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.let-7 ͉ miR-125b ͉ poly(A) ͉ translation

Rapid degradation of c-fos proto-oncogene mRNA is crucial for transient c-fos gene expression. Experiments were performed to investigate the cellular mechanisms responsible for the extremely short half-life of human c-los mRNA in growth-factor-stimulated fibroblasts. These experiments demonstrate the existence of two distinct cellular pathways for rapid c-fos mRNA degradation. Each of these pathways recognizes a different, functionally independent instability determinant within the c-fos transcript. One instability determinant, which is located within the c-fos 3'-untranslated region, is a 75-nucleotide AU-rich segment. Insertion of this element into ~-globin mRNA markedly reduces the half-life of that normally long-lived message. Nevertheless, specific deletion of the AU-rich element from c-fos mRNA has little effect on the transcript's cytoplasmic halflife due to the presence of the other c-fos instability determinant, which is located in the protein-coding segment of the c-fos message. Examination of mRNA decay in cells treated with transcription inhibitors indicates that one c-fos mRNA degradation pathway is dependent on RNA synthesis, whereas the other is not.

The mechanisms by which c-fos mRNA is targeted for decay have been examined. Rapid removal of the poly(A) tail occurs before the transcribed portion of the c-/os message is degraded. Identification of the determinants that mediate c-/os message deadenylation reveals that they coincide directly with previously characterized determinants of c-/os mRNA instability, one in the protein-coding region and the other an AU-rich element (ARE) in the 3'-untranslated region. Insertion of either of these c-/os instability elements into the stable p-globin message confers the property of rapid deadenylation. Mutation of the ARE indicates that this sequence controls two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require intact AUUUA pentanucleotides within the ARE, and subsequent degradation of the transcribed portion of the message, which appears to be dependent on the AUUUA pentanucleotides. These results indicate that structurally distinct instability determinants within the transcribed portion of labile messages can function by promoting rapid removal of the poly(A) tail as a first step in the decay process.

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.

Labile mRNAs that encode cytokine and immediate-early gene products often contain AU-rich sequences within their 3 untranslated region (UTR). These AU-rich sequences appear to be key determinants of the short half-lives of these mRNAs, although the sequence features of these elements and the mechanism by which they target mRNAs for rapid decay have not been fully defined. We have examined the features of AU-rich elements (AREs) that are crucial for their function as determinants of mRNA instability in mammalian cells by testing the ability of various mutant c-fos AREs and synthetic AREs to direct rapid mRNA deadenylation and decay when inserted within the 3 UTR of the normally stable ␤-globin mRNA. Evidence is presented that the pentamer AUUUA, which previously was suggested to be the minimal determinant of instability present in mammalian AREs, cannot direct rapid mRNA deadenylation and decay. Instead, the nonamer UUAUUUAUU is the elemental AU-rich sequence motif that destabilizes mRNA. Removal of one uridine residue from either end of the nonamer (UUAUUUAU or UAUUUAUU) results in a decrease of potency of the element, while removal of a uridine residue from both ends of the nonamer (UAUUUAU) eliminates detectable destabilizing activity. The inclusion of an additional uridine residue at both ends of the nonamer (UUUAUUUAUUU) does not further increase the efficacy of the element. Taken together, these findings suggest that the nonamer UUAUUUAUU is the minimal AU-rich motif that effectively destabilizes mRNA. Additional ARE potency is achieved by combining multiple copies of this nonamer in a single mRNA 3 UTR. Furthermore, analysis of poly(A) shortening rates for ARE-containing mRNAs reveals that the UUAUUUAUU sequence also accelerates mRNA deadenylation and suggests that the UUAUUUAUU motif targets mRNA for rapid deadenylation as an early step in the mRNA decay process.

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 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.

The 5'-untranslated region of the long-lived Escherichia coli ompA transcript functions as an mRNA stabilizer capable of prolonging the lifetime in E. coli of a number of heterologous messages to which it is fused. To elucidate the structural basis of differential mRNA stability in bacteria, the domains of the ompA 5'-untranslated region that allow it to protect mRNA from degradation have been identified by mutational analysis. The presence of a stem-loop no more than 2-4 nucleotides from the extreme 5' terminus of this RNA segment is crucial to its stabilizing influence, whereas the sequence of the stem-loop is relatively unimportant. The potential to form a hairpin very close to the 5' end is a feature common to a number of stable prokaryotic messages. Moreover, the lifetime of a normally labile message {bla mRNA) can be prolonged in £. coli by adding a simple hairpin structure at its 5' terminus. Accelerated degradation of ompA mRNA in the absence of a 5'-terminal stem-loop appears to start downstream of the 5' end. We propose that E. coli messages beginning with a single-stranded RNA segment of significant length are preferentially targeted by a degradative tibonuclease that interacts with the mRNA 5' terminus before cleaving internally at one or more distal sites.