DIS3L2 knockdown impairs key oncogenic properties of colorectal cancer cells via the mTOR signaling pathway.

DIS3L2 degrades different types of RNAs in an exosome-independent manner including mRNAs and several types of non-coding RNAs. DIS3L2-mediated degradation is preceded by the addition of nontemplated uridines at the 3'end of its targets by the terminal uridylyl transferases 4 and 7. Most of the literature that concerns DIS3L2 characterizes its involvement in several RNA degradation pathways, however, there is some evidence that its dysregulated activity may contribute to cancer development. In the present study, we characterize the role of DIS3L2 in human colorectal cancer (CRC). Using the public RNA datasets from The Cancer Genome Atlas (TCGA), we found higher DIS3L2 mRNA levels in CRC tissues versus normal colonic samples as well as worse prognosis in patients with high DIS3L2 expression. In addition, our RNA deep-sequencing data revealed that knockdown (KD) of DIS3L2 induces a strong transcriptomic disturbance in SW480 CRC cells. Moreover, gene ontology (GO) analysis of significant upregulated transcripts displays enrichment in mRNAs encoding proteins involved in cell cycle regulation and cancer-related pathways, which guided us to evaluate which specific hallmarks of cancer are differentially regulated by DIS3L2. To do so, we employed four CRC cell lines (HCT116, SW480, Caco-2 and HT-29) differing in their mutational background and oncogenicity. We demonstrate that depletion of DIS3L2 results in reduced cell viability of highly oncogenic SW480 and HCT116 CRC cells, but had little or no impact in the more differentiated Caco-2 and HT-29 cells. Remarkably, the mTOR signaling pathway, crucial for cell survival and growth, is downregulated after DIS3L2 KD, whereas AZGP1, an mTOR pathway inhibitor, is upregulated. Furthermore, our results indicate that depletion of DIS3L2 disturbs metastasis-associated properties, such as cell migration and invasion, only in highly oncogenic CRC cells. Our work reveals for the first time a role for DIS3L2 in sustaining CRC cell proliferation and provides evidence that this ribonuclease is required to support the viability and invasive behavior of dedifferentiated CRC cells.

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.

Nonsense-Mediated mRNA Decay in Development, Stress and Cancer.

Nonsense-mediated mRNA decay (NMD) is a well characterized eukaryotic mRNA degradation pathway, responsible for the identification and degradation of transcripts harboring translation termination codons in premature contexts. Transcriptome-wide studies revealed that NMD is not only an mRNA surveillance pathway as initially thought, but is also a post-transcriptional regulatory mechanism of gene expression, as it fine-tunes the transcript levels of many wild-type genes. Hence, NMD contributes to the regulation of many essential biological processes, including pathophysiological mechanisms. In this chapter we discuss the importance of NMD and of its regulation to organism development and its link to the cellular stress responses, like the unfolded protein response (UPR) and the integrated stress response (ISR). Additionally, we describe how tumor cells have explored both NMD functions to promote tumorigenesis. Using published data and databases, we have also performed a network-based approach that further supports the link between NMD and these (patho) physiological processes.

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.

Retraction for Ramos et al., MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia.

Volume 195, no. 16, p. 3514­3523, 2013. A number of problems related to images published in this paper have been brought to our attention. Figure 1D contains duplicated images in lanes S and LE, and Fig. 4D and 6B contain images previously published in articles in this journal and in Microbiology and Microbial Pathogenesis, i.e., the following: C. G. Ramos, S. A. Sousa, A. M. Grilo, J. R. Feliciano, and J. H. Leitão, J. Bacteriol. 193:1515­1526, 2011. doi:10.1128/JB.01374-11. S. A. Sousa, C. G. Ramos, L. M. Moreira, and J. H. Leitão, Microbiology 156:896­908, 2010. doi:10.1099/mic.0.035139-0. C. G. Ramos, S. A. Sousa, A. M. Grilo, L. Eberl, and J. H. Leitão, Microb. Pathog. 48:168­177, 2010. doi: 10.1016/j.micpath.2010.02.006. Therefore, we retract the paper. We deeply regret this situation and apologize for any inconvenience to the editors and readers of Journal of Bacteriology, Microbial Pathogenesis, and Microbiology.

Experimental identification of small non-coding regulatory RNAs in the opportunistic human pathogen Burkholderia cenocepacia J2315.

Small non-coding regulatory RNAs (sRNAs) play important roles in regulating gene expression at the post-transcriptional level and often require the RNA chaperone Hfq. The human opportunistic pathogen Burkholderia cenocepacia J2315 encodes two distinct RNA chaperones, Hfq and Hfq2. The present work describes the experimental identification and validation of 24 sRNAs from B. cenocepacia J2315, based on the co-purification of sRNAs with the bacterium Hfq protein, followed by conversion into cDNA, cloning, computational analysis of sequences and validation by Northern blot analysis. The sRNAs here reported escaped identification by previous studies based on transcriptomics or bioinformatic analyses. Results presented indicate that 3 sRNAs are exclusive to bacteria of the Burkholderia cepacia complex and have no homologues in other bacteria, while the other 21 share homology, at different extents, to sRNAs of other bacterial species.

MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia.

Burkholderia cenocepacia J2315 is a highly epidemic and transmissible clinical isolate of the Burkholderia cepacia complex (Bcc), a group of bacteria causing life-threatening respiratory infections among cystic fibrosis patients. This work describes the functional analysis of the 136-nucleotide (nt)-long MtvR small noncoding RNA (sRNA) from the Bcc member B. cenocepacia J2315, with homologues restricted to the genus Burkholderia. Bioinformatic target predictions revealed a total of 309 mRNAs to be putative MtvR targets. The mRNA levels corresponding to 17 of 19 selected genes were found to be affected when MtvR was either overexpressed or silenced. Analysis of the interaction between MtvR and the hfq mRNA, one of its targets, showed that the sRNA binds exclusively to the 5' untranslated region (UTR) of the hfq mRNA. This interaction resulted in decreased protein synthesis, suggesting a negative regulatory effect of MtvR on the RNA chaperone Hfq. Bacterial strains with MtvR silenced or overexpressed exhibited pleiotropic phenotypes related to growth and survival after several stresses, swimming and swarming motilities, biofilm formation, resistance to antibiotics, and ability to colonize and kill the nematode Caenorhabditis elegans. Together, the results indicate that the MtvR sRNA is a major posttranscriptional regulator in B. cenocepacia.

Tau mRNA Metabolism in Neurodegenerative Diseases: A Tangle Journey.

Tau proteins are known to be mainly involved in regulation of microtubule dynamics. Besides this function, which is critical for axonal transport and signal transduction, tau proteins also have other roles in neurons. Moreover, tau proteins are turned into aggregates and consequently trigger many neurodegenerative diseases termed tauopathies, of which Alzheimer's disease (AD) is the figurehead. Such pathological aggregation processes are critical for the onset of these diseases. Among the various causes of tau protein pathogenicity, abnormal tau mRNA metabolism, expression and dysregulation of tau post-translational modifications are critical steps. Moreover, the relevance of tau function to general mRNA metabolism has been highlighted recently in tauopathies. In this review, we mainly focus on how mRNA metabolism impacts the onset and development of tauopathies. Thus, we intend to portray how mRNA metabolism of, or mediated by, tau is associated with neurodegenerative diseases.

Experimental supporting data on DIS3L2 over nonsense-mediated mRNA decay targets in human cells.

In this article, we present supportive data related to the research article "A role for DIS3L2 over natural nonsense-mediated mRNA decay targets in human cells" [1], where interpretation of the data presented here is available. Indeed, here we analyze the impact of the DIS3L2 exoribonuclease over nonsense-mediated mRNA decay (NMD)-targets. Specifically, we present data on: a) the expression of various reporter human ß-globin mRNAs, monitored by Northern blot and RT-qPCR, before and after altering DIS3L2 levels in HeLa cells, and b) the gene expression levels of deregulated transcripts generated by re-analyzing publicly available data from UPF1-depleted HeLa cells that were further cross-referenced with a dataset of transcripts upregulated in DIS3L2-depleted cells. These analyses revealed that DIS3L2 regulates the levels of a subset of NMD-targets. These data can be valuable for researchers interested in the NMD mechanism.

Genetics of personalized medicine: cancer and rare diseases.

The 21st annual meeting of the Portuguese Society of Human Genetics (SPGH), organized by Luísa Romão, Ana Sousa and Rosário Pinto Leite, was held in Caparica, Portugal, from the 16th to the 18th of November 2017. Having entered an era in which personalized medicine is emerging as a paradigm for disease diagnosis, treatment and prevention, the program of this meeting intended to include lectures by leading national and international scientists presenting exceptional findings on the genetics of personalized medicine. Various topics were discussed, including cancer genetics, transcriptome dynamics and novel therapeutics for cancers and rare disorders that are designed to specifically target molecular alterations in individual patients. Several panel discussions were held to emphasize (ethical) issues associated with personalized medicine, including genetic cancer counseling.

The role of alternative splicing coupled to nonsense-mediated mRNA decay in human disease.

Alternative pre-mRNA splicing (AS) affects gene expression as it generates proteome diversity. Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation-termination codons (PTCs), preventing the production of truncated proteins that could result in disease. Several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs. In addition, it is known that several regulated AS events do not lead to generation of protein products, as they lead to transcripts that carry PTCs and thus, they are committed to NMD. Indeed, an estimated one-third of naturally occurring, alternatively spliced mRNAs is targeted for NMD, being AS coupled to NMD (AS-NMD) an efficient strategy to regulate gene expression. In this review, we will focus on how AS mechanism operates and how can be coupled to NMD to fine-tune gene expression levels. Furthermore, we will demonstrate the physiological significance of the interplay among AS and NMD in human disease, such as cancer and neurological disorders. The understanding of how AS-NMD orchestrates expression of vital genes is of utmost importance for the advance in diagnosis, prognosis and treatment of many human disorders.

Regulation of Hfq mRNA and protein levels in Escherichia coli and Pseudomonas aeruginosa by the Burkholderia cenocepacia MtvR sRNA.

Small non-coding RNAs (sRNAs) are important players of gene expression regulation in bacterial pathogens. MtvR is a 136-nucleotide long sRNA previously identified in the human pathogen Burkholderia cenocepacia J2315 and with homologues restricted to bacteria of the Burkholderia cepacia complex. In this work we have investigated the effects of expressing MtvR in Escherichia coli and Pseudomonas aeruginosa. Results are presented showing that MtvR negatively regulates the hfq mRNA levels in both bacterial species. In the case of E. coli, this negative regulation is shown to involve binding of MtvR to the 5'-UTR region of the hfqEc mRNA. Results presented also show that expression of MtvR in E. coli and P. aeruginosa originates multiple phenotypes, including reduced resistance to selected stresses, biofilm formation ability, and increased susceptibility to various antibiotics.

The novel cis-encoded small RNA h2cR is a negative regulator of hfq2 in Burkholderia cenocepacia.

Small non-coding regulatory RNAs (sRNAs) post-transcriptionally affect multiple phenotypes in prokaryotes and eukaryotes, yet most of the underlying regulatory mechanisms and the nature of the target mRNAs remain unclear. Here we report the identification and functional analysis of the novel cis-encoded sRNA h2cR, from the human opportunistic pathogen Burkholderia cenocepacia J2315. The sRNA was found to negatively regulate the hfq2 mRNA, through binding to part of the 5'-UTR region of the hfq2 mRNA, resulting in accelerated hfq2 mRNA decay and reduced protein levels in exponentially growing cells. Both the h2cR transcript and the hfq2 mRNA are stabilized by the other B. cenocepacia RNA chaperone, Hfq. Infection experiments using the nematode Caenorhabditis elegans revealed that down-regulation of Hfq2 by h2cR decreases the B. cenocepacia ability to colonize and persist within the nematode, suggesting a role for h2cR on bacterial persistence in the host.