Post-transcriptional control in the regulation of polyhydroxyalkanoates synthesis

Funding Information: Funding: Work at ITQB NOVA was financially supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie ID: 867437, and projects UIDB/04612/2020 and UIDP/04612/2020 (Molecular, Structural and Cellular Microbiology), funded by FEDER through COMPETE 2020—Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT—Fundação para a Ciência e a Tecnologia; Work at NOVA School of Science and Technology FCT I.P., was funded by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy-i4HB. Funding Information: Work at ITQB NOVA was financially supported by the European Union?s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie ID: 867437, and projects UIDB/04612/2020 and UIDP/04612/2020 (Molecular, Structural and Cellular Microbiology), funded by FEDER through COMPETE 2020?Programa Operacional Competitividade e Internacionaliza??o (POCI) and by national funds through FCT?Funda??o para a Ci?ncia e a Tecnologia; Work at NOVA School of Science and Technology FCT I.P., was funded by national funds from FCT?Funda??o para a Ci?ncia e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences?UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy-i4HB. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.The large production of non-degradable petrol-based plastics has become a major global issue due to its environmental pollution. Biopolymers produced by microorganisms such as polyhy-droxyalkanoates (PHAs) are gaining potential as a sustainable alternative, but the high cost associated with their industrial production has been a limiting factor. Post-transcriptional regulation is a key step to control gene expression in changing environments and has been reported to play a major role in numerous cellular processes. However, limited reports are available concerning the regulation of PHA accumulation in bacteria, and many essential regulatory factors still need to be identified. Here, we review studies where the synthesis of PHA has been reported to be regulated at the post-transcriptional level, and we analyze the RNA-mediated networks involved. Finally, we discuss the forthcoming research on riboregulation, synthetic, and metabolic engineering which could lead to improved strategies for PHAs synthesis in industrial production, thereby reducing the costs currently associated with this procedure.publishersversionpublishe

Assessing the involvement of Dis3L1 in mammalian quality control pathways

The exosome is an evolutionarily conserved protein complex that is involved in all aspects of RNA metabolism, namely, RNA decay, processing and quality control. The only catalytic subunit of the core exosome is a 3´end exoribonuclease from the RNase II family of enzymes. In humans, two different homologues of this protein were identified, Dis3 and Dis3L1. While Dis3 mainly localizes in the nucleoplasm and has endonucleolytic activity, Dis3L1 is strictly cytoplasmic and has no endonucleolytic activity. The rapid decay of aberrant transcripts is not completely understood, but it is known that involves both 5’ to 3’ and 3’ to 5’ degradation. Despite that it localizes in the same compartment where NMD generally occurs, nothing is known about the role of Dis3L1 in quality control processes. In this work, we assessed the involvement of Dis3L1 in the 3’ to 5’ degradation of reporter human b-globin transcripts with premature termination and nonstop codons

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

An open chat with… Cecília Maria Arraiano

Professor Cecília Maria Arraiano directs a research group named ‘Control of Gene Expression’ at Instituto de Tecnologia Química e Biológica, Universidade NOVA de Lisboa, Oeiras, Portugal. She started her scientific journey at the University of Lisbon, where she graduated in Biology, before completing her PhD in Genetics as a Fulbright‐Hays Fellow at the University of Georgia, Athens, USA. After a postdoc in the USA, she returned to Lisbon to establish her own lab. She has authored close to 200 publications mainly in the field of RNA degradation mechanisms, with a focus on enzymes and RNA chaperones that mediate RNA decay in microorganisms. She has received several prizes and is an active member of prestigious organizations. Namely, she is an EMBO member, Fellow of the European Academy of Microbiology, Fellow of the American Academy of Microbiology, and member of the Portuguese Academy of Sciences. In addition, Prof Arraiano has chaired the FEBS Working Group on Women in Science from 2014 to 2022. In this fascinating interview, she discusses her research, her experience working in the USA and Portugal, and the importance of initiatives to support women in science

New Perspectives on BolA: A Still Mysterious Protein Connecting Morphogenesis, Biofilm Production, Virulence, Iron Metabolism, and Stress Survival

The BolA-like protein family is widespread among prokaryotes and eukaryotes. BolA was originally described in E. coli as a gene induced in the stationary phase and in stress conditions. The BolA overexpression makes cells spherical. It was characterized as a transcription factor modulating cellular processes such as cell permeability, biofilm production, motility, and flagella assembly. BolA is important in the switch between motile and sedentary lifestyles having connections with the signaling molecule c-di-GMP. BolA was considered a virulence factor in pathogens such as Salmonella Typhimurium and Klebsiella pneumoniae and it promotes bacterial survival when facing stresses due to host defenses. In E. coli, the BolA homologue IbaG is associated with resistance to acidic stress, and in Vibrio cholerae, IbaG is important for animal cell colonization. Recently, it was demonstrated that BolA is phosphorylated and this modification is important for the stability/turnover of BolA and its activity as a transcription factor. The results indicate that there is a physical interaction between BolA-like proteins and the CGFS-type Grx proteins during the biogenesis of Fe-S clusters, iron trafficking and storage. We also review recent progress regarding the cellular and molecular mechanisms by which BolA/Grx protein complexes are involved in the regulation of iron homeostasis in eukaryotes and prokaryotes

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 5′ linear domain of MicA.

<p>Northern blot analysis of MicA in Δ<i>mic</i>A cells or its isogenic derivative lacking RNase III (Δ<i>mic</i>A Δ<i>rnc</i>), expressing <i>in trans</i> either the wild-type MicA (from the pMicA-WT plasmid) or the 5′ mutated MicA variant (from the pMicA-5′mut plasmid). RNA was extracted from stationary phase cultures and MicA stability was measured as described in <i>Material and Methods</i>.</p

Plasmids used in this work.

<p>Plasmids used in this work.</p

Strains used in this work.

<p>Strains used in this work.</p