Interplay of Exoribonucleases, Hfq and Small RNAs Structural Determinants in the Control of Gene Expression

Dissertation presented to obtain the Ph.D degree in BiologySmall non-coding RNAs (sRNAs) are regulatory molecules that typically are not translated into proteins. These molecules are often highly structured and very stable and can affect many genetic pathways in all domains of life. Bacterial small regulatory RNAs (sRNAs) parallel microRNAs in their ability to control multiple targets. Small RNAs can bind to proteins or to mRNA targets. The sRNAs that act by an antisense mechanism can have full (cis-encoded) or partial complementarity (trans-encoded) with their targets. Most of the trans-encoded sRNAs studied so far in Escherichia coli bind the RNA chaperone Hfq. The 5’ end of antisense RNAs is usually found to be critical for the interaction with targets, generally inhibiting translation and promoting mRNA decay. RNases are key elements in the control of RNA levels in the cell and not surprisingly are also critical in the regulation of sRNAs. In E. coli there are three 3’-5’exoribonucleases that accomplish most of the mRNA exodegradative activity: ribonuclease II (RNase II), ribonuclease R (RNase R) and polynucleotide phosphorylase (PNPase).(...

RNase R Controls Membrane Fatty Acid Composition in Streptococcus pneumoniae

Previous 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 Arnr 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.This research was funded by national funds through FCT—Fundação para a Ciência e a Tecnologia—I. P., Project MOSTMICRO-ITQB with refs UIDB/04612/2020 and UIDP/04612/2020, and Project EXPL/BIA-MOL/1244/2021. S.D. and V.P. were financed by FCT contracts according to DL57/2016, respectively SFRH/BPD/84080/2012) and (SFRH/BPD/87188/2012). C.B. had a contract under the FCT project PTDC/BIA BQM/28479/2017.N

Pseudomonas putida KT2440 is naturally endowed to withstand industrial-scale stress conditions

Pseudomonas putida is recognized as a very promising strain for industrial application due to its high redox capacity and frequently observed tolerance towards organic solvents. In this research, we studied the metabolic and transcriptional response of P. putida KT2440 exposed to large-scale heterogeneous mixing conditions in the form of repeated glucose shortage. Cellular responses were mimicked in an experimental setup comprising a stirred tank reactor and a connected plug flow reactor. We deciphered that a stringent response-like transcriptional regulation programme is frequently induced, which seems to be linked to the intracellular pool of 3-hydroxyalkanoates (3-HA) that are known to serve as precursors for polyhydroxyalkanoates (PHA). To be precise, P. putida is endowed with a survival strategy likely to access cellular PHA, amino acids and glycogen in few seconds under glucose starvation to obtain ATP from respiration, thereby replenishing the reduced ATP levels and the adenylate energy charge. Notably, cells only need 0.4% of glucose uptake to build those 3-HA-based energy buffers. Concomitantly, genes that are related to amino acid catabolism and β-oxidation are upregulated during the transient absence of glucose. Furthermore, we provide a detailed list of transcriptional short- and long-term responses that increase the cellular maintenance by about 17% under the industrial-like conditions tested.publishersversionpublishe

Copper Acts Synergistically With Fluconazole in Candida glabrata by Compromising Drug Efflux, Sterol Metabolism, and Zinc Homeostasis

Funding Information: This work was supported by (1) Project LISBOA-01-0145-FEDER-007660 (“Microbiologia Molecular, Estrutural e Celular”) funded by FEDER funds through COMPETE2020 – “Programa Operacional Competitividade e Internacionalização” (POCI); (2) “Fundação para a Ciência e a Tecnologia” (FCT) through programme IF (IF/00124/2015) to CP; (3) the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 810856; (4) COST Action CA15133, supported by COST (European Cooperation in Science and Technology); and (5) PPBI – Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122) co-funded by national funds from OE – “Orçamento de Estado” and by FEDER. AG-C was supported by a FCT Ph.D. fellowship (SFRH/BD/118866/2016), and CA and VP by a FCT contract according to DL57/2016 (SFRH/BPD/74294/2010 and SFRH/BPD/87188/2012, respectively). Publisher Copyright: Copyright © 2022 Gaspar-Cordeiro, Amaral, Pobre, Antunes, Petronilho, Paixão, Matos and Pimentel.The synergistic combinations of drugs are promising strategies to boost the effectiveness of current antifungals and thus prevent the emergence of resistance. In this work, we show that copper and the antifungal fluconazole act synergistically against Candida glabrata, an opportunistic pathogenic yeast intrinsically tolerant to fluconazole. Analyses of the transcriptomic profile of C. glabrata after the combination of copper and fluconazole showed that the expression of the multidrug transporter gene CDR1 was decreased, suggesting that fluconazole efflux could be affected. In agreement, we observed that copper inhibits the transactivation of Pdr1, the transcription regulator of multidrug transporters and leads to the intracellular accumulation of fluconazole. Copper also decreases the transcriptional induction of ergosterol biosynthesis (ERG) genes by fluconazole, which culminates in the accumulation of toxic sterols. Co-treatment of cells with copper and fluconazole should affect the function of proteins located in the plasma membrane, as several ultrastructural alterations, including irregular cell wall and plasma membrane and loss of cell wall integrity, were observed. Finally, we show that the combination of copper and fluconazole downregulates the expression of the gene encoding the zinc-responsive transcription regulator Zap1, which possibly, together with the membrane transporters malfunction, generates zinc depletion. Supplementation with zinc reverts the toxic effect of combining copper with fluconazole, underscoring the importance of this metal in the observed synergistic effect. Overall, this work, while unveiling the molecular basis that supports the use of copper to enhance the effectiveness of fluconazole, paves the way for the development of new metal-based antifungal strategies.publishe

Two-way attack on IAPP proteotoxicity with implications for diabetes

Funding Information: This study was supported by FCT–Fundação para a Ciência e a Tecnologia (grants UIDB/04567/2020 and UIDP/ 04567/2020 to CBIOS, PTDC/BIA-MOL/31104/2017, and PhD grants PD/BD/135504/2018 to AFR and UI/BD/151421/2021 to SF. RM is funded by FCT Scientific Employment Stimulus contract with the reference number CEEC/04567/ CBIOS/2020. Authors also acknowledge COFAC/ILIND – Cooperativa De Formação e Animação Cultural CRL/Instituto Lusófono de Investigação e Desenvolvimento (grant COFAC/ILIND/CBIOS/2/2021). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofunded by Fundação para a Ciência e Tecnologia (FCT) / Ministério da Ciência e do Ensino Superior, through national funds, and by FEDER under the PT2020 Partnership Agreement, is acknowledged (UIDB/04462/2020 and UIDP/04462/2020). CNS acknowledge the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 804229. JAB gratefully acknowledges FCT-Fundação para a Ciência e a Tecnologia, I.P. through MOSTMICRO-ITQB R&D Unit-UIDB/04612/2020 and LS4FUTURE Associated Laboratory-LA/P/0087/2020, and by the framework of Article 23 of Decree-Law No.57/2017 of August 29. Publisher Copyright: Copyright © 2022 Raimundo, Ferreira, Pobre, Lopes-da-Silva, Brito, dos Santos, Saraiva, dos Santos and Menezes.Introduction: Diabetes is one of the major metabolic diseases worldwide. Despite being a complex systemic pathology, the aggregation and deposition of Islet Amyloid Polypeptide (IAPP), or amylin, is a recognized histopathological marker of the disease. Although IAPP proteotoxicity represents an important trigger of β-cell dysfunction and ultimately death, its exploitation as a therapeutic tool remains underdeveloped. The bioactivity of (poly)phenols towards inhibition of pathological protein aggregation is well known, however, most of the identified molecules have limited bioavailability. Methods: Using a strategy combining in silico, cell-free and cell studies, we scrutinized a unique in-house collection of (poly)phenol metabolites predicted to appear in the human circulation after (poly)phenols ingestion. Results: We identified urolithin B as a potent inhibitor of IAPP aggregation and a powerful modulator of cell homeostasis pathways. Urolithin B was shown to affect IAPP aggregation pattern, delaying the formation of amyloid fibrils and altering their size and morphology. The molecular mechanisms underlying urolithin B-mediated protection include protein clearance pathways, mitochondrial function, and cell cycle ultimately rescuing IAPP-mediated cell dysfunction and death. Discussion: In brief, our study uncovered urolithin B as a novel small molecule targeting IAPP pathological aggregation with potential to be exploited as a therapeutic tool for mitigating cellular dysfunction in diabetes. Resulting from the colonic metabolism of dietary ellagic acid in the human body, urolithin B bioactivity has the potential to be explored in nutritional, nutraceutical, and pharmacological perspectives.publishersversionpublishe

PNPase is involved in the coordination of mRNA degradation and expression in stationary phase cells of Escherichia coli

Background: Exoribonucleases are crucial for RNA degradation in Escherichia coli but the roles of RNase R and PNPase and their potential overlap in stationary phase are not well characterized. Here, we used a genome-wide approach to determine how RNase R and PNPase affect the mRNA half-lives in the stationary phase. The genome-wide mRNA half-lives were determined by a dynamic analysis of transcriptomes after transcription arrest. We have combined the analysis of mRNA half-lives with the steady-state concentrations (transcriptome) to provide an integrated overview of the in vivo activity of these exoribonucleases at the genome-scale. Results: The values of mRNA half-lives demonstrated that the mRNAs are very stable in the stationary phase and that the deletion of RNase R or PNPase caused only a limited mRNA stabilization. Intriguingly the absence of PNPase provoked also the destabilization of many mRNAs. These changes in mRNA half-lives in the PNPase deletion strain were associated with a massive reorganization of mRNA levels and also variation in several ncRNA concentrations. Finally, the in vivo activity of the degradation machinery was found frequently saturated by mRNAs in the PNPase mutant unlike in the RNase R mutant, suggesting that the degradation activity is limited by the deletion of PNPase but not by the deletion of RNase R. Conclusions: This work had identified PNPase as a central player associated with mRNA degradation in stationary phase

Small RNA Modules Confer Different Stabilities and Interact Differently with Multiple Targets

<div><p>Bacterial Hfq-associated small regulatory RNAs (sRNAs) parallel animal microRNAs in their ability to control multiple target mRNAs. The small non-coding MicA RNA represses the expression of several genes, including major outer membrane proteins such as <em>omp</em>A, <em>tsx</em> and <em>ecn</em>B. In this study, we have characterised the RNA determinants involved in the stability of MicA and analysed how they influence the expression of its targets. Site-directed mutagenesis was used to construct MicA mutated forms. The 5′linear domain, the structured region with two stem-loops, the A/U-rich sequence or the 3′ poly(U) tail were altered without affecting the overall secondary structure of MicA. The stability and the target regulation abilities of the wild-type and the different mutated forms of MicA were then compared. The 5′ domain impacted MicA stability through an RNase III-mediated pathway. The two stem-loops showed different roles and disruption of stem-loop 2 was the one that mostly affected MicA stability and abundance. Moreover, STEM2 was found to be more important for the <em>in vivo</em> repression of both <em>omp</em>A and <em>ecn</em>B mRNAs while STEM1 was critical for regulation of <em>tsx</em> mRNA levels. The A/U-rich linear sequence is not the only Hfq-binding site present in MicA and the 3′ poly(U) sequence was critical for sRNA stability. PNPase was shown to be an important exoribonuclease involved in sRNA degradation. In addition to the 5′ domain of MicA, the stem-loops and the 3′ poly(U) tail are also shown to affect target-binding. Disruption of the 3′U-rich sequence greatly affects all targets analysed. In conclusion, our results have shown that it is important to understand the “sRNA anatomy” in order to modulate its stability. Furthermore, we have demonstrated that MicA RNA can use different modules to regulate its targets. This knowledge can allow for the engineering of non-coding RNAs that interact differently with multiple targets.</p> </div

Mutagenesis of the A/U-rich domain, a Hfq-binding site in MicA.

<p>(A) Northern blot analysis of MicA in Δ<i>mic</i>A cells expressing <i>in trans</i> either the wild-type MicA (from the pMicA-WT plasmid) or a MicA variant with the A/U-rich domain mutated to a C-rich sequence (from the pMicA-hfq plasmid). Plasmid pMicA-WT was also used to transform a deletion strain of <i>hfq</i>. A smaller form of MicA (designated MicA∗) is only clearly observed in the absence of Hfq; this fragment had been previously identified <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052866#pone.0052866-Andrade2" target="_blank">[13]</a>. A size marker is shown on the left of the gel. The riboprobe used to detect MicA, cross-reacts with a nonspecific band, (that is also detected on the Δ<i>mic</i>A strain, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052866#pone.0052866.s006" target="_blank">Figure S6</a>) that was here used as loading control. A more stringent washing step eliminates this band without affecting the MicA signal, as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052866#pone.0052866-Andrade1" target="_blank">[12]</a>. RNA was extracted from stationary phase cultures. (B) Mutagenesis of the A/U-rich domain of MicA to a C-rich sequence affects the Hfq binding ability to this small RNA. The gel mobility shift assay was performed with a constant amount of radiolabelled MicA-WT or MicA-hfq variant as RNA substrates and increasing amounts of purified Hfq protein, as indicated in the figure. The free RNA and the Hfq-RNA complexes are indicated. The gels were then dried and exposed to a PhosphorImager screen and quantified using ImageQuant software. The results were plot using SigmaPlot software and binding curves were fit. Filled circles represent MicA-WT and open circles represent MicA-hfq variant.</p

Mutagenesis of the 3′ end U-rich domain of MicA.

<p>(A) Effect of mutations in the 3′end U-rich linear sequence in the stability of the MicA RNA. Northern blot analysis of MicA in Δ<i>mic</i>A cells expressing <i>in trans</i> the wild-type MicA (from the pMicA-WT) or the mutated 3′ end variants (from the pMicA-3′mut1 or pMicA-3′mut2 plasmids). Read-through bands are indicated by the symbol (¶). Two different sized forms of MicA can be detected and are marked with arrows on the side of the gel. (B) Northern blot analysis of MicA in Δ<i>mic</i>A cells or its derivative isogenic mutants lacking either PNPase (Δ<i>mic</i>A <i>pnp</i>) or Poly(A) polymerase I (Δ<i>mic</i>A Δ<i>pcn</i>B) expressing <i>in trans</i> either the mutated MicA-3′mut1 or the MicA-3′mut2 variant. RNA was extracted from stationary phase cultures. Upon hybridization of the membrane, a nonspecific band is observed and was here used as loading control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052866#pone.0052866-Andrade1" target="_blank">[12]</a>.</p

Primers used in this work.

<p>Nucleotide changes are indicated in small capitals;</p><p>T7 promotor sequence is underlined.</p><p>“+” precedes LNA-modified nucleotides.</p