RNA-RNA Interactomes of ProQ and Hfq Reveal Overlapping and Competing Roles.

Base pairing RNAs modulate gene expression in all studied organisms. In many bacteria, the base pairing between most small regulatory RNAs (sRNAs) and their targets is mediated by the Hfq RNA chaperone. However, recent studies have shown FinO-domain proteins also bind sRNAs. To examine the global contribution of the FinO-domain ProQ protein in Escherichia coli, we carried out RIL-seq to identify RNA pairs bound to this protein. The RNA-RNA interactome for ProQ contains hundreds of pairs. Intriguingly, a significant fraction of the ProQ-bound RNA pairs are also found associated with Hfq, indicating overlapping, complementary, or competing roles for the two proteins. Characterization of one novel RNA pair bound by both chaperones revealed that while Hfq is required for RNA sponge-mediated downregulation of the sRNA, ProQ can inhibit this regulation. Overall, our results uncover increased complexity in RNA regulatory networks involving RNA chaperone proteins, RNases, sRNAs, and mRNAs.

Identification of Ribonuclease Inhibitors for the Control of Pathogenic Bacteria.

Bacteria are known to be constantly adapting to become resistant to antibiotics. Currently, efficient antibacterial compounds are still available; however, it is only a matter of time until these compounds also become inefficient. Ribonucleases are the enzymes responsible for the maturation and degradation of RNA molecules, and many of them are essential for microbial survival. Members of the PNPase and RNase II families of exoribonucleases have been implicated in virulence in many pathogens and, as such, are valid targets for the development of new antibacterials. In this paper, we describe the use of virtual high-throughput screening (vHTS) to identify chemical compounds predicted to bind to the active sites within the known structures of RNase II and PNPase from Escherichia coli. The subsequent in vitro screening identified compounds that inhibited the activity of these exoribonucleases, with some also affecting cell viability, thereby providing proof of principle for utilizing the known structures of these enzymes in the pursuit of new antibacterials.

Maturation of the E. coli Glu2, Ile1, and Ala1B tRNAs utilizes a complex processing pathway.

Despite significant progress in understanding the diversity of tRNA processing pathways in Escherichia coli, the mechanism for the maturation of tRNAs encoded within the rRNA operons has not received much attention. Here, we show that the Glu2, Ile1, and Ala1B tRNAs, encoded by 10 genes located between the 16S and 23S rRNAs in the seven rRNA operons, are matured via a RNase E-independent processing pathway that utilizes at least six different enzymes. It has been shown that the Glu2 and Ile1-Ala1B pre-tRNAs released by initial RNase III cleavages of the 30S primary rRNA transcripts retain extended 5'-leader (35-139 nt) and 3'-trailer (166-185 nt) sequences. However, the 5' maturation of the tRNAs by RNase P is inhibited until the trailer sequences are shortened to 1-4 nucleotides, initially by a second RNase III cleavage at 31-42 nucleotides downstream of the CCA determinant followed by exonucleolytic trimming. The RNase III cleaved Glu2 and Ile1-Ala1B trailer fragments are degraded via PAP I- dependent exonucleolytic decay. Compared to the six previously characterized tRNA processing pathways, maturation of the Glu2, Ile1, and Ala1B tRNAs is considerably more complex and appears to be distinct from what occurs in Gram-positive bacteria.

Structural and biochemical characteristics of two Staphylococcus epidermidis RNase J paralogs RNase J1 and RNase J2.

RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.

Ribonuclease II preserves chloroplast RNA homeostasis by increasing mRNA decay rates, and cooperates with polynucleotide phosphorylase in 3' end maturation.

Ribonuclease R (RNR1) and polynucleotide phosphorylase (cpPNPase) are the two known 3'→5' exoribonucleases in Arabidopsis chloroplasts, and are involved in several aspects of rRNA and mRNA metabolism. In this work, we show that mutants lacking both RNR1 and cpPNPase exhibit embryo lethality, akin to the non-viability of the analogous double mutant in Escherichia coli. We were successful, however, in combining an rnr1 null mutation with weak pnp mutant alleles, and show that the resulting chlorotic plants display a global reduction in RNA abundance. Such a counterintuitive outcome following the loss of RNA degradation activity suggests a major importance of RNA maturation as a determinant of RNA stability. Detailed analysis of the double mutant demonstrates that the enzymes catalyze a two-step maturation of mRNA 3' ends, with RNR1 polishing 3' termini created by cpPNPase. The bulky quaternary structure of cpPNPase compared with RNR1 could explain this activity split between the two enzymes. In contrast to the double mutants, the rnr1 single mutant overaccumulates most mRNA species when compared with the wild type. The excess mRNAs in rnr1 are often present in non-polysomal fractions, and half-life measurements demonstrate a substantial increase in the stability of most mRNA species tested. Together, our data reveal the cooperative activity of two 3'→5' exoribonucleases in chloroplast mRNA 3' end maturation, and support the hypothesis that RNR1 plays a significant role in the destabilization of mRNAs unprotected by ribosomes.

Quorum sensing and QsvR tightly control the transcription of vpa0607 encoding an active RNase II-type protein in Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a Gram-negative, halophilic bacterium, is a leading cause of acute gastroenteritis in humans. AphA and OpaR are the master quorum sensing (QS) regulators operating at low cell density (LCD) and high cell density (HCD), respectively. QsvR is an AraC-type protein that integrates into the QS system to control gene expression by directly controlling the transcription of aphA and opaR. However, the regulation of QsvR itself remains unclear to date. In this study, we show that vpa0607 and qsvR are transcribed as an operon, vpa0607-qsvR. AphA indirectly activates the transcription of vpa0607 at LCD, whereas OpaR and QsvR directly repress vpa0607 transcription at HCD, leading to the highest expression levels of vpa0607 occurs at LCD. Moreover, VPA0607 acts as an active RNase II-type protein in V. parahaemolyticus and feedback inhibits the expression of QsvR at the post-transcriptional level. Taken together, this work deepens our understanding of the regulation of QsvR and enriches the integration mechanisms of QsvR with the QS system in V. parahaemolyticus.

How hydrolytic exoribonucleases impact human disease: Two sides of the same story.

RNAs are extremely important molecules inside the cell, which perform many different functions. For example, messenger RNAs, transfer RNAs and ribosomal RNAs are involved in protein synthesis, whereas noncoding RNAs have numerous regulatory roles. Ribonucleases (RNases) are the enzymes responsible for the processing and degradation of all types of RNAs, having multiple roles in every aspect of RNA metabolism. However, the involvement of RNases in disease is still not well understood. This review focuses on the involvement of the RNase II/RNB family of 3'-5' exoribonucleases in human disease. This can be attributed to direct effects, whereby mutations in the eukaryotic enzymes of this family [defective in sister chromatid joining (Dis3; or Rrp44), Dis3-like exonuclease 1 (Dis3L1; or Dis3L) and Dis3-like exonuclease 2 (Dis3L2)] are associated with a disease, or indirect effects, whereby mutations in the prokaryotic counterparts of RNase II/RNB family (RNase II and/or RNase R) affect the physiology and virulence of several human pathogens. In this review, we compare the structural and biochemical characteristics of the members of the RNase II/RNB family of enzymes. The outcomes of mutations impacting enzymatic function are revisited, in terms of both the direct and indirect effects on disease. Furthermore, we also describe the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exoribonuclease and its importance to combat the COVID-19 pandemic. As a result, RNases may be a good therapeutic target to reduce bacterial and viral pathogenicity. These are the two perspectives on RNase II/RNB family enzymes that are presented in this review.

In Vitro Characterization of the Prokaryotic Counterparts of the Exosome Complex.

The same basic set of enzymatic activities exhibited by the eukaryotic RNA exosome are also found in prokaryotes. Bacteria have two predominant and distinct 3'→5' exoribonuclease activities: one is characterized by processive hydrolysis, derived from RNase II and RNase R, and the other by processive phosphorolysis, derived from PNPase. In this chapter we describe methods for (1) the overexpression and purification of these three proteins; and (2) their in vitro biochemical and enzymatic characterization-including RNA binding. The labeling and preparation of a set of specific RNA substrates is also described.

The Bacterial Counterparts of the Eukaryotic Exosome: An Evolutionary Perspective.

There are striking similarities between the processes of RNA degradation in bacteria and eukaryotes, which rely on the same basic set of enzymatic activities. In particular, enzymes that catalyze 3'→5' RNA decay share evolutionary relationships across the three domains of life. Over the years, a large body of biochemical and structural data has been generated that elucidated the mechanism of action of these enzymes. In this overview, to trace the evolutionary origins of the multisubunit RNA exosome complex, we compare the structural and functional characteristics of the eukaryotic and prokaryotic exoribonucleolytic activities.

Major 3'-5' Exoribonucleases in the Metabolism of Coding and Non-coding RNA.

3'-5' exoribonucleases are key enzymes in the degradation of superfluous or aberrant RNAs and in the maturation of precursor RNAs into their functional forms. The major bacterial 3'-5' exoribonucleases responsible for both these activities are PNPase, RNase II and RNase R. These enzymes are of ancient nature with widespread distribution. In eukaryotes, PNPase and RNase II/RNase R enzymes can be found in the cytosol and in mitochondria and chloroplasts; RNase II/RNase R-like enzymes are also found in the nucleus. Humans express one PNPase (PNPT1) and three RNase II/RNase R family members (Dis3, Dis3L and Dis3L2). These enzymes take part in a multitude of RNA surveillance mechanisms that are critical for translation accuracy. Although active against a wide range of both coding and non-coding RNAs, the different 3'-5' exoribonucleases exhibit distinct substrate affinities. The latest studies on these RNA degradative enzymes have contributed to the identification of additional homologue proteins, the uncovering of novel RNA degradation pathways, and to a better comprehension of several disease-related processes and response to stress, amongst many other exciting findings. Here, we provide a comprehensive and up-to-date overview on the function, structure, regulation and substrate preference of the key 3'-5' exoribonucleases involved in RNA metabolism.

Characterizing the Role of Exoribonucleases in the Control of Microbial Gene Expression: Differential RNA-Seq.

Differential RNA-Seq is a next-generation technology method to determine the significant transcriptomic differences between two and more samples. With this method it is possible to analyze the total RNA content of different samples making it the best global analysis method currently available to study the roles of exoribonucleases in the cell. These enzymes are responsible for the RNA processing and degradation in the cells and therefore affect the total RNA pool in ways not yet fully understood. In Escherichia coli there are three main degradative exoribonucleases RNase II, RNase R, and PNPase that degrade the RNA from the 3' to the 5'-end. These enzymes have several roles in the cell and even though they are degradative enzymes RNase II and PNPase can also protect some RNAs from degradation and PNPase can also act as an RNA polymerase under some conditions. The multiplicity of roles of these exoribonucleases leads to a very high number of transcripts that are affected by their absence in the cell. With the differential RNA-Seq it is possible to obtain a much deeper understanding of how these enzymes work and regulate the bacterial gene expression. In this chapter we have described a differential RNA-Seq data analysis protocol applied to the study of exoribonucleases. We also included the protocol for experimental validation of the RNA-Seq data using qPCR and motility assays. Although the methods described in this chapter were applied to the study of the exoribonucleases, they can also be used for other differential RNA-Seq studies.

Effect of antimalarials treatment on rat liver lysosomal function-Anin vivo study.

Effects of treatmentin vivo with the antimalarials:chloroquine (CQ), primaquine (PQ) and quinine(Q) on lysosomal enzymes and lysosomal membrane integrity were examined. Treatment with the three antimalarials showed an apparent increase in the membrane stability. CQ treatment resulted in increase in both the 'free' and 'total' activities of all the enzymes i.e. acid phosphatase, RNase II, DNase II and cathepsin D. PQ treatment lowered the 'free' and 'total' activities of acid phosphatase and cathepsin D, but the DNase II activities increased. Treatment with Q resulted in increased 'free' and 'total' activities of RNase II and DNase II. While 'free' activities of acid phosphatase and cathepsin D were low; the 'total' activities increased significantly. Our results suggest that a generalized increase in free nucleases activities following prolonged treatment with antimalarials may lead to cell damage and/or necrosis.

YoeB toxin is activated during thermal stress.

Type II toxin-antitoxin (TA) modules are thought to mediate stress-responses by temporarily suppressing protein synthesis while cells redirect transcription to adapt to environmental change. Here, we show that YoeB, a ribosome-dependent mRNase toxin, is activated in Escherichia coli cells grown at elevated temperatures. YoeB activation is dependent on Lon protease, suggesting that thermal stress promotes increased degradation of the YefM antitoxin. Though YefM is efficiently degraded in response to Lon overproduction, we find that Lon antigen levels do not increase during heat shock, indicating that another mechanism accounts for temperature-induced YefM proteolysis. These observations suggest that YefM/YoeB functions in adaptation to temperature stress. However, this response is distinct from previously described models of TA function. First, YoeB mRNase activity is maintained over several hours of culture at 42°C, indicating that thermal activation is not transient. Moreover, heat-activated YoeB does not induce growth arrest nor does it suppress global protein synthesis. In fact, E. coli cells proliferate more rapidly at elevated temperatures and instantaneously accelerate their growth rate in response to acute heat shock. We propose that heat-activated YoeB may serve a quality control function, facilitating the recycling of stalled translation complexes through ribosome rescue pathways.