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.

Nonsense suppression therapies in human genetic diseases.

About 11% of all human disease-associated gene lesions are nonsense mutations, resulting in the introduction of an in-frame premature translation-termination codon (PTC) into the protein-coding gene sequence. When translated, PTC-containing mRNAs originate truncated and often dysfunctional proteins that might be non-functional or have gain-of-function or dominant-negative effects. Therapeutic strategies aimed at suppressing PTCs to restore deficient protein function-the so-called nonsense suppression (or PTC readthrough) therapies-have the potential to provide a therapeutic benefit for many patients and in a broad range of genetic disorders, including cancer. These therapeutic approaches comprise the use of translational readthrough-inducing compounds that make the translational machinery recode an in-frame PTC into a sense codon. However, most of the mRNAs carrying a PTC can be rapidly degraded by the surveillance mechanism of nonsense-mediated decay (NMD), thus decreasing the levels of PTC-containing mRNAs in the cell and their availability for PTC readthrough. Accordingly, the use of NMD inhibitors, or readthrough-compound potentiators, may enhance the efficiency of PTC suppression. Here, we review the mechanisms of PTC readthrough and their regulation, as well as the recent advances in the development of novel approaches for PTC suppression, and their role in personalized medicine.

Conserved Double Translation Initiation Site for Δ160p53 Protein Hints at Isoform's Key Role in Mammalian Physiology.

p53 is the most commonly mutated gene in human cancers. Two fundamental reasons for this are its long protein isoforms protect from cancer, while its shorter C-terminal isoforms can support cancer and metastasis. Previously, we have shown that the Δ160p53 protein isoform enhances survival and the invasive character of cancer cells. Here, we identified a translation initiation site nine codons downstream of codon 160-the known initiation codon for the translation of Δ160p53-that is recognized by the translation machinery. When translation failed to initiate from AUG160 due to mutation, it initiated from AUG169 instead, producing similar levels of a similar protein, Δ169p53, which promoted cell survival as efficiently as Δ160p53 following DNA damage. Interestingly, almost all mammalian species with an orthologue to human AUG160 also possess one for AUG169, while none of the non-mammalian species lacking AUG160 have AUG169, even if that region of the p53 gene is well conserved. In view of our findings, we do not believe that Δ169p53 acts as a different p53 protein isoform; instead, we propose that the double translation initiation site strengthens the translation of these products with a critical role in cell homeostasis. Future studies will help verify if this is a more general mechanism for the expression of essential proteins in mammals.

Nonsense-mediated RNA decay and its bipolar function in cancer.

Nonsense-mediated decay (NMD) was first described as a quality-control mechanism that targets and rapidly degrades aberrant mRNAs carrying premature termination codons (PTCs). However, it was found that NMD also degrades a significant number of normal transcripts, thus arising as a mechanism of gene expression regulation. Based on these important functions, NMD regulates several biological processes and is involved in the pathophysiology of a plethora of human genetic diseases, including cancer. The present review aims to discuss the paradoxical, pro- and anti-tumorigenic roles of NMD, and how cancer cells have exploited both functions to potentiate the disease. Considering recent genetic and bioinformatic studies, we also provide a comprehensive overview of the present knowledge of the advantages and disadvantages of different NMD modulation-based approaches in cancer therapy, reflecting on the challenges imposed by the complexity of this disease. Furthermore, we discuss significant advances in the recent years providing new perspectives on the implications of aberrant NMD-escaping frameshifted transcripts in personalized immunotherapy design and predictive biomarker optimization. A better understanding of how NMD differentially impacts tumor cells according to their own genetic identity will certainly allow for the application of novel and more effective personalized treatments in the near future.

Perspective in Alternative Splicing Coupled to Nonsense-Mediated mRNA Decay.

Alternative splicing (AS) of precursor mRNA (pre-mRNA) is a cellular post-transcriptional process that generates protein isoform diversity. Nonsense-mediated RNA decay (NMD) is an mRNA surveillance pathway that recognizes and selectively degrades transcripts containing premature translation-termination codons (PTCs), thereby preventing the production of truncated proteins. Nevertheless, NMD also fine-tunes the gene expression of physiological mRNAs encoding full-length proteins. Interestingly, around one third of all AS events results in PTC-containing transcripts that undergo NMD. Numerous studies have reported a coordinated action between AS and NMD, in order to regulate the expression of several genes, especially those coding for RNA-binding proteins (RBPs). This coupling of AS to NMD (AS-NMD) is considered a gene expression tool that controls the ratio of productive to unproductive mRNA isoforms, ultimately degrading PTC-containing non-functional mRNAs. In this review, we focus on the mechanisms underlying AS-NMD, and how this regulatory process is able to control the homeostatic expression of numerous RBPs, including splicing factors, through auto- and cross-regulatory feedback loops. Furthermore, we discuss the importance of AS-NMD in the regulation of biological processes, such as cell differentiation. Finally, we analyze interesting recent data on the relevance of AS-NMD to human health, covering its potential roles in cancer and other disorders.

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.

Cap-independent translation ensures mTOR expression and function upon protein synthesis inhibition.

The mechanistic/mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates cellular signals from the nutrient and energy status to act, namely, on the protein synthesis machinery. While major advances have emerged regarding the regulators and effects of the mTOR signaling pathway, little is known about the regulation of mTOR gene expression. Here, we show that the human mTOR transcript can be translated in a cap-independent manner, and that its 5' untranslated region (UTR) is a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that mTOR is able to bypass the cap requirement for translation both in normal and hypoxic conditions. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell-cycle progression into S phase. These results suggest a novel regulatory mechanism for mTOR gene expression that integrates the global protein synthesis changes induced by translational inhibitory conditions.

Translational Regulation by Upstream Open Reading Frames and Human Diseases.

Short upstream open reading frames (uORFs) are cis-acting elements located within the 5'-leader sequence of transcripts and are defined by an initiation codon in-frame with a termination codon located upstream or downstream of its main ORF (mORF) initiation codon. Recent genome-wide ribosome profiling studies have confirmed the widespread presence of uORFs and have shown that many uORFs can initiate with non-AUG codons. uORFs can impact gene expression of the downstream mORF by triggering mRNA decay or by regulating translation. Thus, disruption or creation of uORFs can elicit the development of several genetic diseases. Here, we review the mechanisms by which AUG- and non-AUG uORFs regulate translation. We also show some examples of uORF deregulation in human genetic diseases, focusing mainly on cancer. The knowledge of how uORF deregulation drives the onset of a disease, points out the need to screen the 5'-leader sequences of the transcripts in search for potential disease-related variants. This information will be relevant for the implementation of new diagnostic and/or therapeutic tools.

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.

eIF3: a factor for human health and disease.

The eukaryotic initiation factor 3 (eIF3) is one of the most complex translation initiation factors in mammalian cells, consisting of several subunits (eIF3a to eIF3m). It is crucial in translation initiation and termination, and in ribosomal recycling. Accordingly, deregulated eIF3 expression is associated with different pathological conditions, including cancer. In this manuscript, we discuss the interactome and function of each subunit of the human eIF3 complex. Furthermore, we review how altered levels of eIF3 subunits correlate with neurodegenerative disorders and cancer onset and development; in addition, we evaluate how such misregulation may also trigger infection cascades. A deep understanding of the molecular mechanisms underlying eIF3 role in human disease is essential to develop new eIF3-targeted therapeutic approaches and thus, overcome such conditions.

More than just scanning: the importance of cap-independent mRNA translation initiation for cellular stress response and cancer.

The scanning model for eukaryotic mRNA translation initiation states that the small ribosomal subunit, along with initiation factors, binds at the cap structure at the 5' end of the mRNA and scans the 5' untranslated region (5'UTR) until an initiation codon is found. However, under conditions that impair canonical cap-dependent translation, the synthesis of some proteins is kept by alternative mechanisms that are required for cell survival and stress recovery. Alternative modes of translation initiation include cap- and/or scanning-independent mechanisms of ribosomal recruitment. In most cap-independent translation initiation events there is a direct recruitment of the 40S ribosome into a position upstream, or directly at, the initiation codon via a specific internal ribosome entry site (IRES) element in the 5'UTR. Yet, in some cellular mRNAs, a different translation initiation mechanism that is neither cap- nor IRES-dependent seems to occur through a special RNA structure called cap-independent translational enhancer (CITE). Recent evidence uncovered a distinct mechanism through which mRNAs containing N 6-methyladenosine (m6A) residues in their 5'UTR directly bind eukaryotic initiation factor 3 (eIF3) and the 40S ribosomal subunit in order to initiate translation in the absence of the cap-binding proteins. This review focuses on the important role of cap-independent translation mechanisms in human cells and how these alternative mechanisms can either act individually or cooperate with other cis-acting RNA regulons to orchestrate specific translational responses triggered upon several cellular stress states, and diseases such as cancer. Elucidation of these non-canonical mechanisms reveals the complexity of translational control and points out their potential as prospective novel therapeutic targets.

Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). The level of sensitivity of a PTC-containing mRNA to NMD is multifactorial. We have previously shown that human ß-globin mRNAs carrying PTCs in close proximity to the translation initiation AUG codon escape NMD. This was called the 'AUG-proximity effect'. The present analysis of nonsense codons in the human α-globin mRNA illustrates that the determinants of the AUG-proximity effect are in fact quite complex, reflecting the ability of the ribosome to re-initiate translation 3' to the PTC and the specific sequence and secondary structure of the translated ORF. These data support a model in which the time taken to translate the short ORF, impacted by distance, sequence, and structure, not only modulates translation re-initiation, but also impacts on the exact boundary of AUG-proximity protection from NMD.

Translation of the human erythropoietin transcript is regulated by an upstream open reading frame in response to hypoxia.

Erythropoietin (EPO) is a key mediator hormone for hypoxic induction of erythropoiesis that also plays important nonhematopoietic functions. It has been shown that EPO gene expression regulation occurs at different levels, including transcription and mRNA stabilization. In this report, we show that expression of EPO is also regulated at the translational level by an upstream open reading frame (uORF) of 14 codons. As judged by comparisons of protein and mRNA levels, the uORF acts as a cis-acting element that represses translation of the main EPO ORF in unstressed HEK293, HepG2, and HeLa cells. However, in response to hypoxia, this repression is significantly released, specifically in HeLa cells, through a mechanism that involves processive scanning of ribosomes from the 5' end of the EPO transcript and enhanced ribosome bypass of the uORF. In addition, we demonstrate that in HeLa cells, hypoxia induces the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) concomitantly with a significant increase of EPO protein synthesis. These findings provide a framework for understanding that production of high levels of EPO induced by hypoxia also involves regulation at the translational level.

Gene expression regulation by upstream open reading frames and human disease.

Upstream open reading frames (uORFs) are major gene expression regulatory elements. In many eukaryotic mRNAs, one or more uORFs precede the initiation codon of the main coding region. Indeed, several studies have revealed that almost half of human transcripts present uORFs. Very interesting examples have shown that these uORFs can impact gene expression of the downstream main ORF by triggering mRNA decay or by regulating translation. Also, evidence from recent genetic and bioinformatic studies implicates disturbed uORF-mediated translational control in the etiology of many human diseases, including malignancies, metabolic or neurologic disorders, and inherited syndromes. In this review, we will briefly present the mechanisms through which uORFs regulate gene expression and how they can impact on the organism's response to different cell stress conditions. Then, we will emphasize the importance of these structures by illustrating, with specific examples, how disturbed uORF-mediated translational control can be involved in the etiology of human diseases, giving special importance to genotype-phenotype correlations. Identifying and studying more cases of uORF-altering mutations will help us to understand and establish genotype-phenotype associations, leading to advancements in diagnosis, prognosis, and treatment of many human disorders.

Interaction of PABPC1 with the translation initiation complex is critical to the NMD resistance of AUG-proximal nonsense mutations.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3'-5' linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.

mRNA Metabolism in Health and Disease.

Eukaryotic gene expression involves several interlinked steps, in which messenger RNAs (mRNAs), which code for proteins, are the key intermediates [...].

Internal Ribosome Entry Site (IRES)-Mediated Translation and Its Potential for Novel mRNA-Based Therapy Development.

Many conditions can benefit from RNA-based therapies, namely, those targeting internal ribosome entry sites (IRESs) and their regulatory proteins, the IRES trans-acting factors (ITAFs). IRES-mediated translation is an alternative mechanism of translation initiation, known for maintaining protein synthesis when canonical translation is impaired. During a stress response, it contributes to cell reprogramming and adaptation to the new environment. The relationship between IRESs and ITAFs with tumorigenesis and resistance to therapy has been studied in recent years, proposing new therapeutic targets and treatments. In addition, IRES-dependent translation initiation dysregulation is also related to neurological and cardiovascular diseases, muscular atrophies, or other syndromes. The participation of these structures in the development of such pathologies has been studied, yet to a far lesser extent than in cancer. Strategies involving the disruption of IRES-ITAF interactions or the modification of ITAF expression levels may be used with great impact in the development of new therapeutics. In this review, we aim to comprehend the current data on groups of human pathologies associated with IRES and/or ITAF dysregulation and their application in the designing of new therapeutic approaches using them as targets or tools. Thus, we wish to summarise the evidence in the field hoping to open new promising lines of investigation toward personalised treatments.

Gene Variants Involved in Nonsense-Mediated mRNA Decay Suggest a Role in Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental condition with unclear etiology. Many genes have been associated with ASD risk, but the underlying mechanisms are still poorly understood. An important post-transcriptional regulatory mechanism that plays an essential role during neurodevelopment, the Nonsense-Mediated mRNA Decay (NMD) pathway, may contribute to ASD risk. In this study, we gathered a list of 46 NMD factors and regulators and investigated the role of genetic variants in these genes in ASD. By conducting a comprehensive search for Single Nucleotide Variants (SNVs) in NMD genes using Whole Exome Sequencing data from 1828 ASD patients, we identified 270 SNVs predicted to be damaging in 28.7% of the population. We also analyzed Copy Number Variants (CNVs) from two cohorts of ASD patients (N = 3570) and discovered 38 CNVs in 1% of cases. Importantly, we discovered 136 genetic variants (125 SNVs and 11 CNVs) in 258 ASD patients that were located within protein domains required for NMD. These gene variants are classified as damaging using in silico prediction tools, and therefore may interfere with proper NMD function in ASD. The discovery of NMD genes as candidates for ASD in large patient genomic datasets provides evidence supporting the involvement of the NMD pathway in ASD pathophysiology.

Control of human beta-globin mRNA stability and its impact on beta-thalassemia phenotype.

Messenger RNA (mRNA) stability is a critical determinant that affects gene expression. Many pathways have evolved to modulate mRNA stability in response to developmental, physiological and/or environmental stimuli. Eukaryotic mRNAs have a considerable range of half-lives, from as short as a few minutes to as long as several days. Human globin mRNAs constitute an example of highly stable mRNAs. However, a wide variety of naturally occurring mutations that result in the clinical syndrome of thalassemia can trigger accelerated mRNA decay thus controlling mRNA quality prior to translation. Distinct surveillance mechanisms have been described as being targeted for specific defective globin mRNAs. Here, we review mRNA stability mechanisms implicated in the control of ß-globin gene expression and the surveillance pathways that prevent translation of aberrant ß-globin mRNAs. In addition, we emphasize the importance of these pathways in modulating the severity of the ß-thalassemia phenotype.