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SARS‐CoV‐2 virus has triggered a global pandemic with devastating consequences. The understanding of fundamental aspects of this virus is of extreme importance. In this work, we studied the viral ribonuclease nsp14, one of the most interferon antagonists from SARS‐CoV‐2. Nsp14 is a multifunctional protein with two distinct activities, an N‐terminal 3’‐to‐5’ exoribonuclease (ExoN) and a C‐terminal N7‐methyltransferase (N7‐MTase), both critical for coronaviruses life cycle, indicating nsp14 as a prominent target for the development of antiviral drugs. In coronaviruses, nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein. We have performed a biochemical characterization of nsp14‐nsp10 complex from SARS‐CoV‐2. We confirm the 3’‐5’ exoribonuclease and MTase activities of nsp14 and the critical role of nsp10 in upregulating the nsp14 ExoN activity. Furthermore, we demonstrate that SARS‐CoV‐2 nsp14 N7‐MTase activity is functionally independent of the ExoN activity and nsp10. A model from SARS‐CoV‐2 nsp14‐nsp10 complex allowed mapping key nsp10 residues involved in this interaction. Our results show that a stable interaction between nsp10 and nsp14 is required for the nsp14‐mediated ExoN activity of SARS‐CoV‐2. We 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 residues in other coronaviruses, which can have important implications regarding the specific pathogenesis of SARS‐CoV‐2. This work unraveled a basis for discovering inhibitors targeting specific amino acids in order to disrupt the assembly of this complex and interfere with coronaviruses replication., In this report, we have shown that SARS‐CoV‐2 nsp14 is a multifunctional protein with two distinct activities, an N‐terminal 3’‐to‐5’ exoribonuclease (ExoN) and a C‐terminal N7‐methyltransferase (N7‐MTase). Nsp10 upregulates the nsp14 ExoN activity and does not interfere with nsp14 N7‐MTase. A stable interaction between these proteins is required for an active nsp14‐mediated ExoN activity.

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. This project has received funding from the European Union’s Horizon 2020 research and innovation programme. BterBioPlastics - 867437 publishersversion published

The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological RNAs that encode full-length proteins. NMD results from improper translation termination at stop codons, and thus, it is a cytoplasmic and translation-dependent process. In human cells, mRNA decay inherent to NMD involves an endonucleolytic cleavage near the stop codon 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. However, DIS3L2 exoribonuclease activity is independent of the exosome. DIS3L1 and DIS3L2 exoribonucleases localize in the same compartment where NMD occurs, however little is known about their role in this process. In order to unveil the role of DIS3L2 in NMD, we performed its knockdown in HeLa cells and measured the mRNA levels of various natural NMD-targets. Our results show that some NMD-targets accumulate in DIS3L2-depleted cells. In addition, mRNA half-life analysis indicated that these NMD-targets are direct DIS3L2 substrates. Besides, we observed that DIS3L2 acts over full-length transcripts, being DIS3L2-mediated decay dependent on the activity of the terminal uridylyl transferases (TUTases) Zcchc6/11 (TUT7/4). Together, our findings establish the role of DIS3L2 and uridylation over human NMD-targets. PTFC/BIM-MEC/3749/2014; UID/MULTI/04046/2013 N/A

The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological RNAs that encode full-length proteins. Also, NMD regulates the levels of many physiological PTC-free 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 achieved by a process not yet completely understood that promotes the decay of the mRNAs in 5’-to-3’ and 3’-to-5’ by the XRN1 and exosome, respectively. 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 the Perlman syndrome-associated exoribonuclease DIS3L2. Conversely, to its counterparts, DIS3L2 activity is independent of the exosome. In order to unveil the role of DIS3L2 in NMD, we performed its knockdown in HeLa cells and measured the mRNA levels of various natural NMD-targets. Our results show that DIS3L2 is involved in NMD-targets decay. Besides that, DIS3L2 acts directly on NMD-targets and interacts with the key NMD factor UPF1. We also show that DIS3L2-mediated decay depends on the activity of the terminal uridylyl transferases (TUTases) 4 and 7, which adds non-templated uridines to the mRNAs 3’ end, marking these mRNAs for DIS3L2 degradation. Together, our findings establish a direct role of DIS3L2 in NMD in an uridylation-dependent manner. This work was partially supported by Fundacão para a Ciência e a Tecnologia (PTDC/BIM-MEC/3749/2014 and UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC). N/A