Biology subjects

Arraiano, C. M.

Publications and source records attributed to Arraiano, C. M..

8 recordsLinked to original sources

bioRxiv2026

RNase III influences microaerobic symbiotic pathways and RNA regulation in Sinorhizobium meliloti

Bacterial ribonucleases (RNases) are central components of post-transcriptional networks underlying environmental adaptation. However, their contribution to the ecological specialization of bacteria with complex lifestyles, such as nitrogen-fixing legume symbionts, remains poorly understood. Here, we investigated the role of the double-stranded RNase III ortholog (SmRNase III) in Sinorhizobium meliloti, the symbiotic partner of alfalfa (Medicago sativa L.). Loss of SmRNase III function affected the expression of nearly 30% of protein-coding genes and 12% of annotated non-coding RNAs (sRNAs). Remarkably, more than 70% of these changes occurred under the microaerobic conditions typical of symbiotic root nodules. Many SmRNase III-dependent transcripts encode pathways supporting microaerobic metabolism and nitrogen fixation in endosymbiotic bacteroids. Analysis of sequencing read coverage revealed putative consensus cleavage signatures biased toward mRNA 5' untranslated regions, suggesting preferential processing at these sites. Altered expression of sRNAs and/or their predicted mRNA targets further supports a role for SmRNase III in sRNA-mediated silencing. Consistently, in vivo and in vitro assays demonstrated that SmRNase III is required for the repression of nifK (encoding the {beta}-subunit of the nitrogenase MoFe protein) and dctA (encoding a major dicarboxylate transporter) by the antisense sRNA asNifK1 and the trans-sRNA AbcR1, respectively. Our findings reveal a major impact of SmRNase III on shaping the symbiotic transcriptome of S. meliloti and provide a foundation for deeper investigation into the mechanisms and regulatory roles of RNase III activity in rhizobia.

microbiology↗

bioRxiv2025

Structural and mechanistic insights into Dis3L2 mediated degradation of structured RNA

The RNase II/RNB family of exoribonucleases is present in all domains of life and includes three main eukaryotic members, the Dis3-like proteins (Dis3, Dis3L1, Dis3L2). At the cellular level, Dis3L2 is distinguished by the unique preference for uridylated RNA substrates and the highest efficiency in degrading double-stranded RNA. Defects in these enzymes have been linked to some types of cancers and overgrowth disorders in humans. In this work, we used the Dis3L2 protein from the model organism Schizosaccharomyces pombe (SpDis3L2) to better understand the mechanism of action of Dis3-like exoribonucleases, and to elucidate how single amino acid substitutions in these proteins can affect the biochemical properties of the enzymes, potentially contributing to the molecular basis of the related human diseases. We determined the crystal structure of SpDis3L2 bound to a U13 RNA, in which the protein displays a typical vase-like conformation, accommodating 6 nucleotides of the RNA 3-end. Furthermore, we constructed two SpDis3L2 protein variants, harbouring single amino acid substitutions mimicking the ones already found in human patients, to test their catalytic activity in vitro. We highlight the A756R SpDis3L2 variant, which loses the ability to degrade double-stranded RNA substrates and accumulates intermediary degradation products when degrading single-stranded RNA substrates. As such, A756 seems to be a key residue responsible for the normal cellular function of Dis3L2, specifically regarding its important role in the degradation of structured RNA substrates.

biochemistry↗

bioRxiv2023

RNase R Controls Membrane Fatty Acid Composition in Streptococcus pneumoniae

AO_SCPLOWBSTRACTC_SCPLOWPrevious studies on RNase R have highlighted significant effects of this ribonuclease in several processes of Streptococcus pneumoniae biology. In this work we have studied the global impact of RNase R by comparing the transcriptional landscape of a deleted RNase R mutant to that of the wild-type strain, and this led us investigate specific targets affected by RNase R. RNA-Seq showed that RNase R deletion affects transcripts from several different biological processes. Of particular interest, elimination of RNase R results in overexpression of most of the genes encoding the components of type II fatty acid biosynthesis (FAS-II) cluster. We demonstrate that RNase R governs the turnover of most of genes from this pathway, affecting the outcome of the whole FAS-II cluster, and leading to an unbalanced membrane fatty acid composition. Our results show that the membrane of the deleted strain contains a higher proportion of unsaturated and long-chained fatty acids than the wild type strain. This leads to a higher fluidity of the {Delta}rnr mutant membrane, which is probably related with the increased sensitivity to detergent observed in this strain. We demonstrate that RNase R expression is induced in cells challenged with H2O2, which is suggestive of a role for this ribonuclease on the regulation of membrane homeostasis under oxidative stress. Reprogramming of membrane fluidity is an adaptative cell response crucial for bacterial survival in constantly changing environmental conditions. The fact that RNase R controls the expression of several essential genes to the fatty acid synthesis unveils a new important function of this enzyme.

molecular biology↗

bioRxiv2021

RNase R, a new virulence determinant of Streptococcus pneumoniae

Pneumococcal infections have increasingly high mortality rates despite the availability of vaccines and antibiotics. The increase of bacterial resistance to antibiotics urges the discovery of new alternative therapeutics. Therefore, the identification of new virulence determinants, and the understanding of the molecular mechanisms behind pathogenesis and pneumococcal-host interactions has become of paramount importance in the search of new targets for drug development. The exoribonuclease RNase R has been involved in virulence in a growing number of pathogens. In this work, we have used Galleria mellonella as an infection model to demonstrate that the presence of ribonuclease R increases the pneumococcus virulence. Although the absence of RNase R does not affect exponential growth, the ability of the RNase R deleted strain to replicate in the hemolymph is compromised. Larvae infected with the RNase R mutant strain show an increased expression level of antimicrobial peptides, and have a lower bacterial load in the haemolymph in the later stages of infection, leading to a higher survival rate. Interestingly RNase R carrying pneumococci suffer a sudden drop in bacterial numbers immediately after infection, resembling the eclipse phase observed after intravenous inoculation in mice. Together our results suggest that RNase R might be involved in the ability of pneumococci to evade the host immune response, probably by interfering with internalisation and/or replication inside the larval hemocytes.

molecular biology↗

bioRxiv2021

New targets for drug design: Importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2

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 coronavirus (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.

microbiology↗

bioRxiv2019

Dis3L2 regulates cellular proliferation through a PI3-Kinase dependent signalling pathway

Dis3L2 is a highly conserved 3-5 exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms tumour. Here, we have generated a new dis3L2 null mutant and a UAS-nuclease-dead line in Drosophila to demonstrate that the catalytic activity of Dis3L2 is required to control proliferation in imaginal discs. Using RNA-seq on dis3L2 mutant wing discs, we show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth observed in dis3L2 null mutants. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. We also show that loss of DIS3L2 in human HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells we show that loss of DIS3L2 results in an increase in the activity of the PI3-Kinase/AKT signalling pathway, which we subsequently show to also have a role in driving the proliferation phenotype in Drosophila. Our work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth.

molecular biology↗

bioRxiv2019

Tailor-made sRNAs: a toolbox to control metabolic targets

Pseudomonas putida is a highly attractive production system for industrial needs. Modulation of gene expression is an urgent need to redesign P. putida metabolism for its improvement as biocatalyst at industrial level. We report the construction of a small RNA-based system with potential to be used for different purposes in synthetic biology. Due to their modular composition, design facilities and ability in tuning gene expression, sRNAs constitute a powerful tool in genetic and metabolic engineering. In the toolbox presented here, the synthetic sRNA is specifically directed to any region of a chosen target. The expression of the synthetic sRNAs is shown to differentially modulate the level of endogenous and reporter genes. The antisense interaction of the sRNA with the mRNA results in different outcomes. Depending on the particularity of each sRNA-target mRNA pair, we managed to demonstrate the duality of this system, able either to repress or overexpress a given gene. This system combines a high specificity with a wide applicability due to its ability to modulate the expression of virtually any given gene. By plugging-in and -out genetic circuits, this tailor-made regulatory system can be used to redesign P. putida metabolism, fulfilling an important industrial gap in synthetic biology.

synthetic biology↗

bioRxiv2019

A role for DIS3L2 over human nonsense-mediated mRNA decay targets

The nonsense-mediated decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5 and 3 ends. This is done by a process not yet completely understood that recruits decapping and 5-to-3 exonuclease activities, as well as deadenylating and 3-to-5 exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. DIS3L1 and DIS3L2 exoribonucleases localize in the same compartment where NMD occurs, but little is known about their role in this process. In order to unveil the role of DIS3L2 in NMD, here we show that some NMD-targets accumulate in DIS3L2-depleted cells. mRNA half-life analysis further supports that these NMD-targets are in fact DIS3L2 substrates. Besides, we observed that DIS3L2 acts over full-length transcripts, through a process that also involves UPF1. Moreover, DIS3L2-mediated decay is dependent on the activity of the terminal uridylyl transferases Zcchc6/11 (TUT7/4). Together, our findings establish a role for DIS3L2 and uridylation in NMD.

molecular biology↗