Dis3L2 regulates cell proliferation and tissue growth through a conserved mechanism.

Dis3L2 is a highly conserved 3'-5' exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms' tumour of the kidney. Using Drosophila melanogaster as a model system, we have generated a new dis3L2 null mutant together with wild-type and nuclease-dead genetic lines in Drosophila to demonstrate that the catalytic activity of Dis3L2 is required to control cell proliferation. To understand the cellular pathways regulated by Dis3L2 to control proliferation, we used RNA-seq on dis3L2 mutant wing discs to show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth. 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 demonstrate that loss of DIS3L2 in human kidney 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 PI3-Kinase/AKT signalling pathway, which we subsequently show to contribute towards 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.

Ribonucleases control distinct traits of Pseudomonas putida lifestyle.

The role of archetypal ribonucleases (RNases) in the physiology and stress endurance of the soil bacterium and metabolic engineering platform Pseudomonas putida KT2440 has been inspected. To this end, variants of this strain lacking each of the most important RNases were constructed. Each mutant lacked either one exoribonuclease (PNPase, RNase R) or one endoribonuclease (RNase E, RNase III, RNase G). The global physiological and metabolic costs of the absence of each of these enzymes were then analysed in terms of growth, motility and morphology. The effects of different oxidative chemicals that mimic the stresses endured by this microorganism in its natural habitats were studied as well. The results highlighted that each ribonuclease is specifically related with different traits of the environmental lifestyle that distinctively characterizes this microorganism. Interestingly, the physiological responses of P. putida to the absence of each enzyme diverged significantly from those known previously in Escherichia coli. This exposed not only species-specific regulatory functions for otherwise known RNase activities but also expanded the panoply of post-transcriptional adaptation devices that P. putida can make use of for facing hostile environments.

Prediction of novel non-coding RNAs relevant for the growth of Pseudomonas putida in a bioreactor.

Pseudomonas putida is a micro-organism with great potential for industry due to its stress-endurance traits and easy manipulation of the metabolism. However, optimization is still required to improve production yields. In the last years, manipulation of bacterial small non-coding RNAs (ncRNAs) has been recognized as an effective tool to improve the production of industrial compounds. So far, very few ncRNAs are annotated in P. putida beyond the generally conserved. In the present study, P. putida was cultivated in a two-compartment scale-down bioreactor that simulates large-scale industrial bioreactors. We performed RNA-Seq of samples collected at distinct locations and time-points to predict novel and potentially important ncRNAs for the adaptation of P. putida to bioreactor stress conditions. Instead of using a purely genomic approach, we have rather identified regions of putative ncRNAs with high expression levels using two different programs (Artemis and sRNA detect). Only the regions identified with both approaches were considered for further analysis and, in total, 725 novel ncRNAs were predicted. We also found that their expression was not constant throughout the bioreactor, showing different patterns of expression with time and position. This is the first work focusing on the ncRNAs whose expression is triggered in a bioreactor environment. This information is of great importance for industry, since it provides possible targets to engineer more effective P. putida strains for large-scale production.

Tailor-made sRNAs: a plasmid tool to control the expression of target mRNAs in Pseudomonas putida.

Pseudomonas putida is a highly attractive production system for industrial needs. However, for its improvement as a biocatalyst at the industrial level, modulation of its gene expression is urgently needed. We report the construction of a plasmid expressing a small RNA-based system with the potential to be used for different purposes. Due to the small RNAs modular composition, the design facilities and ability to tune gene expression, they constitute a powerful tool in genetic and metabolic engineering. In the tool presented here, customized sRNAs are expressed from a plasmid and specifically directed to any region of a chosen target. Expression of these customized sRNAs is shown to differentially modulate the level of endogenous and heterologous reporter genes. The antisense interaction of the sRNA with the mRNA produces different outcomes. Depending on the particularity of each sRNA-target mRNA pair, we demonstrate the duality of this system, which is able either to decrease or increase the expression of the same given gene. This system combines high specificity with the potential to be widely applied, due to its predicted ability to modulate the expression of virtually any given gene. This plasmid can be used to redesign P. putida metabolism, fulfilling an important industrial gap.

A role for DIS3L2 over natural nonsense-mediated mRNA decay targets in human cells.

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. However, little is known about their role in NMD. Here, we show that some NMD-targets are DIS3L2 substrates in human cells. In addition, 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.

Reprogramming bacteria with RNA regulators.

The revolution of genomics and growth of systems biology urged the creation of synthetic biology, an engineering discipline aiming at recreating and reprogramming cellular functions for industrial needs. There has been a huge effort in synthetic biology to develop versatile and programmable genetic regulators that would enable the precise control of gene expression. Synthetic RNA components have emerged as a solution, offering a diverse range of programmable functions, including signal sensing, gene regulation and the modulation of molecular interactions. Owing to their compactness, structure and way of action, several types of RNA devices that act on DNA, RNA and protein have been characterized and applied in synthetic biology. RNA-based approaches are more 'economical' for the cell, since they are generally not translated. These RNA-based strategies act on a much shorter time scale than transcription-based ones and can be more efficient than protein-based mechanisms. In this review, we explore these RNA components as building blocks in the RNA synthetic biology field, first by explaining their natural mode of action and secondly discussing how these RNA components have been exploited to rewire bacterial regulatory circuitry.

DIS3 isoforms vary in their endoribonuclease activity and are differentially expressed within haematological cancers.

DIS3 (defective in sister chromatid joining) is the catalytic subunit of the exosome, a protein complex involved in the 3'-5' degradation of RNAs. DIS3 is a highly conserved exoribonuclease, also known as Rrp44. Global sequencing studies have identified DIS3 as being mutated in a range of cancers, with a considerable incidence in multiple myeloma. In this work, we have identified two protein-coding isoforms of DIS3. Both isoforms are functionally relevant and result from alternative splicing. They differ from each other in the size of their N-terminal PIN (PilT N-terminal) domain, which has been shown to have endoribonuclease activity and tether DIS3 to the exosome. Isoform 1 encodes a full-length PIN domain, whereas the PIN domain of isoform 2 is shorter and is missing a segment with conserved amino acids. We have carried out biochemical activity assays on both isoforms of full-length DIS3 and the isolated PIN domains. We find that isoform 2, despite missing part of the PIN domain, has greater endonuclease activity compared with isoform 1. Examination of the available structural information allows us to provide a hypothesis to explain this altered behaviour. Our results also show that multiple myeloma patient cells and all cancer cell lines tested have higher levels of isoform 1 compared with isoform 2, whereas acute myeloid leukaemia and chronic myelomonocytic leukaemia patient cells and samples from healthy donors have similar levels of isoforms 1 and 2. Taken together, our data indicate that significant changes in the ratios of the two isoforms could be symptomatic of haematological cancers.

The Implication of mRNA Degradation Disorders on Human DISease: Focus on DIS3 and DIS3-Like Enzymes.

RNA degradation is considered a critical posttranscriptional regulatory checkpoint, maintaining the correct functioning of organisms. When a specific RNA transcript is no longer required in the cell, it is signaled for degradation through a number of highly regulated steps. Ribonucleases (or simply RNases) are key enzymes involved in the control of RNA stability. These enzymes can perform the RNA degradation alone or cooperate with other proteins in RNA degradation complexes. Important findings over the last years have shed light into eukaryotic RNA degradation by members of the RNase II/RNB family of enzymes. DIS3 enzyme belongs to this family and represents one of the catalytic subunits of the multiprotein complex exosome. This RNase has a diverse range of functions, mainly within nuclear RNA metabolism. Humans encode two other DIS3-like enzymes: DIS3L (DIS3L1) and DIS3L2. DIS3L1 also acts in association with the exosome but is strictly cytoplasmic. In contrast, DIS3L2 acts independently of the exosome and shows a distinctive preference for uridylated RNAs. These enzymes have been shown to be involved in important cellular processes, such as mitotic control, and associated with human disorders like cancer. This review shows how the impairment of function of each of these enzymes is implicated in human disease.

The exoribonuclease Dis3L2 defines a novel eukaryotic RNA degradation pathway.

The final step of cytoplasmic mRNA degradation proceeds in either a 5'-3' direction catalysed by Xrn1 or in a 3'-5' direction catalysed by the exosome. Dis3/Rrp44, an RNase II family protein, is the catalytic subunit of the exosome. In humans, there are three paralogues of this enzyme: DIS3, DIS3L, and DIS3L2. In this work, we identified a novel Schizosaccharomyces pombe exonuclease belonging to the conserved family of human DIS3L2 and plant SOV. Dis3L2 does not interact with the exosome components and localizes in the cytoplasm and in cytoplasmic foci, which are docked to P-bodies. Deletion of dis3l2(+) is synthetically lethal with xrn1Δ, while deletion of dis3l2(+) in an lsm1Δ background results in the accumulation of transcripts and slower mRNA degradation rates. Accumulated transcripts show enhanced uridylation and in vitro Dis3L2 displays a preference for uridylated substrates. Altogether, our results suggest that in S. pombe, and possibly in most other eukaryotes, Dis3L2 is an important factor in mRNA degradation. Therefore, this novel 3'-5' RNA decay pathway represents an alternative to degradation by Xrn1 and the exosome.

The 3'-5' exoribonuclease Dis3 regulates the expression of specific microRNAs in Drosophila wing imaginal discs.

Dis3 is a highly conserved exoribonuclease which degrades RNAs in the 3'-5' direction. Mutations in Dis3 are associated with a number of human cancers including multiple myeloma and acute myeloid leukemia. In this work, we have assessed the effect of a Dis3 knockdown on Drosophila imaginal disc development and on expression of mature microRNAs. We find that Dis3 knockdown severely disrupts the development of wing imaginal discs in that the flies have a "no wing" phenotype. Use of RNA-seq to quantify the effect of Dis3 knockdown on microRNA expression shows that Dis3 normally regulates a small subset of microRNAs, with only 11 (10.1%) increasing in level ≥ 2-fold and 6 (5.5%) decreasing in level ≥ 2-fold. Of these microRNAs, miR-252-5p is increased 2.1-fold in Dis3-depleted cells compared to controls while the level of the miR-252 precursor is unchanged, suggesting that Dis3 can act in the cytoplasm to specifically degrade this mature miRNA. Furthermore, our experiments suggest that Dis3 normally interacts with the exosomal subunit Rrp40 in the cytoplasm to target miR-252-5p for degradation during normal wing development. Another microRNA, miR-982-5p, is expressed at lower levels in Dis3 knockdown cells, while the miR-982 precursor remains unchanged, indicating that Dis3 is involved in its processing. Our study therefore reveals an unexpected specificity for this ribonuclease toward microRNA regulation, which is likely to be conserved in other eukaryotes and may be relevant to understanding its role in human disease.

The virulence of Salmonella enterica Serovar Typhimurium in the insect model galleria mellonella is impaired by mutations in RNase E and RNase III.

Salmonella enterica serovar Typhimurium is a Gram-negative bacterium able to invade and replicate inside eukaryotic cells. To cope with the host defense mechanisms, the bacterium has to rapidly remodel its transcriptional status. Regulatory RNAs and ribonucleases are the factors that ultimately control the fate of mRNAs and final protein levels in the cell. There is growing evidence of the direct involvement of these factors in bacterial pathogenicity. In this report, we validate the use of a Galleria mellonela model in S. Typhimurium pathogenicity studies through the parallel analysis of a mutant with a mutation in hfq, a well-established Salmonella virulence gene. The results obtained with this mutant are similar to the ones reported in a mouse model. Through the use of this insect model, we demonstrate a role for the main endoribonucleases RNase E and RNase III in Salmonella virulence. These ribonuclease mutants show an attenuated virulence phenotype, impairment in motility, and reduced proliferation inside the host. Interestingly, the two mutants trigger a distinct immune response in the host, and the two mutations seem to have an impact on distinct bacterial functions.

Regulation of the small regulatory RNA MicA by ribonuclease III: a target-dependent pathway.

MicA is a trans-encoded small non-coding RNA, which downregulates porin-expression in stationary-phase. In this work, we focus on the role of endoribonucleases III and E on Salmonella typhimurium sRNA MicA regulation. RNase III is shown to regulate MicA in a target-coupled way, while RNase E is responsible for the control of free MicA levels in the cell. We purified both Salmonella enzymes and demonstrated that in vitro RNase III is only active over MicA when in complex with its targets (whether ompA or lamB mRNAs). In vivo, MicA is demonstrated to be cleaved by RNase III in a coupled way with ompA mRNA. On the other hand, RNase E is able to cleave unpaired MicA and does not show a marked dependence on its 5' phosphorylation state. The main conclusion of this work is the existence of two independent pathways for MicA turnover. Each pathway involves a distinct endoribonuclease, having a different role in the context of the fine-tuned regulation of porin levels. Cleavage of MicA by RNase III in a target-dependent fashion, with the concomitant decay of the mRNA target, strongly resembles the eukaryotic RNAi system, where RNase III-like enzymes play a pivotal role.

How hydrolytic exoribonucleases impact human disease: Two sides of the same story.

RNAs are extremely important molecules inside the cell, which perform many different functions. For example, messenger RNAs, transfer RNAs and ribosomal RNAs are involved in protein synthesis, whereas noncoding RNAs have numerous regulatory roles. Ribonucleases (RNases) are the enzymes responsible for the processing and degradation of all types of RNAs, having multiple roles in every aspect of RNA metabolism. However, the involvement of RNases in disease is still not well understood. This review focuses on the involvement of the RNase II/RNB family of 3'-5' exoribonucleases in human disease. This can be attributed to direct effects, whereby mutations in the eukaryotic enzymes of this family [defective in sister chromatid joining (Dis3; or Rrp44), Dis3-like exonuclease 1 (Dis3L1; or Dis3L) and Dis3-like exonuclease 2 (Dis3L2)] are associated with a disease, or indirect effects, whereby mutations in the prokaryotic counterparts of RNase II/RNB family (RNase II and/or RNase R) affect the physiology and virulence of several human pathogens. In this review, we compare the structural and biochemical characteristics of the members of the RNase II/RNB family of enzymes. The outcomes of mutations impacting enzymatic function are revisited, in terms of both the direct and indirect effects on disease. Furthermore, we also describe the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exoribonuclease and its importance to combat the COVID-19 pandemic. As a result, RNases may be a good therapeutic target to reduce bacterial and viral pathogenicity. These are the two perspectives on RNase II/RNB family enzymes that are presented in this review.

Synergies between RNA degradation and trans-translation in Streptococcus pneumoniae: cross regulation and co-transcription of RNase R and SmpB.

BACKGROUND: Ribonuclease R (RNase R) is an exoribonuclease that recognizes and degrades a wide range of RNA molecules. It is a stress-induced protein shown to be important for the establishment of virulence in several pathogenic bacteria. RNase R has also been implicated in the trans-translation process. Transfer-messenger RNA (tmRNA/SsrA RNA) and SmpB are the main effectors of trans-translation, an RNA and protein quality control system that resolves challenges associated with stalled ribosomes on non-stop mRNAs. Trans-translation has also been associated with deficiencies in stress-response mechanisms and pathogenicity. RESULTS: In this work we study the expression of RNase R in the human pathogen Streptococcus pneumoniae and analyse the interplay of this enzyme with the main components of the trans-translation machinery (SmpB and tmRNA/SsrA). We show that RNase R is induced after a 37°C to 15°C temperature downshift and that its levels are dependent on SmpB. On the other hand, our results revealed a strong accumulation of the smpB transcript in the absence of RNase R at 15°C. Transcriptional analysis of the S. pneumoniae rnr gene demonstrated that it is co-transcribed with the flanking genes, secG and smpB. Transcription of these genes is driven from a promoter upstream of secG and the transcript is processed to yield mature independent mRNAs. This genetic organization seems to be a common feature of Gram positive bacteria, and the biological significance of this gene cluster is further discussed. CONCLUSIONS: This study unravels an additional contribution of RNase R to the trans-translation system by demonstrating that smpB is regulated by this exoribonuclease. RNase R in turn, is shown to be under the control of SmpB. These proteins are therefore mutually dependent and cross-regulated. The data presented here shed light on the interactions between RNase R, trans-translation and cold-shock response in an important human pathogen.

Developing New Tools to Fight Human Pathogens: A Journey through the Advances in RNA Technologies.

A long scientific journey has led to prominent technological advances in the RNA field, and several new types of molecules have been discovered, from non-coding RNAs (ncRNAs) to riboswitches, small interfering RNAs (siRNAs) and CRISPR systems. Such findings, together with the recognition of the advantages of RNA in terms of its functional performance, have attracted the attention of synthetic biologists to create potent RNA-based tools for biotechnological and medical applications. In this review, we have gathered the knowledge on the connection between RNA metabolism and pathogenesis in Gram-positive and Gram-negative bacteria. We further discuss how RNA techniques have contributed to the building of this knowledge and the development of new tools in synthetic biology for the diagnosis and treatment of diseases caused by pathogenic microorganisms. Infectious diseases are still a world-leading cause of death and morbidity, and RNA-based therapeutics have arisen as an alternative way to achieve success. There are still obstacles to overcome in its application, but much progress has been made in a fast and effective manner, paving the way for the solid establishment of RNA-based therapies in the future.

The nsp15 Nuclease as a Good Target to Combat SARS-CoV-2: Mechanism of Action and Its Inactivation with FDA-Approved Drugs.

The pandemic caused by SARS-CoV-2 is not over yet, despite all the efforts from the scientific community. Vaccination is a crucial weapon to fight this virus; however, we still urge the development of antivirals to reduce the severity and progression of the COVID-19 disease. For that, a deep understanding of the mechanisms involved in viral replication is necessary. nsp15 is an endoribonuclease critical for the degradation of viral polyuridine sequences that activate host immune sensors. This enzyme is known as one of the major interferon antagonists from SARS-CoV-2. In this work, a biochemical characterization of SARS-CoV-2 nsp15 was performed. We saw that nsp15 is active as a hexamer, and zinc can block its activity. The role of conserved residues from SARS-CoV-2 nsp15 was investigated, and N164 was found to be important for protein hexamerization and to contribute to the specificity to degrade uridines. Several chemical groups that impact the activity of this ribonuclease were also identified. Additionally, FDA-approved drugs with the capacity to inhibit the in vitro activity of nsp15 are reported in this work. This study is of utmost importance by adding highly valuable information that can be used for the development and rational design of therapeutic strategies.

The world of ribonucleases from pseudomonads: a short trip through the main features and singularities.

The development of synthetic biology has brought an unprecedented increase in the number molecular tools applicable into a microbial chassis. The exploration of such tools into different bacteria revealed not only the challenges of context dependency of biological functions but also the complexity and diversity of regulatory layers in bacterial cells. Most of the standardized genetic tools and principles/functions have been mostly based on model microorganisms, namely Escherichia coli. In contrast, the non-model pseudomonads lack a deeper understanding of their regulatory layers and have limited molecular tools. They are resistant pathogens and promising alternative bacterial chassis, making them attractive targets for further studies. Ribonucleases (RNases) are key players in the post-transcriptional control of gene expression by degrading or processing the RNA molecules in the cell. These enzymes act according to the cellular requirements and can also be seen as the recyclers of ribonucleotides, allowing a continuous input of these cellular resources. This makes these post-transcriptional regulators perfect candidates to regulate microbial physiology. This review summarizes the current knowledge and unique properties of ribonucleases in the world of pseudomonads, taking into account genomic context analysis, biological function and strategies to use ribonucleases to improve biotechnological processes.

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

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.

The Bacterial Counterparts of the Eukaryotic Exosome: An Evolutionary Perspective.

There are striking similarities between the processes of RNA degradation in bacteria and eukaryotes, which rely on the same basic set of enzymatic activities. In particular, enzymes that catalyze 3'→5' RNA decay share evolutionary relationships across the three domains of life. Over the years, a large body of biochemical and structural data has been generated that elucidated the mechanism of action of these enzymes. In this overview, to trace the evolutionary origins of the multisubunit RNA exosome complex, we compare the structural and functional characteristics of the eukaryotic and prokaryotic exoribonucleolytic activities.

In Vitro Characterization of the Prokaryotic Counterparts of the Exosome Complex.

The same basic set of enzymatic activities exhibited by the eukaryotic RNA exosome are also found in prokaryotes. Bacteria have two predominant and distinct 3'→5' exoribonuclease activities: one is characterized by processive hydrolysis, derived from RNase II and RNase R, and the other by processive phosphorolysis, derived from PNPase. In this chapter we describe methods for (1) the overexpression and purification of these three proteins; and (2) their in vitro biochemical and enzymatic characterization-including RNA binding. The labeling and preparation of a set of specific RNA substrates is also described.