A novel endoribonuclease, RNase LS, in Escherichia coli

The dmd gene of bacteriophage T4 is required for the stability of late-gene mRNAs. When this gene is mutated, late genes are globally silenced because of rapid degradation of their mRNAs. Our previous work suggested that a novel Escherichia coli endonuclease, RNase LS, is responsible for the rapid degradation of mRNAs. In this study, we demonstrated that rnlA (formerly yfjN) is essential for RNase LS activity both in vivo and in vitro. In addition, we investigated a role of RNase LS in the RNA metabolism of E. coli cells under vegetative growth conditions. A mutation in rnlA reduced the decay rate of many E. coli mRNAs, although there are differences in the mutational effects on the stabilization of different mRNAs. In addition, we found that a 307-nucleotide fragment with an internal sequence of 23S rRNA accumulated to a high level in rnlA mutant cells. These results strongly suggest that RNase LS plays a role in the RNA metabolism of E. coli as well as phage T4.     

Poly(A) shortening and degradation of the 3'' A+U-rich sequences of human c-myc mRNA in a cell-free system.

The early steps in the degradation of human c-myc mRNA were investigated, using a previously described cell-free mRNA decay system. The first detectable step was poly(A) shortening, which generated a pool of oligoadenylated mRNA molecules. In contrast, the poly(A) of a stable mRNA, gamma globin, was not excised, even after prolonged incubation. The second step, degradation of oligoadenylated c-myc mRNA, generated decay products whose 3' termini were located within the A+U-rich portion of the 3' untranslated region. These products disappeared soon after they were formed, consistent with rapid degradation of the 3' region. In contrast, the 5' region, corresponding approximately to c-myc exon 1, was stable in vitro. The data indicate a sequential degradation pathway in which 3' region cleavages occur only after most or all of the poly(A) is removed. To account for rapid deadenylation, we suggest that the c-myc poly(A)-poly(A)-binding protein complex is readily dissociated, generating a protein-depleted poly(A) tract that is no longer resistant to nucleases.     

The small RNA RyhB activates the translation of shiA mRNA encoding a permease of shikimate, a compound involved in siderophore synthesis

RyhB is a small RNA (sRNA) that downregulates about 20 genes involved in iron metabolism. It is expressed under low iron conditions and pairs with specific mRNAs to trigger their rapid degradation by the RNA degradosome. In contrast to this, another study has suggested that RyhB also activates several genes by increasing their mRNA level. Among these activated genes is shiA, which encodes a permease of shikimate, an aromatic compound participating in the biosynthesis of siderophores. Here, we demonstrate in vivo and in vitro that RyhB directly pairs at the 5'-untranslated region (5'-UTR) of the shiA mRNA to disrupt an intrinsic inhibitory structure that sequesters the ribosome-binding site (Shine-Dalgarno) and the first translation codon. This is the first demonstration of direct gene activation by RyhB, which has been exclusively described in degradation of mRNAs. Our physiological results indicate that the transported compound of the ShiA permease, shikimate, is important under conditions of RyhB expression, that is, iron starvation. This is demonstrated by growth assays in which shikimate or the siderophore enterochelin correct the growth defect observed for a ryhB mutant in iron-limited media.     

AU binding proteins recruit the exosome to degrade ARE-containing mRNAs.

Inherently unstable mammalian mRNAs contain AU-rich elements (AREs) within their 3' untranslated regions. Although found 15 years ago, the mechanism by which AREs dictate rapid mRNA decay is not clear. In yeast, 3'-to-5' mRNA degradation is mediated by the exosome, a multisubunit particle. We have purified and characterized the human exosome by mass spectrometry and found its composition to be similar to its yeast counterpart. Using a cell-free RNA decay system, we demonstrate that the mammalian exosome is required for rapid degradation of ARE-containing RNAs but not for poly(A) shortening. The mammalian exosome does not recognize ARE-containing RNAs on its own. ARE recognition requires certain ARE binding proteins that can interact with the exosome and recruit it to unstable RNAs, thereby promoting their rapid degradation.     

Adaptation of Saccharomyces cerevisiae to high hydrostatic pressure causing growth inhibition

Genome-wide mRNA expression profiles of Saccharomyces cerevisiae growing under hydrostatic pressure were characterized. We selected a hydrostatic pressure of 30 MPa at 25 degrees C because yeast cells were able to grow under these conditions, while cell size and complexity were increased after decompression. Functional characterization of pressure-induced genes suggests that genes involved in protein metabolism and membrane metabolism were induced. The response to 30 MPa was significantly different from that observed under lethal conditions because protein degradation was not activated under 30 MPa pressure. Strongly induced genes those that contribute to membrane metabolism and which are also induced by detergents, oils, and membrane stabilizers.     

GW body disassembly triggered by siRNAs independently of their silencing activity

GW bodies (or P-bodies) are cytoplasmic granules containing proteins involved in both mRNA degradation and storage, including the RNA interference machinery. Their mechanism of assembly and function are still poorly known although their number depends upon the flux of mRNA to be stored or degraded. We show here that silencing of the translational regulator CPEB1 leads to their disappearance, as reported for other GW body components. Surprisingly, the same results were obtained with several siRNAs targeting genes encoding proteins unrelated to mRNA metabolism. The disappearance of GW bodies did not correlate with the silencing activity of the siRNA and did not inhibit further silencing by siRNA. Importantly, in most cases, GW bodies were rapidly reinduced by arsenite, indicating that their assembly was not prevented by the inhibition of the targeted or off-target genes. We therefore propose that some siRNA sequences affect mRNA metabolism so as to diminish the amount of mRNA directed to the GW bodies. As an exception, GW bodies were not reinduced following Rck/p54 depletion by interference, indicating that this component is truly required for the GW body assembly. Noteworthy, Rck/p54 was dispensable for the assembly of stress granules, in spite of their close relationship with the GW bodies.     

Messenger Ribonucleic Acid Synthesis and Degradation in Escherichia coli During Inhibition of Translation

Various aspects of the coupling between the movement of ribosomes along messenger ribonucleic acids (mRNA) and the synthesis and degradation of mRNA have been investigated. Decreasing the rate of movement of ribosomes along an mRNA does not affect the rate of movement of some, and possibly most, of the RNA polymerases transcribing the gene coding for that mRNA. Inhibiting translation with antibiotics such as chloramphenicol, tetracycline, or fusidic acid protects extant mRNA from degradation, presumably by immobilizing ribosomes, whereas puromycin exposes mRNA to more rapid degradation than normal. The promoter distal (3') portion of mRNA, synthesized after ribosomes have been immobilized by chloramphenicol on the promoter proximal (5') portion of the mRNA, is subsequently degraded.     

Sok antisense RNA from plasmid R1 is functionally inactivated by RNase E and polyadenylated by poly(A) polymerase I

The hok/sok system of plasmid R1, which mediates plasmid stabilization by the killing of plasmid-free cells, codes for two RNA species, Sok antisense RNA and hok mRNA. Sok RNA, which is unstable, inhibits translation of the stable hok mRNA. The 64 nt Sok RNA folds into a single stem-loop domain with an 11 nt unstructured 5' domain. The initial recognition reaction between Sok RNA and hok mRNA takes place between the 5' domain and the complementary region in hok mRNA. In this communication we examine the metabolism of Sok antisense RNA. We find that RNase E cleaves the RNA 6 nt from its 5' end and that this cleavage initiates Sok RNA decay. The RNase E cleavage occurs in the part of Sok RNA that is responsible for the initial recognition of the target loop in hok mRNA and thus leads to functional inactivation of the antisense. The major RNase E cleavage product (denoted pSok_-6) is rapidly degraded by polynucleotide phosphorylase (PNPase). Thus, the RNase E cleavage tags pSok₋₆ for further rapid degradation by PNPase from its 3' end. We also show that Sok RNA is polyadenylated by poly(A) polymerase I (PAP I), and that the poly(A)-tailing is prerequisite for the rapid 3'-exonucleolytic degradation by PNPase.     

Non-dairy lactic fermentations: the cereal world*

Sourdough is the foremost cereal fermentation performed in a variety of technologies with almost any cereal. The lactobacilli studied most intensely include Lactobacillus sanfranciscensis, L. reuteri and L. pontis isolated from traditional and modern rye and wheat fermentations. Molecular biology tools are available for their rapid identification and monitoring throughout a process. The currently available insight on their metabolism can be used to explain their prevalence in this environment and their interactions. Key genes of the sugar degradation pathway were cloned and characterised from L. sanfranciscensis. In addition some strains were found to have special properties including the production of antagonistic compounds or the adhesion to human intestinal cells.     

细菌mRNA的降解机制

在细菌中,mRNA降解具有重要的意义,它不仅可以再循环核苷酸,而且还可以根据生长条件的变化调控基因表达.细菌mRNA的降解机制可以分为3种：① mRNA的一般降解途径|② mRNA的质量控制途径|③ 小RNA介导的降解途径. 这些途径有些与真核生物的mRNA降解途径存在很大差异,有些在真核生物中消失了. 另外,mRNA降解途径还可以直接调控细菌致病因子的表达,这使得细菌mRNA的降解途径很有希望成为药物研发的新靶标,或疫苗制备的新平台,以应对越来越严重的细菌耐药性问题.本文综述了细菌mRNA的降解机制,并对其应用前景进行了展望.     

Evidence for a 3'-5' decay pathway for c-myc mRNA in mammalian cells.

Many mRNAs in mammalian cells decay via a sequential pathway involving rapid conversion of polyadenylated molecules to a poly(A)-deficient state followed by rapid degradation of the poly(A)-deficient molecules. However, the rapidity of this latter step(s) has precluded further analyses of the decay pathways involved. Decay intermediates derived from degradation of poly(A)-deficient molecules could offer clues regarding decay pathways, but these intermediates have not been readily detected. Cell-free mRNA decay systems have proven useful in analyses of decay pathways because decay intermediates are rather stable in vitro. Cell-free systems indicate that many mRNAs decay by a sequential 3'-5' pathway because 3'-terminal decay intermediates form following deadenylation. However, if 3'-terminal, in vitro decay intermediates reflect a biologically significant aspect of mRNA turnover, then similar intermediates should be present in cells. Here, I have compared the in vivo and in vitro decay of mRNA encoded by the c-myc proto-oncogene. Its decay both in vivo and in vitro occurs by rapid removal of the poly(A) tract and generation of a 3'-terminal decay intermediate. These data strongly suggest that a 3'-5' pathway contributes to turnover of c-myc mRNA in cells. It is likely that 3'-5' decay represents a major turnover pathway in mammalian cells.