The degradation of mRNA in Escherichia coli is thought to occur through a series of endonucleolytic and exonucleolytic steps. By constructing a series of multiple mutants containing the pnp-7 (polynucleotide phosphorylase), rnb-S00 (RNase II), and ams-l (altered message stability) alleles, it was possible to study general mRNA turnover as well as the degradation of specific mRNAs. Of most interest was the ams-1 pnp-7 rnb-500 triple mutant in which the half-life of total pulse-labeled RNA increased three-to fourfold at the nonpermissive temperature. RNA-DNA hybridization analysis of several specific mRNAs such as trxA (thioredoxin), ssb (single-stranded-DNA-binding protein), uvrD (DNA helicase II), cat (chloramphenicol acetyltransferase), nusA (N utilization substance), and pnp (polynucleotide phosphorylase) demonstrated twoto fourfold increases in their chemical half-lives. A new method for high-resolution Northern (RNA) analysis showed that the trxA and cat mRNAs are degraded into discrete fragments which are significantly stabilized only in the triple mutant. A model for mRNA turnover is discussed.

The amount of a messenger RNA available for protein synthesis depends on the efficiency of its transcription and stability. The mechanisms of degradation that determine the stability of mRNAs in bacteria have been investigated extensively during the last decade and have begun to be better understood. Several endo- and exoribonucleases involved in the mRNA metabolism have been characterized as well as structural features of mRNA which account for its stability have been determined. The most important recent developments have been the discovery that the degradosome-a multiprotein complex containing an endoribonuclease (RNase E), an exoribonuclease (polynucleotide phosphorylase), and a DEAD box helicase (RhlB)-has a central role in mRNA degradation and that oligo(A) tails synthesized by poly(A) polymerase facilitate the degradation of mRNAs and RNA fragments. Moreover, the phosphorylation status and the base pairing of 5' extremities, together with 3' secondary structures of transcriptional terminators, contribute to the stability of primary transcripts. Degradation of mRNAs can follow several independent pathways. Interestingly, poly(A) tails and multienzyme complexes also control the stability and the degradation of eukaryotic mRNAs. These discoveries have led to the development of refined models of mRNA degradation.

The polycistronic mRNA of the histidine operon is subject to a processing event that generates a rather stable transcript encompassing the five distal cistrons. The molecular mechanisms by which such a transcript is produced were investigated in Escherichia coil strains carrying mutations in several genes for exo-and endonucleases. The experimental approach made use of S1 nuclease protection assays on in vivo synthesized transcripts, site-directed mutagenesis and construction of chimeric plasmids, dissection of the processing reaction by RNA mobility retardation experiments, and in vitro RNA degradation assays with cellular extracts. We have found that processing requires (1) a functional endonuclease E; (2) target site(s) for this activity in the RNA region upstream of the 5' end of the processed transcript that can be substituted by another well-characterized me-dependent cleavage site; (3) efficient translation initiation of the first cistron immediately downstream of the 5' end; and (4) a functional endonuclease P that seems to act on the processing products generated by ribonuclease E. This is the first evidence that ribonuclease P, an essential ribozyme required for the biosynthesis of tRNA, may also be involved in the segmental stabilization of a mRNA.[Key Words: Escherichia coli; Salmonella typhimurium; his operon; mRNA processing; translation; RNase E; RNase P] Received June 2, 1994; revised version accepted October 18, 1994. mRNA decay in bacteria is mediated by the coordinated action of exonucleases and endonucleases (for review, see Petersen 1992). Polynucleotide phosphorylase (PNPase) and ribonuclease II (RNase II) are the two major 3'--* 5' exonucleases involved in mRNA turnover (Donovan and Kushner 1986). Endoribonuclease III (RNase III) does not affect bulk mRNA stability (Babitzke et al. 1993) and is mostly involved in processing of the 30S rRNA precursor even though RNase III can initiate the functional decay of a few specific mRNAs (Court 1993). Endoribonuclease E (RNase E)was initially characterized as the enzyme that processes the precursor of 5S rRNA (Ghora and Apirion 1978). RNase E is the only endoribonuclease implicated thus far in general mRNA turnover (Arraiano et al. 1988;Mudd et al. 1990b;Babitzke and Kushner 1991). Many mRNAs were shown to contain sites whose cleavage was abolished in strains harboring temperature-sensitive rne mutations at the nonpermissive temperature (Kokoska et al. 1990;Mudd et al. 1990a;Gross 1991 1991; Patel and Dunn 1992;Arraiano et al. 1993;Klug 1993;Mudd and Higgins 1993;Carpousis et al. 1994). rne-dependent cleavages might produce relatively stable processed transcripts, such as the T4 gene 32 (Mudd et al. 1988) and the dicF (Faubladier et al. 1990) and the ghX mRNAs (Brunet al. 1990). In different studies ribonuclease P (RNase P) and other nucleases responsible for stable RNA processing did not seem to be involved in mRNA degradation (Deutscher 1988).We have previously identified in Salmonella typhimurium a rather stable 3900-nucleotide-long mRNA molecule...

Small non-coding RNAs (sRNAs) are critical post-transcriptional regulators of gene expression. Distinct RNA-binding proteins (RBPs) influence the processing, stability and activity of bacterial small RNAs. The vast majority of bacterial sRNAs interact with mRNA targets, affecting mRNA stability and/or its translation rate. The assistance of RNA-binding proteins facilitates and brings accuracy to sRNA-mRNA basepairing and the RNA chaperones Hfq and ProQ are now recognized as the most prominent RNA matchmakers in bacteria. These RBPs exhibit distinct high affinity RNA-binding surfaces, promoting RNA strand interaction between a trans-encoding sRNA and its mRNA target. Nevertheless, some organisms lack ProQ and/or Hfq homologs, suggesting the existence of other RBPs involved in sRNA function. Along this line of thought, the global regulator CsrA was recently shown to facilitate the access of an sRNA to its target mRNA and may represent an additional factor involved in sRNA function. Ribonucleases (RNases) can be considered a class of RNA-binding proteins with nucleolytic activity that are responsible for RNA maturation and/or degradation. Presently RNase E, RNase III, and PNPase appear to be the main players not only in sRNA turnover but also in sRNA processing. Here we review the current knowledge on the most important bacterial RNA-binding proteins affecting sRNA activity and sRNA-mediated networks.

Organic acids are recognized as one of the most prevalent compounds in ecosystems, thus the transport and assimilation of these molecules represent an adaptive advantage for organisms. The AceTr family members are associated with the active transport of organic acids, namely acetate and succinate. The phylogenetic analysis shows this family is dispersed in the tree of life. However, in eukaryotes, it is almost limited to microbes, though reaching a prevalence close to 100% in fungi, with an essential role in spore development. Aiming at deepening the knowledge in this family, we studied the acetate permease AceP from Methanosarcina acetivorans, as the first functionally characterized archaeal member of this family. Furthermore, we demonstrate that the yeast Gpr1 from Yarrowia lipolytica is an acetate permease, whereas the Ady2 closest homologue in Saccharomyces cerevisiae, Fun34, has no role in acetate uptake. In this work, we describe the functional role of the AceTr conserved motif NPAPLGL(M/S). We further unveiled the role of the amino acid residues R122 and Q125 of SatP as essential for protein activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has triggered a global pandemic with devastating consequences for health-care and social-economic systems. Thus, the understanding of fundamental aspects of SARS-CoV-2 is of extreme importance.In this work, we have focused our attention on the viral ribonuclease (RNase) nsp14, since this protein was considered one of the most interferon antagonists from SARS-CoV-2, and affects viral replication. This RNase is a multifunctional protein that harbors two distinct activities, an N-terminal 3’-to-5’ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both with critical roles in coronaviruses life cycle. Namely, SARS-CoV-2 nsp14 ExoN knockout mutants are non-viable, indicating nsp14 as a prominent target for the development of antiviral drugs.Nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein, which has a pleiotropic function during viral replication. In this study, we have performed the first biochemical characterization of the complex nsp14-nsp10 from SARS-CoV-2. Here we confirm the 3’-5’ exoribonuclease and MTase activities of nsp14 in this new Coronavirus, and the critical role of nsp10 in upregulating the nsp14 ExoN activity in vitro. Furthermore, we demonstrate that SARS-CoV-2 nsp14 N7-MTase activity is functionally independent of the ExoN activity. The nsp14 MTase activity also seems to be independent of the presence of nsp10 cofactor, contrarily to nsp14 ExoN.Until now, there is no available structure for the SARS-CoV-2 nsp14-nsp10 complex. As such, we have modelled the SARS-CoV-2 nsp14-nsp10 complex based on the 3D structure of the complex from SARS-CoV (PDB ID 5C8S). We also have managed to map key nsp10 residues involved in its interaction with nsp14, all of which are also shown to be essential for stimulation of the nsp14 ExoN activity. This reinforces the idea that a stable interaction between nsp10 and nsp14 is strictly required for the nsp14-mediated ExoN activity of SARS-CoV-2, as observed for SARS-CoV.We have studied the role of conserved DEDD catalytic residues of SARS-CoV-2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function contrasting to the functionality of these conserved catalytic residues in SARS-CoV, and in the Middle East respiratory syndrome coronavírus (MERS). The differences here revealed can have important implications regarding the specific pathogenesis of SARS-CoV-2.The nsp10-nsp14 interface is a recognized attractive target for antivirals against SARS-CoV-2 and other coronaviruses. This work has unravelled a basis for discovering inhibitors targeting the specific amino acids here reported, in order to disrupt the assembly of this complex and interfere with coronaviruses replication.

Gene regulation was long thought to be controlled almost entirely by proteins that bind to DNA and RNA. Over the last years, it has become clear that small non-coding RNAs (sRNAs) are important in almost every facet of gene regulation. Understanding how they are matured and degraded has therefore become of maximum importance, in order to know how to "regulate the regulators." Ribonucleases perform a key role in the biogenesis and processing of sRNAs, as well as in controlling their cellular levels through regulation of their turnover. Accordingly, RNases can have a major impact on sRNAs regulatory pathways. In this review, we present an overview of what is presently known about the main RNases, as well as other factors involved in sRNA processing and turnover, in essence contributing to the assembly of the increasing number of pieces in the puzzling global mechanism of sRNA regulation. Although the primary focus will be on bacterial sRNAs, parallels will be made with the siRNAs and miRNAs in eukaryotes.

The intracellular pathogen serovar Typhimurium has emerged as a major cause of foodborne illness, representing a severe clinical and economic concern worldwide. The capacity of this pathogen to efficiently infect and survive inside the host depends on its ability to synchronize a complex network of virulence mechanisms. Therefore, the identification of new virulence determinants has become of paramount importance in the search of new targets for drug development. BolA-like proteins are widely conserved in all kingdoms of life. In, this transcription factor has a critical regulatory role in several mechanisms that are tightly related to bacterial virulence. Therefore, in the present work we used the well-established infection model to evaluate the role of BolA protein in Typhimurium virulence. We have shown that BolA is an important player in Typhimurium pathogenesis. Specifically, the absence of BolA leads to a defective virulence capacity that is most likely related to the remarkable effect of this protein on Typhimurium evasion of the cellular response. Furthermore, it was demonstrated that BolA has a critical role in bacterial survival under harsh conditions since BolA conferred protection against acidic and oxidative stress. Hence, we provide evidence that BolA is a determining factor in the ability of to survive and overcome host defense mechanisms, and this is an important step in progress to an understanding of the pathways underlying bacterial virulence. BolA has been described as an important protein for survival in the late stages of bacterial growth and under harsh environmental conditions. High levels of BolA in stationary phase and under stresses have been connected with a plethora of phenotypes, strongly suggesting its important role as a master regulator. Here, we show that BolA is a determining factor in the ability of to survive and overcome host defense mechanisms, and this is an important step in progress to an understanding of the pathways underlying bacterial virulence. This work constitutes a relevant step toward an understanding of the role of BolA protein and may have an important impact on future studies in other organisms. Therefore, this study is of utmost importance for understanding the genetic and molecular bases involved in the regulation of virulence and may contribute to future industrial and public health care applications.