Sandra C. Viegas

The critical role of RNA processing and degradation in the control of gene expression

The continuous degradation and synthesis of prokaryotic mRNAs not only give rise to the metabolic changes that are required as cells grow and divide but also rapid adaptation to new environmental conditions. In bacteria, RNAs can be degraded by mechanisms that act independently, but in parallel, and that target different sites with different efficiencies. The accessibility of sites for degradation depends on several factors, including RNA higher-order structure, protection by translating ribosomes and polyadenylation status. Furthermore, RNA degradation mechanisms have shown to be determinant for the post-transcriptional control of gene expression. RNases mediate the processing, decay and quality control of RNA. RNases can be divided into endonucleases that cleave the RNA internally or exonucleases that cleave the RNA from one of the extremities. Just in Escherichia coli there are >20 different RNases. RNase E is a single-strand-specific endonuclease critical for mRNA decay in E. coli. The enzyme interacts with the exonuclease polynucleotide phosphorylase (PNPase), enolase and RNA helicase B (RhlB) to form the degradosome. However, in Bacillus subtilis, this enzyme is absent, but it has other main endonucleases such as RNase J1 and RNase III. RNase III cleaves double-stranded RNA and family members are involved in RNA interference in eukaryotes. RNase II family members are ubiquitous exonucleases, and in eukaryotes, they can act as the catalytic subunit of the exosome. RNases act in different pathways to execute the maturation of rRNAs and tRNAs, and intervene in the decay of many different mRNAs and small noncoding RNAs. In general, RNases act as a global regulatory network extremely important for the regulation of RNA levels.

The exoribonuclease Dis3L2 defines a novel eukaryotic RNA degradation pathway

The final step of cytoplasmic mRNA degradation proceeds in either a 5′‐3′ direction catalysed by Xrn1 or in a 3′‐5′ direction catalysed by the exosome. Dis3/Rrp44, an RNase II family protein, is the catalytic subunit of the exosome. In humans, there are three paralogues of this enzyme: DIS3, DIS3L, and DIS3L2. In this work, we identified a novel Schizosaccharomyces pombe exonuclease belonging to the conserved family of human DIS3L2 and plant SOV. Dis3L2 does not interact with the exosome components and localizes in the cytoplasm and in cytoplasmic foci, which are docked to P‐bodies. Deletion of dis3l2+ is synthetically lethal with xrn1Δ, while deletion of dis3l2+ in an lsm1Δ background results in the accumulation of transcripts and slower mRNA degradation rates. Accumulated transcripts show enhanced uridylation and in vitro Dis3L2 displays a preference for uridylated substrates. Altogether, our results suggest that in S. pombe, and possibly in most other eukaryotes, Dis3L2 is an important factor in mRNA degradation. Therefore, this novel 3′‐5′ RNA decay pathway represents an alternative to degradation by Xrn1 and the exosome.

Regulation of the small regulatory RNA MicA by ribonuclease III: a target-dependent pathway

MicA is a trans-encoded small non-coding RNA, which downregulates porin-expression in stationary-phase. In this work, we focus on the role of endoribonucleases III and E on Salmonella typhimurium sRNA MicA regulation. RNase III is shown to regulate MicA in a target-coupled way, while RNase E is responsible for the control of free MicA levels in the cell. We purified both Salmonella enzymes and demonstrated that in vitro RNase III is only active over MicA when in complex with its targets (whether ompA or lamB mRNAs). In vivo, MicA is demonstrated to be cleaved by RNase III in a coupled way with ompA mRNA. On the other hand, RNase E is able to cleave unpaired MicA and does not show a marked dependence on its 5′ phosphorylation state. The main conclusion of this work is the existence of two independent pathways for MicA turnover. Each pathway involves a distinct endoribonuclease, having a different role in the context of the fine-tuned regulation of porin levels. Cleavage of MicA by RNase III in a target-dependent fashion, with the concomitant decay of the mRNA target, strongly resembles the eukaryotic RNAi system, where RNase III-like enzymes play a pivotal role.

The role of RNases in the regulation of small RNAs

Ribonucleases (RNases) are key factors in the control of biological processes, since they modulate the processing, degradation and quality control of RNAs. This review gives many illustrative examples of the role of RNases in the regulation of small RNAs (sRNAs). RNase E and PNPase have been shown to degrade the free pool of sRNAs. RNase E can also be recruited to cleave mRNAs when they are interacting with sRNAs. RNase III cleaves double-stranded structures, and can cut both the sRNA and its RNA target when they are hybridized. Overall, ribonucleases act as conductors in the control of sRNAs. Therefore, it is very important to further understand their role in the post-transcriptional control of gene expression.

Regulating the regulators: How ribonucleases dictate the rules in the control of small non-coding RNAs.

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.

SATP (YaaH), a succinate–acetate transporter protein in Escherichia coli

In the present paper we describe a new carboxylic acid transporter in Escherichia coli encoded by the gene yaaH. In contrast to what had been described for other YaaH family members, the E. coli transporter is highly specific for acetic acid (a monocarboxylate) and for succinic acid (a dicarboxylate), with affinity constants at pH 6.0 of 1.24±0.13 mM for acetic acid and 1.18±0.10 mM for succinic acid. In glucose-grown cells the ΔyaaH mutant is compromised for the uptake of both labelled acetic and succinic acids. YaaH, together with ActP, described previously as an acetate transporter, affect the use of acetic acid as sole carbon and energy source. Both genes have to be deleted simultaneously to abolish acetate transport. The uptake of acetate and succinate was restored when yaaH was expressed in trans in ΔyaaH ΔactP cells. We also demonstrate the critical role of YaaH amino acid residues Leu¹³¹ and Ala¹⁶⁴ on the enhanced ability to transport lactate. Owing to its functional role in acetate and succinate uptake we propose its assignment as SatP: the Succinate-Acetate Transporter Protein.

Synergies between RNA degradation and trans-translation in Streptococcus pneumoniae: cross regulation and co-transcription of RNase R and SmpB

BackgroundRibonuclease R (RNase R) is an exoribonuclease that recognizes and degrades a wide range of RNA molecules. It is a stress-induced protein shown to be important for the establishment of virulence in several pathogenic bacteria. RNase R has also been implicated in the trans-translation process. Transfer-messenger RNA (tmRNA/SsrA RNA) and SmpB are the main effectors of trans-translation, an RNA and protein quality control system that resolves challenges associated with stalled ribosomes on non-stop mRNAs. Trans-translation has also been associated with deficiencies in stress-response mechanisms and pathogenicity.ResultsIn this work we study the expression of RNase R in the human pathogen Streptococcus pneumoniae and analyse the interplay of this enzyme with the main components of the trans-translation machinery (SmpB and tmRNA/SsrA). We show that RNase R is induced after a 37°C to 15°C temperature downshift and that its levels are dependent on SmpB. On the other hand, our results revealed a strong accumulation of the smpB transcript in the absence of RNase R at 15°C. Transcriptional analysis of the S. pneumoniae rnr gene demonstrated that it is co-transcribed with the flanking genes, secG and smpB. Transcription of these genes is driven from a promoter upstream of secG and the transcript is processed to yield mature independent mRNAs. This genetic organization seems to be a common feature of Gram positive bacteria, and the biological significance of this gene cluster is further discussed.ConclusionsThis study unravels an additional contribution of RNase R to the trans-translation system by demonstrating that smpB is regulated by this exoribonuclease. RNase R in turn, is shown to be under the control of SmpB. These proteins are therefore mutually dependent and cross-regulated. The data presented here shed light on the interactions between RNase R, trans-translation and cold-shock response in an important human pathogen.

Effect of the increased stability of the penicillin amidase mRNA on the protein expression levels

Several factors at transcriptional, post‐transcriptional or post‐translational level determine the fate of a target protein and can severely restrict its yield. Here, we focus on the post‐transcriptional regulation of the biosynthesis of the periplasmic protein, penicillin amidase (PA). The PA mRNA stability was determined under depleted RNase conditions in strains carrying single or multiple RNase deletions. Single deletion of the endonuclease RNase E yielded, as the highest, a fourfold stabilization of the PA mRNA. This effect, however, was reduced twice at post‐translational level. The RNase II, generating secondary exonucleolytic cleavages in the mRNA, although not significantly influencing the PA mRNA decay, led also to an increase of the amount of mature PA. The non‐proportional correlation between increased mRNA longevity and amount of active enzyme propose that the rational strategies for yield improvement must be based on a simultaneous tuning of more than one yield restricting factor.

Surprises in the 3′‐end: ‘U’ can decide too!

RNA molecules are subjected to post‐transcriptional modifications that might determine their maturation, activity, localization and stability. These alterations can occur within the RNA molecule or at its 5′‐ or 3′‐ extremities, and are essential for gene regulation and proper function of the RNA. One major type of modification is the 3′‐end addition of nontemplated nucleotides. Polyadenylation is the most well studied type of 3′‐RNA modification, both in eukaryotes and prokaryotes. The importance of 3′‐oligouridylation has recently gained attention through the discovery of several types of uridylated‐RNAs, by the existence of enzymes that specifically add poly(U) tails and others that preferentially degrade these tails. Namely, Dis3L2 is a 3′–5′ exoribonuclease from the RNase II/RNB family that has been shown to act preferentially on oligo(U)‐tailed transcripts. Our understanding of this process is still at the beginning, but it is already known to interfere in the regulation of diverse RNA species in most eukaryotes. Now that we are aware of the prevalence of RNA uridylation and the techniques available to globally evaluate the 3′‐terminome, we can expect to make rapid progress in determining the extent of terminal oligouridylation in different RNA populations and unravel its impact on RNA decay mechanisms. Here, we sum up what is known about 3′‐RNA modification in the different cellular compartments of eukaryotic cells, the conserved enzymes that perform this 3′‐end modification and the effectors that are selectively activated by this process.

The acetate uptake transporter family motif “NPAPLGL(M/S)” is essential for substrate uptake

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.