Nonsense-mediated mRNA Decay Pathway Research Articles

Molecular docking and ADME evaluation of plant-based bioactive molecules targeting nonsense-mediated mRNA decay pathway factors to modulate tumorigenesis

Cancer is a global health challenge that requires continuous efforts to discover effective anticancer drugs. Phytochemicals are compounds found in plants that often have medicinal properties. They possess a wide range of bioactive properties, including anticancer activity. Their multiple mechanisms of action in different physiological processes in humans make them promising candidates in the anticancer therapeutics development. The presence of these compounds makes plants valuable resources for traditional medicine and modern pharmaceutical research as well. Natural products from plants and marine sources are being used to find new anticancer agents. In humans, different cellular pathways are involved in the tumorigenesis process. Many studies have shown the role of the nonsense-mediated mRNA decay (NMD) pathway in the process of tumorigenesis. This NMD pathway is controlled by multiple proteins. In this study, we conducted a molecular docking analysis of 50 phytochemicals against the human NMD factor up-frameshit2 (UPF2) protein. The results of the molecular docking experiment and ADME properties indicate that 4 of these molecules (Genistein, Trihydroxyflavone, Baicalein and Epigallocatechin) have the potential to modulate the NMD pathway. Furthermore, these molecules comply with Lipinski's rule of five. The effects of these 4 phytochemicals may be further evaluated using in vitro and in vivo methods for novel anticancer therapeutic development.

Bioinformatics-Driven Refinement of the Commonly Used TPI Nonsense-Mediated Decay Reporter System.

The cellular nonsense-mediated decay (NMD) pathway recognizes and degrades mRNAs with unusual structural features, such as long 3' UTRs or overlapping reading frames, and therefore serves as a transcript quality control mechanism. A broad spectrum of today's knowledge about the nonsense-mediated mRNA decay pathway has been discovered using NMD reporter systems, mostly consisting of multiple exons, with a wild type (WT) and a premature termination codon (PTC) containing variant. In a preliminary NMD study, we used the seven-exon triose phosphate isomerase (TPI) reporter and observed that in this well-known NMD reporter, surprisingly, not all splice sites are used constitutively, but additional cryptic splice sites are used. As this is more frequently observed in the construction of minigenes, especially when unknown splicing regulatory elements are removed, e.g. by shortening introns, this may affect the reliability of such reporters. To demonstrate how such minigenes can be improved in general with respect to constitutive splice site recognition, we restored an intron length in the TPI reporter or made bioinformatic adjustments to splice regulatory elements (SREs) or intrinsic strength of the splice sites themselves. As a result, this NMD reporter could be made more robust and specific for the evaluation of NMD sensitivity within a single transcript. The modifications of the TPI reporter shown here as examples can generally be used for the transfer of cellular multiexon transcripts to minigenes.

Structure of the Nmd4-Upf1 complex supports conservation of the nonsense-mediated mRNA decay pathway between yeast and humans.

The nonsense-mediated mRNA decay (NMD) pathway clears eukaryotic cells of mRNAs containing premature termination codons (PTCs) or normal stop codons located in specific contexts. It therefore plays an important role in gene expression regulation. The precise molecular mechanism of the NMD pathway has long been considered to differ substantially from yeast to metazoa, despite the involvement of universally conserved factors such as the central ATP-dependent RNA-helicase Upf1. Here, we describe the crystal structure of the yeast Upf1 bound to its recently identified but yet uncharacterized partner Nmd4, show that Nmd4 stimulates Upf1 ATPase activity and that this interaction contributes to the elimination of NMD substrates. We also demonstrate that a region of Nmd4 critical for the interaction with Upf1 in yeast is conserved in the metazoan SMG6 protein, another major NMD factor. We show that this conserved region is involved in the interaction of SMG6 with UPF1 and that mutations in this region affect the levels of endogenous human NMD substrates. Our results support the universal conservation of the NMD mechanism in eukaryotes.

Effects of functional defects in the NMD pathway on rice phenotype and transcriptome.

Nonsense-mediated mRNA decay (NMD) is an important RNA quality control pathway. It aids in degrading harmful erroneous mRNA, thereby preserving a stable and healthy internal environment. In this study, we employed CRISPR/Cas9 and amiRNA technology to generate knock out or knock down mutants of realted genes in the rice NMD pathway. Through transcriptome sequencing and observing phenotype changes, the study explored the impact of NMD pathway defects on rice gene expression and alternative splicing. The results suggest that even partial defects will induce phenotypic changes such as plant height and pollen vitality to different degrees, showing necessity of NMD factors. Gene expression analysis reveals that most differentially expressed genes are upregulated in the mutants, with ko-upf1-like and kd-upf1 defects having a more significant impact than kd-upf2 and kd-upf3. Specifically, NMD pathway defects result in increased expression levels of rice defense response-related genes and decreased expression levels of secondary metabolism-related genes, with a wider range of affected genes observed in 60-day-old senescence mutants. Transcript analysis indicates that different NMD related genes defects alter hundreds of alternative splicing events, mostly enriched in genes involving alternative splicing regulatory pathways. Approximately half of these events are shared among different mutants, and a substantial number of affected transcripts show NMD target features. NMD could affect both the transcript abundance and their splicing subtypes to regulate the defense response and early-senescence associated pathways, which plays a vital role in rice growth and reproduction.

Therapeutic Nonsense Suppression Modalities: From Small Molecules to Nucleic Acid-Based Approaches.

Nonsense mutations are genetic mutations that create premature termination codons (PTCs), leading to truncated, defective proteins in diseases such as cystic fibrosis, neurofibromatosis type 1, Dravet syndrome, Hurler syndrome, Beta thalassemia, inherited bone marrow failure syndromes, Duchenne muscular dystrophy, and even cancer. These mutations can also trigger a cellular surveillance mechanism known as nonsense-mediated mRNA decay (NMD) that degrades the PTC-containing mRNA. The activation of NMD can attenuate the consequences of truncated, defective, and potentially toxic proteins in the cell. Since approximately 20% of all single-point mutations are disease-causing nonsense mutations, it is not surprising that this field has received significant attention, resulting in a remarkable advancement in recent years. In fact, since our last review on this topic, new examples of nonsense suppression approaches have been reported, namely new ways of promoting the translational readthrough of PTCs or inhibiting the NMD pathway. With this review, we update the state-of-the-art technologies in nonsense suppression, focusing on novel modalities with therapeutic potential, such as small molecules (readthrough agents, NMD inhibitors, and molecular glue degraders); antisense oligonucleotides; tRNA suppressors; ADAR-mediated RNA editing; targeted pseudouridylation; and gene/base editing. While these various modalities have significantly advanced in their development stage since our last review, each has advantages (e.g., ease of delivery and specificity) and disadvantages (manufacturing complexity and off-target effect potential), which we discuss here.

Small molecule NMD and MDM2 inhibitors synergistically trigger apoptosis in HeLa cells

The nonsense-mediated mRNA decay (NMD) pathway and the p53 pathway, linked to tumorgenesis and are also promising targets for cancer treatment. NMD plays an important role in RNA quality control, while the p53 pathway is involved in cancer suppression. However, their individual and combined effects on cervical cancer (CC) are poorly understood. In this study, we evaluated the impacts of NMD inhibitor, MDM2 inhibitor, and their combination on cell apoptosis, cell cycle, and p53 target genes in HPV-18-positive HeLa cells. Our findings revealed that XR-2failed to activate p53 or induce apoptosis in HeLa cells, whereas SMG1i repressed cell proliferation at high concentrations. Notably the combination of these two agents significantly inhibited cell proliferation, arrested the cell cycle, and triggered cell apoptosis. Mechanistically, MDM2 inhibitor and NMD inhibitor likely exert a synergistically through the truncated E6 protein. These results underscore the potential of employing a combination of MDM2 inhibitor and NMD inhibitor as a promising candidate for the clinical treatment of HPV-infected tumors.

Fine-Tuning Amyloid Precursor Protein Expression through Nonsense-Mediated mRNA Decay.

Studies on genetic robustness recently revealed transcriptional adaptation (TA) as a mechanism by which an organism can compensate for genetic mutations through activation of homologous genes. Here, we discovered that genetic mutations, introducing a premature termination codon (PTC) in the amyloid precursor protein-b (appb) gene, activated TA of two other app family members, appa and amyloid precursor-like protein-2 (aplp2), in zebrafish. The observed transcriptional response of appa and aplp2 required degradation of mutant mRNA and did not depend on Appb protein level. Furthermore, TA between amyloid precursor protein (APP) family members was observed in human neuronal progenitor cells; however, compensation was only present during early neuronal differentiation and could not be detected in a more differentiated neuronal stage or adult zebrafish brain. Using knockdown and chemical inhibition, we showed that nonsense-mediated mRNA decay (NMD) is involved in degradation of mutant mRNA and that Upf1 and Upf2, key proteins in the NMD pathway, regulate the endogenous transcript levels of appa, appb, aplp1, and aplp2 In conclusion, our results suggest that the expression level of App family members is regulated by the NMD pathway and that mutations destabilizing app/APP mRNA can induce genetic compensation by other family members through TA in both zebrafish and human neuronal progenitors.

A Novel Frameshift Variant of the ELF4 Gene in a Patient with Autoinflammatory Disease: Clinical Features, Transcriptomic Profiling and Functional Studies.

We described the diagnosis and treatment of a patient with autoinflammatory disease, named "Deficiency in ELF4, X-linked (DEX)". A novel ELF4 variant was discovered and its pathogenic mechanism was elucidated. The data about clinical, laboratory and endoscopic features, treatment, and follow-up of a patient with DEX were analyzed. Whole exome sequencing and Sanger sequencing were performed to identify potential pathogenic variants. The mRNA and protein levels of ELF4 were analyzed by qPCR and Western blotting, respectively. The association of ELF4 frameshift variant with nonsense-mediated mRNA decay (NMD) in the pathogenesis DEX was examined. Moreover, RNA-seq was performed to identify the key molecular events triggered by ELF4 variant. The relationship between ELF4 and IFN-β activity was validated using a dual-luciferase reporter assay and a ChIP-qPCR assay. An 11-year-old boy presented with a Behçet's-like phenotype. The laboratory abnormality was the most obvious in elevated inflammatory indicators. Endoscopy revealed multiple ileocecal ulcers. Intestinal histopathology showed inflammatory cell infiltrations. The patient was treated with long-term immunosuppressant and TNF-α blocker (adalimumab), which reaped an excellent response over 16 months of follow-up. Genetic analysis identified a maternal hemizygote frameshift variant (c.1022del, p.Q341Rfs*30) in ELF4 gene in the proband. The novel variant decreased the mRNA level of ELF4 via the NMD pathway. Mechanistically, insufficient expression of ELF4 disturbed the immune system, leading to immunological disorders and pathogen susceptibility, and disrupted ELF4-activating IFN-βresponses. This analysis detailed the clinical characteristics of a Chinese patient with DEX who harbored a novel ELF4 frameshift variant. For the first time, we used patient-derived cells and carried out transcriptomic analysis to delve into the mechanism of ELF4 variant in DEX.

Physiological Consequences of Nonsense-Mediated Decay and Its Role in Adaptive Responses.

The evolutionarily conserved nonsense-mediated mRNA decay (NMD) pathway is a quality control mechanism that degrades aberrant mRNA containing one or more premature termination codons (PTCs). Recent discoveries indicate that NMD also differentially regulates mRNA from wild-type protein-coding genes despite lacking PTCs. Together with studies showing that NMD is involved in development and adaptive responses that influence health and longevity, these findings point to an expanded role of NMD that adds a new layer of complexity in the post-transcriptional regulation of gene expression. However, the extent of its control, whether different types of NMD play different roles, and the resulting physiological outcomes remain unclear and need further elucidation. Here, we review different branches of NMD and what is known of the physiological outcomes associated with this type of regulation. We identify significant gaps in the understanding of this process and the utility of genetic tools in accelerating progress in this area.

Nonsense-mediated mRNA decay of metal-binding activator MAC1 is dependent on copper levels and 3'-UTR length in Saccharomyces cerevisiae.

The nonsense-mediated mRNA decay (NMD) pathway was initially identified as a surveillance pathway that degrades mRNAs containing premature termination codons (PTCs). NMD is now also recognized as a post-transcriptional regulatory pathway that regulates the expression of natural mRNAs. Earlier studies demonstrated that regulation of functionally related natural mRNAs by NMD can be differential and condition-specific in Saccharomyces cerevisiae. Here, we investigated the regulation of MAC1 mRNAs by NMD in response to copper as well as the role the MAC1 3'-UTR plays in this regulation. MAC1 is a copper-sensing transcription factor that regulates the high-affinity copper uptake system. MAC1 expression is activated upon copper deprivation. We found that MAC1 mRNAs are regulated by NMD under complete minimal (CM) but escaped NMD under low and high copper conditions. Mac1 protein regulated gene, CTR1 is not regulated by NMD in conditions where MAC1 mRNAs are NMD sensitive. We also found that the MAC1 3'-UTR is the NMD targeting feature on the mRNAs, and that MAC1 mRNAs lacking 3'-UTRs were stabilized during copper deprivation. Our results demonstrate a mechanism of regulation for a metal-sensing transcription factor, at both the post-transcriptional and post-translational levels, where MAC1 mRNA levels are regulated by NMD and copper, while the activity of Mac1p is controlled by copper levels.

CRISPR/Cas9 screen reveals that targeting TRIM34 enhances ferroptosis sensitivity and augments immunotherapy efficacy in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a prevalent malignancy characterized by complex heterogeneity and drug resistance. Resistance to ferroptosis is closely related to the progression of HCC. While HCC tumors vary in their sensitivity to ferroptosis, the precise factors underlying this heterogeneity remain unclear. In this study, we sought to elucidate the mechanisms that contribute to ferroptosis resistance in HCC. Whole-genome CRISPR/Cas9 screen using a subtoxic concentration (IC20) of ferroptosis inducer erastin in the HCC cell line Huh7 revealed TRIM34 as a critical driver of ferroptosis resistance in HCC. Further investigation revealed that TRIM34 suppresses ferroptosis in HCC cells, promoting their proliferation, migration, and invasion both in vitro and in vivo. Furthermore, TRIM34 expression is elevated in HCC tumor tissues, correlating with a poor prognosis. Mechanistically, TRIM34 directly interacts with Up-frameshift 1 (UPF1), a core component of the nonsense-mediated mRNA decay (NMD) pathway, to promote its ubiquitination and degradation. This interaction suppresses GPX4 transcript degradation, thus promoting the protein levels of this critical ferroptosis suppressor in HCC. In light of the close crosstalk between ferroptosis and the adaptive immune response in cancer, HCC cells with targeting knockdown of TRIM34 exhibited an improved response to anti-PD-1 treatment. Taken together, the TRIM34/UPF1/GPX4 axis mediates ferroptosis resistance in HCC, thereby promoting malignant phenotypes. Targeting TRIM34 may thus represent a promising new strategy for HCC treatment.

Abstract 5625: Targeting the nonsense mediated mRNA decay pathway to prevent the degradation of highly immunogenic frameshift mutated transcripts

Abstract Introduction: Frameshift insertions/deletions (fs-indels) can lead to the generation of immunogenic non-self-peptides which contribute to anti-tumor immune responses. However, fs-indels often generate premature termination codons (PTCs), leading to mRNA degradation by the nonsense mediated mRNA decay (NMD) pathway. Importantly, some PTC-containing transcripts naturally escape NMD degradation and produce highly immunogenic neoantigens. Therefore, we hypothesize that NMD inhibition could be used to increase the cellular pool of fs-indels derived neoantigens in cancer cells, and enhance anti-tumor immunogenicity. NMD also regulates other cellular processes by degrading a subset of non-PTC-containing self-transcripts. Therefore, by focusing NMD inhibition towards the degradation of PTC-transcripts only, toxicity may be limited in a clinical setting. We investigate how targeting different NMD proteins affects the degradation of fs-transcripts across several cancers types. To assess the clinical relevance of NMD inhibition, we study how it specifically affects p53 fs-transcripts, since p53 is the most mutated gene in cancer and fs-mutant p53 has been shown to be highly immunogenic. Methods: Core NMD proteins were depleted across a panel of cell lines from different cancer types and transcriptomics, flow cytometry, qPCR and western blot were used to evaluate changes in the expression of NMD-targeted transcripts and NMD members. Proteomics and immunopeptidomics were used to study protein expression and HLA-peptide presentation arising from fs-transcripts. A Translating RNA Imaging by Coat protein Knock-off (TRICK) assay was developed to study the translation of NMD-targeted p53 mutants upon NMD inhibition. This assay can precisely quantify the translation of p53 mutant RNA upon different treatments and cellular stresses at single-molecule resolution. Results: We found an increase in expression of PTC-transcripts upon silencing of multiple NMD members. Our data suggests that some NMD members are better targets for inhibition of NMD PTC-surveillance, whilst others are more appropriate for inhibition of NMD-mediated gene expression regulation. These data support the recently proposed hypothesis that NMD is a branched pathway, where each branch targets specific subsets of transcripts depending on the cell type. These results are clinically relevant as the method by which NMD is inhibited could help optimize the generation of immunogenic neoantigens whilst minimizing toxicity. The TRICK assay has shown that inhibiting NMD surveillance reduces the degradation of p53 fs-transcripts and increases their protein expression. Conclusion: We have identified NMD pathway members that can be targeted to efficiently inhibit the surveillance role of NMD and prevent the degradation of fs-transcripts that could generate highly immunogenic neoantigens. Citation Format: Shanila Fernandez Patel, Roberto Vendramin, Danwen Qian, Yue Zhao, Lorena Ligammari, Krupa Thakkar, Jun Murai, Eva Grönroos, Jeremy Carlton, Kevin Litchfield. Targeting the nonsense mediated mRNA decay pathway to prevent the degradation of highly immunogenic frameshift mutated transcripts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5625.

Abstract 6737: Pharmacologic modulation of nonsense-mediated decay induces anti-tumor immunogenicity in ex vivo patient tumors

Abstract Introduction: Immunogenic neoantigens derived from somatic tumor mutations are essential to initiate and sustain robust anti-tumor immune responses. Frameshift insertion/deletions (fs-indels) are a key source of immunogenic neoantigens. However, fs-indels often result in the introduction of premature termination codons (PTCs), leading to mRNA degradation by the nonsense-mediated mRNA decay (NMD) pathway. In human malignancies, NMD activity is increased in response to the heightened burden of somatic fs-indel mutations and effectively prevents the generation of highly immunogenic tumor-specific neoantigens, thereby promoting immune evasion. Approach: Here, we pharmacologically targeted SMG1, a core component of the NMD pathway, for the first time in patient tumor and normal samples as well as in a panel of human cancer cell lines and patient-derived organoids, to prevent the degradation of highly immunogenic frame-shifted transcripts. We analyzed the changes in transcriptome, proteome and immunopeptidome following SMG1 inhibition (SMG1i). We then made use of tumor-T cell co-cultures and Patient-Derived Tumor Fragments (PDTFs) to assess the effects of SMG1i on anti-tumor immunogenicity. Ex vivo immunological responses were measured by high-dimensional flow cytometry, cytometric bead array and single cell RNA and TCR sequencing. Results: Our data demonstrates a significant increase in T cell activation, expansion of tumor-reactive intratumoural CD8+ lymphocytes and cytokine secretion following SMG1 inhibition in ex vivo PTDFs (n=15) but not in matched normal-adjacent tissue (n=10). Mechanistically, SMG1 inhibition increased the abundance of frame-shifted mRNA transcripts, and aberrant HLA-presented peptides. Co-culture of tumor cells and patient-derived organoids with CD8+ T cells upon SMG1 inhibition induced potent MHC class I antigen-dependent T cell activation and tumor cell killing. Conclusion: Our findings, in a clinically relevant platform, highlight SMG1 inhibition as a novel cancer immunotherapy approach to promote neoantigen production and to induce anti-tumor immunogenicity irrespective of the tumor type or TMB status. Citation Format: Roberto Vendramin, Hongchang Fu, Yue Zhao, Shanila Fernandez-Patel, Danwen Qian, Lorena Ligammari, Gordon Beattie, Ronen Levy, Polina Greenberg, Osnat Bartok, Krupa Thakkar, Jun Murai, Despoina Karagianni, Chris Sng, Mansi Shah, Felipe Galvez-Cancino, Krijn Djikstra, Sergio Quezada, Yardena Samuels, TRACERx consortium, James Reading, Charles Swanton, Kevin Litchfield. Pharmacologic modulation of nonsense-mediated decay induces anti-tumor immunogenicity in ex vivo patient tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6737.

In Vitro Cross-Linking MS Reveals SMG1-UPF2-SMG7 Assembly as Molecular Partners within the NMD Surveillance.

mRNAs containing premature stop codons are responsible for various genetic diseases as well as cancers. The truncated proteins synthesized from these aberrant mRNAs are seldom detected due to the nonsense-mediated mRNA decay (NMD) pathway. Such a surveillance mechanism detects most of these aberrant mRNAs and rapidly destroys them from the pool of mRNAs. Here, we implemented chemical cross-linking mass spectrometry (CLMS) techniques to trace novel biology consisting of protein-protein interactions (PPIs) within the NMD machinery. A set of novel complex networks between UPF2 (Regulator of nonsense transcripts 2), SMG1 (Serine/threonine-protein kinase SMG1), and SMG7 from the NMD pathway were identified, among which UPF2 was found as a connection bridge between SMG1 and SMG7. The UPF2 N-terminal formed most interactions with SMG7, and a set of residues emerged from the MIF4G-I, II, and III domains docked with SMG1 or SMG7. SMG1 mediated interactions with initial residues of UPF2, whereas SMG7 formed very few interactions in this region. Modelled structures highlighted that PPIs for UPF2 and SMG1 emerged from the well-defined secondary structures, whereas SMG7 appeared from the connecting loops. Comparing the influence of cancer-derived mutations over different CLMS sites revealed that variants in the PPIs for UPF2 or SMG1 have significant structural stability effects. Our data highlights the protein-protein interface of the SMG1, UPF2, and SMG7 genes that can be used for potential therapeutic approaches. Blocking the NMD pathway could enhance the production of neoantigens or internal cancer vaccines, which could provide a platform to design potential peptide-based vaccines.

Regulation of non-canonical proteins from diverse origins through the nonsense-mediated mRNA decay pathway.

Immunotherapy harnesses neoantigens encoded within the human genome, but their therapeutic potential is hampered by low expression, which may be controlled by the nonsense-mediated mRNA decay (NMD) pathway. This study investigates the impact of UPF1-knockdown on the expression of non-canonical/mutant proteins, employing proteogenomic to explore UPF1 role within the NMD pathway. Additionally, we conducted a comprehensive pan-cancer analysis of UPF1 expression and evaluated UPF1 expression in Triple-Negative Breast Cancer (TNBC) tissue in-vivo. Our findings reveal that UPF1-knockdown leads to increased translation of non-canonical/mutant proteins, particularly those originating from retained-introns, pseudogenes, long non-coding RNAs, and unannotated transcript biotypes. Moreover, our analysis demonstrates elevated UPF1 expression in various cancer types, with notably heightened protein levels in patient-derived TNBC tumors compared to adjacent tissues. This study elucidates UPF1 role in mitigating transcriptional noise by degrading transcripts encoding non-canonical/mutant proteins. Targeting this mechanism may reveal a new spectrum of neoantigens accessible to the antigen presentation pathway. Our novel findings provide a strong foundation for the development of therapeutic strategies aimed at targeting UPF1 or modulating the NMD pathway.

Nonsense-mediated mRNA decay pathway in plants under stress: general gene regulatory mechanism and advances.

Nonsense-mediated mRNA decay in eukaryotes is vital to cellular homeostasis. Further knowledge of its putative role in plant RNA metabolism under stress is pivotal to developing fitness-optimizing strategies. Nonsense-mediated mRNA decay (NMD), part of the mRNA surveillance pathway, is an evolutionarily conserved form of gene regulation in all living organisms. Degradation of mRNA-bearing premature termination codons and regulation of physiological RNA levels highlight NMD's role in shaping the cellular transcriptome. Initially regarded as purely a tool for cellular RNA quality control, NMD is now considered to mediate various aspects of plant developmental processes and responses to environmental changes. Here we offer a basic understanding of NMD in eukaryotes by explaining the concept of premature termination codon recognition and NMD complex formation. We also provide a detailed overview of the NMD mechanism and its role in gene regulation. The potential role of effectors, including ABCE1, in ribosome recycling during the translation process is also explained. Recent reports of alternatively spliced variants of corresponding genes targeted by NMD inArabidopsis thalianaare provided in tabular format. Detailed figures are also provided to clarify the NMD concept in plants. In particular, accumulating evidence shows that NMD can serve as a novel alternative strategy for genetic manipulation and can help design RNA-based therapies to combat stress in plants. A key point of emphasis is its function as a gene regulatory mechanism as well as its dynamic regulation by environmental and developmental factors. Overall, a detailed molecular understanding of the NMD mechanism can lead to further diverse applications, such as improving cellular homeostasis in living organisms.

THUMPD2 catalyzes the N2-methylation of U6 snRNA of the spliceosome catalytic center and regulates pre-mRNA splicing and retinal degeneration.

The mechanisms by which the relatively conserved spliceosome manages the enormously large number of splicing events that occur in humans (∼200000 versus ∼300 in yeast) are poorly understood. Here, we show deposition of one RNA modification-N2-methylguanosine (m2G) on the G72 of U6 snRNA (the catalytic center of the spliceosome) promotes efficient pre-mRNA splicing activity in human cells. This modification was identified to be conserved among vertebrates. Further, THUMPD2 was demonstrated as the methyltransferase responsible for U6 m2G72 by explicitly recognizing the U6-specific sequences and structural elements. The knock-out of THUMPD2 eliminated U6 m2G72 and impaired the pre-mRNA splicing activity, resulting in thousands of changed alternative splicing events of endogenous pre-mRNAs in human cells. Notably, the aberrantly spliced pre-mRNA population elicited the nonsense-mediated mRNA decay pathway. We further show that THUMPD2 was associated with age-related macular degeneration and retinal function. Our study thus demonstrates how an RNA epigenetic modification of the major spliceosome regulates global pre-mRNA splicing and impacts physiology and disease.

Nonsense-Mediated mRNA Decay: Mechanistic Insights and Physiological Significance.

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved surveillance mechanism across eukaryotes and also regulates the expression of physiological transcripts, thus involved in gene regulation. It essentially ensures recognition and removal of aberrant transcripts. Therefore, the NMD protects the cellular system by restricting the synthesis of truncated proteins, potentially by eliminating the faulty mRNAs. NMD is an evolutionarily conserved surveillance mechanism across eukaryotes and also regulates the expression of physiological transcripts, thus involved in gene regulation as well. Primarily, the NMD machinery scans and differentiates the aberrant and non-aberrant transcripts. A myriad of cellular dysfunctions arise due to production of truncated proteins, so the NMD core proteins, the up-frameshift factors (UPFs) recognizes the faulty mRNAs and further recruits factors resulting in the mRNA degradation. NMD exhibits astounding variability in its ability in regulating cellular mechanisms including both pathological and physiological events. But, the detailed underlying molecular mechanisms in NMD remains blurred and require extensive investigation to gain insights on cellular homeostasis. The complexity in understanding of NMD pathway arises due to the involvement of numerous proteins, molecular interactions and their functioning in different steps of this process. Moreover methods such as alternative splicing generates numerous isoforms of mRNA, so it makes difficulties in understanding the impact of alternative splicing on the efficiency of NMD functioning. Role of NMD in cancer development is very complex. Studies have shown that in some cases cancer cells use NMD pathway as a tool to exploit the NMD mechanism to maintain tumor microenvironment. A greater level of understanding about the intricate mechanism of how tumor used NMD pathway for their benefits, a strategy can be developed for targeting and inhibiting NMD factors involved in pro-tumor activity. There are very little amount of information available about the NMD pathway, how it discriminate mRNAs that are targeted by NMD from those that are not. This review highlights our current understanding of NMD, specifically the regulatory mechanisms and attempts to outline less explored questions that warrant further investigations. Taken as a whole, a detailed molecular understanding of the NMD mechanism could lead to wide-ranging applications for improving cellular homeostasis and paving out strategies in combating pathological disorders leaping forward toward achieving United Nations sustainable development goals (SDG 3: Good health and well-being).

Identification and bioinformatics analysis of a novel mutation in PLA2G6 gene in a patient with neurodegenerative disorder

PLA2G6-associated neurodegeneration disorders are a group of conditions that result from mutations in the PLA2G6 gene. These disorders can cause a range of symptoms affecting the brain and nervous system, such as movement problems, cognitive decline, and vision loss. In this case, Whole Exome Sequencing (WES) was conducted on a 9-year-old boy born to consanguineous parents, who have an increased risk of passing on recessive genetic disorders. Although the boy did not exhibit any clinical symptoms until he was seven months old, his condition deteriorated severely by the age of two, rendering him unable to sit or move around, and he did not develop the ability to speak. DNAs were extracted from the blood samples of the proband (the 9-year-old boy) and his parents. The proband's DNA was sequenced using WES to identify potential disease-causing mutations. To confirm the WES findings, PCR-Sanger sequencing analysis was performed on the DNAs obtained from the proband (the 9-year-old boy) and his parents. The effect of the mutation on the protein structure was studied using the I-TASSER web-tool, which uses computational methods to predict protein structure and function based on amino acid sequences.The analysis of WES data revealed a homozygous dinucleotide TG insertion at position c.1362–1363 in exon 10 of the PLA2G6 gene (NC_000022.11(NM_003560.4):c.1362_1363insTG). This insertion introduced a premature stop codon at position 455 (p.Met455Ter) in the protein sequence. The validation of the WES results using PCR-Sanger sequencing confirmed the presence of the mutation in the proband and revealed that both parents were carriers of the mutation. The premature stop codon, located 65 nucleotides before the exon-junction complex, is likely to be detected by the nonsense-mediated mRNA decay (NMD) pathway, leading to the degradation of the mRNA. The use of the I-TASSER web-tool further supported the findings by demonstrating significant structural and functional changes in the mutant protein. Observing a very severe phenotype in our case supports the idea that the mutation was highly deleterious.

Translation-coupled mRNA quality control mechanisms.

mRNA surveillance pathways are essential for accurate gene expression and to maintain translation homeostasis, ensuring the production of fully functional proteins. Future insights into mRNA quality control pathways will enable us to understand how cellular mRNA levels are controlled, how defective or unwanted mRNAs can be eliminated, and how dysregulation of these can contribute to human disease. Here we review translation-coupled mRNA quality control mechanisms, including the non-stop and no-go mRNA decay pathways, describing their mechanisms, shared trans-acting factors, and differences. We also describe advances in our understanding of the nonsense-mediated mRNA decay (NMD) pathway, highlighting recent mechanistic findings, the discovery of novel factors, as well as the role of NMD in cellular physiology and its impact on human disease.