNA degradation in E. coli and suggest a possible sequence of events that follow pyrophosphate removal.

Enterohaemorrhagic Escherichia coli harbours a pathogenicity island encoding a type 3 secretion system used to translocate effector proteins into the cytosol of intestinal epithelial cells and subvert their function. The structural proteins of the translocon are encoded in a major espADB mRNA processed from a precursor. The translocon mRNA should be highly susceptible to RNase E cleavage because of its AU-rich leader region and monophosphorylated 5'-terminus, yet it manages to avoid rapid degradation. Here, we report that the espADB leader region contains a strong Shine-Dalgarno element (SD2) and a translatable mini-ORF of six codons. Disruption of SD2 so as to weaken ribosome binding significantly reduces the concentration and stability of esp mRNA, whereas codon substitutions that impair translation of the mini-ORF have no such effect. These findings suggest that occupancy of SD2 by ribosomes, but not mini-ORF translation, helps to protect espADB mRNA from degradation, likely by hindering RNase E access to the AU-rich leader region.

Many Escherichia coli mRNAs are degraded by a 5'-end-dependent mechanism in which RppH-catalyzed conversion of the 5'-terminal triphosphate to a monophosphate triggers rapid endonucleolytic cleavage by RNase E. However, little is understood about what governs the decay rates of these transcripts. We investigated the decay of three such messages-rpsT P1, yfcZ, and ydfG-to characterize the rate-determining step in their degradation. The steady-state ratio of monophosphorylated to triphosphorylated rpsT P1 and yfcZ mRNA indicates that their decay rate is limited by cleavage of the monophosphorylated intermediate, making RNase E critical for their rapid turnover. Conversely, the decay rate of ydfG is limited by generation of the monophosphorylated intermediate; therefore, either RNase E or its less abundant paralog RNase G is sufficient for rapid ydfG degradation. Although all three transcripts are stabilized when RppH is absent, overproducing RppH does not accelerate their decay, nor does RppH overproduction appear to influence the longevity of most other messages that it targets. The failure of excess RppH to hasten rpsT P1 and yfcZ degradation despite increasing the percentage of each that is monophosphorylated is consistent with the observation that pyrophosphate removal is not the rate-limiting step in their decay. In contrast, neither the ydfG decay rate nor the fraction of ydfG transcripts that are monophosphorylated increases when the cellular concentration of RppH is raised, suggesting that, for some RppH targets, the rate of formation of the monophosphorylated intermediate is limited by an ancillary factor or by a step that precedes pyrophosphate removal.

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