Rute G. Matos

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.

New insights into the mechanism of RNA degradation by ribonuclease II: identification of the residue responsible for setting the RNase II end-product

RNase II is a key exoribonuclease involved in the maturation, turnover, and quality control of RNA. RNase II homologues are components of the exosome, a complex of exoribonucleases. The structure of RNase II unraveled crucial aspects of the mechanism of RNA degradation. Here we show that mutations in highly conserved residues at the active site affect the activity of the enzyme. Moreover, we have identified the residue that is responsible for setting the end product of RNase II. In addition, we present for the first time the models of two members of the RNase II family, RNase R from Escherichia coli and human Rrp44, also called Dis3. Our findings improve the present model for RNA degradation by the RNase II family of enzymes.

Determination of key residues for catalysis and RNA-cleavage specificity: one mutation turns RNase II into a "super"-enzyme

RNase II is the prototype of a ubiquitous family of enzymes that are crucial for RNA metabolism. In Escherichia coli this protein is a single-stranded-specific 3′-exoribonuclease with a modular organization of four functional domains. In eukaryotes, the RNase II homologue Rrp44 (also known as Dis3) is the catalytic subunit of the exosome, an exoribonuclease complex essential for RNA processing and decay. In this work we have performed a functional characterization of several highly conserved residues located in the RNase II catalytic domain to address their precise role in the RNase II activity. We have constructed a number of RNase II mutants and compared their activity and RNA binding to the wild type using different single- or double-stranded substrates. The results presented in this study substantially improve the RNase II model for RNA degradation. We have identified the residues that are responsible for the discrimination of cleavage of RNA versus DNA. We also show that the Arg-500 residue present in the RNase II active site is crucial for activity but not for RNA binding. The most prominent finding presented is the extraordinary catalysis observed in the E542A mutant that turns RNase II into a “super-enzyme.”

RNase II: The finer details of the Modus operandi of a molecular killer

The RNase II family of enzymes is ubiquitous and has crucial roles in the processing, degradation and quality control of all types of RNA. These exoribonucleases processively degrade RNA from the 3’-end releasing 5’-nucleotide monophosphates. In prokaryotes, RNase II and RNase R have two N-terminal CSD and one C-terminal S1 domain involved in RNA binding, and a central catalytic RNB domain. In eukaryotes, Rrp44p/Dis3, is a RNase II-like protein with similar modular organization, that is the only catalytically active nuclease in the exosome, a complex crucial for RNA metabolism. Here we review recent progresses in the understanding of the degradation mechanism of RNase II, based on mutational analysis and their characterization regarding catalysis and RNA affinity. We have given particular emphasis on E. coli RNase II but the synergies between the functional and structural studies have shown that our findings have implications in the understanding the similar mode of action of other RNase II family members.

Swapping the domains of exoribonucleases RNase II and RNase R: Conferring upon RNase II the ability to degrade ds RNA

RNase II and RNase R are the two E. coli exoribonucleases that belong to the RNase II super family of enzymes. They degrade RNA hydrolytically in the 3′ to 5′ direction in a processive and sequence independent manner. However, while RNase R is capable of degrading structured RNAs, the RNase II activity is impaired by dsRNAs. The final end‐product of these two enzymes is also different, being 4 nt for RNase II and 2 nt for RNase R. RNase II and RNase R share structural properties, including 60% of amino acid sequence similarity and have a similar modular domain organization: two N‐terminal cold shock domains (CSD1 and CSD2), one central RNB catalytic domain, and one C‐terminal S1 domain. We have constructed hybrid proteins by swapping the domains between RNase II and RNase R to determine which are the responsible for the differences observed between RNase R and RNase II. The results obtained show that the S1 and RNB domains from RNase R in an RNase II context allow the degradation of double‐stranded substrates and the appearance of the 2 nt long end‐product. Moreover, the degradation of structured RNAs becomes tail‐independent when the RNB domain from RNase R is no longer associated with the RNA binding domains (CSD and S1) of the genuine protein. Finally, we show that the RNase R C‐terminal Lysine‐rich region is involved in the degradation of double‐stranded substrates in an RNase II context, probably by unwinding the substrate before it enters into the catalytic cavity. Proteins 2011;

The Bacterial Protein Azurin Impairs Invasion and FAK/Src Signaling in P-Cadherin-Overexpressing Breast Cancer Cell Models

P-cadherin overexpression occurs in about 30% of all breast carcinomas, being a poor prognostic factor for breast cancer patients. In a cellular background of wild-type E-cadherin, we have previously shown that its expression promotes invasion, motility and migration of breast cancer cells due to the induced secretion of metalloproteases (MMPs) to the extracellular medium and to the concomitant shedding of a pro-invasive soluble form of this protein (sP-cad). Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human cancer cells relative to normal cells. Three different breast cancer cell lines, MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 were treated with sub-killing doses of azurin. Invasion of these cells was measured using Matrigel Invasion Assays and MTT assays were performed to determine cell viability upon treatment and the effects on cadherins expression was determined by Western blot and Immunofluorescence. Gelatin Zymography was used to determine activity of MMP2 in the conditioned media of azurin treated and untreated cells and the phosphorylation levels of intracellular signaling proteins were determined by Western blot. The invasive phenotype of these breast cancer cells was significantly reduced by azurin. Azurin (50–100 µM) also caused a specific decrease on P-cadherin protein levels from 30–50% in MCF-7/AZ.Pcad and SUM149 breast cancer cell lines, but the levels of E-cadherin remain unaltered. More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins. Our data show that azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling. Therefore, azurin could possibly be considered a therapeutic tool to treat poor-prognosis breast carcinomas overexpressing P-cadherin in a wild type E-cadherin context.

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.

Sinorhizobium meliloti YbeY is an endoribonuclease with unprecedented catalytic features, acting as silencing enzyme in riboregulation

Abstract Structural and biochemical features suggest that the almost ubiquitous bacterial YbeY protein may serve catalytic and/or Hfq-like protective functions central to small RNA (sRNA)-mediated regulation and RNA metabolism. We have biochemically and genetically characterized the YbeY ortholog of the legume symbiont Sinorhizobium meliloti (SmYbeY). Co-immunoprecipitation (CoIP) with a FLAG-tagged SmYbeY yielded a poor enrichment in RNA species, compared to Hfq CoIP-RNA uncovered previously by a similar experimental setup. Purified SmYbeY behaved as a monomer that indistinctly cleaved single- and double-stranded RNA substrates, a unique ability among bacterial endoribonucleases. SmYbeY-mediated catalysis was supported by the divalent metal ions Mg2+, Mn2+ and Ca2+, which influenced in a different manner cleavage efficiency and reactivity patterns, with Ca2+ specifically blocking activity on double-stranded and some structured RNA molecules. SmYbeY loss-of-function compromised expression of core energy and RNA metabolism genes, whilst promoting accumulation of motility, late symbiotic and transport mRNAs. Some of the latter transcripts are known Hfq-binding sRNA targets and might be SmYbeY substrates. Genetic reporter and in vitro assays confirmed that SmYbeY is required for sRNA-mediated down-regulation of the amino acid ABC transporter prbA mRNA. We have thus discovered a bacterial endoribonuclease with unprecedented catalytic features, acting also as gene silencing enzyme.

The RNase R from Campylobacter jejuni has unique features and is involved in the first steps of infection

Background: Members of the RNB family are involved in virulence; nothing is known about the Campylobacter jejuni homologue. Results: Cj-RNase R is active in several conditions. It is also important for adhesion and invasion. Conclusion: RNase R is important for C. jejuni biology and infection. Significance: RNase R could be targeted to reduce infection by this pathogen. Bacterial pathogens must adapt/respond rapidly to changing environmental conditions. Ribonucleases (RNases) can be crucial factors contributing to the fast adaptation of RNA levels to different environmental demands. It has been demonstrated that the exoribonuclease polynucleotide phosphorylase (PNPase) facilitates survival of Campylobacter jejuni in low temperatures and favors swimming, chick colonization, and cell adhesion/invasion. However, little is known about the mechanism of action of other ribonucleases in this microorganism. Members of the RNB family of enzymes have been shown to be involved in virulence of several pathogens. We have searched C. jejuni genome for homologues and found one candidate that displayed properties more similar to RNase R (Cj-RNR). We show here that Cj-RNR is important for the first steps of infection, the adhesion and invasion of C. jejuni to eukaryotic cells. Moreover, Cj-RNR proved to be active in a wide range of conditions. The results obtained lead us to conclude that Cj-RNR has an important role in the biology of this foodborne pathogen.

Characterization of the biochemical properties of Campylobacter jejuni RNase III.

Campylobacter jejuni is a foodborne bacterial pathogen, which is now considered as a leading cause of human bacterial gastroenteritis. The information regarding ribonucleases in C. jejuni is very scarce but there are hints that they can be instrumental in virulence mechanisms. Namely, PNPase (polynucleotide phosphorylase) was shown to allow survival of C. jejuni in refrigerated conditions, to facilitate bacterial swimming, cell adhesion, colonization and invasion. In several microorganisms PNPase synthesis is auto-controlled in an RNase III (ribonuclease III)-dependent mechanism. Thereby, we have cloned, overexpressed, purified and characterized Cj-RNase III (C. jejuni RNase III). We have demonstrated that Cj-RNase III is able to complement an Escherichia coli rnc-deficient strain in 30S rRNA processing and PNPase regulation. Cj-RNase III was shown to be active in an unexpectedly large range of conditions, and Mn2+ seems to be its preferred co-factor, contrarily to what was described for other RNase III orthologues. The results lead us to speculate that Cj-RNase III may have an important role under a Mn2+-rich environment. Mutational analysis strengthened the function of some residues in the catalytic mechanism of action of RNase III, which was shown to be conserved.