Nonsense-mediated Decay Pathway Research Articles

A novel PSMB8 isoform associated with multiple sclerosis lesions induces P-body formation

IntroductionMultiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Current therapies primarily target the inflammatory component of the disease and are highly effective in early stages of MS while limited therapies have an effect in the more chronic progressive stages of MS where resident glia have a larger role. MS lesions tend to be inflammatory even after the initial peripheral immune cell invasion has subsided and this inflammation is known to cause alternative splicing events.MethodsWe used qPCR of normal-appearing white matter and white matter lesions from postmortem MS tissue, in vitro studies, and immunostaining in MS tissue to investigate the alternative splicing of one gene known to be important during recovery in an animal model of MS, PSMB8.ResultsWe found a novel, intron-retained isoform which has not been annotated, upregulated specifically in MS patient white matter lesions. We found that this novel isoform activates the nonsense-mediated decay pathway in primary human astrocytes, the most populous glial cell in the CNS, and is then degraded. Overexpression of this isoform in astrocytes leads to an increased number of processing bodies in vitro, the primary site of mRNA decay. Finally, we demonstrated that MS white matter lesions have a higher burden of processing bodies compared to normal-appearing white matter, predominantly in GFAP-positive astrocytes.DiscussionThe increase in alternative splicing of the PSMB8 gene, the stress that this alternative splicing causes, and the observation that processing bodies are increased in white matter lesions suggests that the lesion microenvironment may lead to increased alternative splicing of many genes. This alternative splicing may blunt the protective or reparative responses of resident glia in and around white matter lesions in MS patients.

Post-transcriptional Regulation of Gene Expression via Unproductive Splicing.

Unproductive splicing is a mechanism of post-transcriptional gene expression control in which premature stop codons are inserted into protein-coding transcripts as a result of regulated alternative splicing, leading to their degradation via the nonsense-mediated decay pathway. This mechanism is especially characteristic of RNA-binding proteins, which regulate each other's expression levels and those of other genes in multiple auto- and cross-regulatory loops. Deregulation of unproductive splicing is a cause of serious human diseases, including cancers, and is increasingly being considered as a prominent therapeutic target. This review discusses the types of unproductive splicing events, the mechanisms of auto- and cross-regulation, nonsense-mediated decay escape, and problems in identifying unproductive splice isoforms. It also provides examples of deregulation of unproductive splicing in human diseases and discusses therapeutic strategies for its correction using antisense oligonucleotides and small molecules.

Functional transcriptome analysis revealed upregulation of MAPK-SMAD signalling pathways in chronic heat stress in crossbred cattle.

Animal geneticists and breeders have the impending challenge of enhancing the resilience of Indian livestock to heat stress through better selection strategies. Climate change's impact on livestock is more intense in tropical countries like India where dairy cattle crossbreeds are more sensitive to heat stress. The main reason for this study was to find the missing relative changes in transcript levels in thermo-neutral and heat stress conditions in crossbred cattle through whole-transcriptome analysis of RNA-Seq data. Differentially expressed genes (DEGs) identified based on the minimum log twofold change value and false discovery rate 0.05 revealed 468 up-regulated genes and 2273 down-regulated significant genes. Functional annotation and pathway analysis of these significant DEGs were compared based on Gene Ontology (Biological process), Kyoto Encyclopedia of Genes and Genome (KEGG), and Reactome pathways using g: Profiler, ShinyGO v0.76, and iDEP.951 web tools. On finding network visualization, the most over-represented and correlated pathways were neuronal and sensory organ development, calcium signalling pathway, Mitogen-activated protein kinase (MAPK) and Smad signalling pathway, Ras-proximate-1, or Ras-related protein 1 (Rap 1) signalling pathway, apoptosis, and oxidative stress. Similarly, down-regulated genes were most expressed in mRNA processing, immune system, B-cell receptor signalling pathway, Nucleotide oligomerization domain(NOD)-like receptors (NLRs) signalling pathway and nonsense-mediated decay (NMD) pathway. The heat stress-responsive genes identified in this study will facilitate our understanding of the molecular basis for climate resilience and heat tolerance in Indian dairy crossbreeds.

Abstract 3986: Exploring nonsense-mediated decay inhibition as a novel approach to improve immunogenicity in small cell lung cancer

Abstract Cancer immunotherapy has revolutionized the field of oncology and is one of the most promising treatments for lung cancer. Small cell lung cancer (SCLC) is highly aggressive and patients often relapse due to chemotherapy resistance. In the recent years, chemotherapy has been combined with immune checkpoint inhibition (ICI) for SCLC treatment, leading to improved responses in some patients, but also leaving a high number of non-responders to address. Paradoxically, SCLC has one of the highest tumor mutational burden, including remarkably abundant frameshifts (FS) which could create highly immunogenic neoantigens (NeoAg). However, FS-harbouring transcripts are often degraded via Nonsense-Mediated Decay (NMD). We therefore aimed to inhibit the NMD pathway to increase the immunogenicity of SCLC. Our lab has so far profiled the mutational landscape of more than 400 SCLC patients and generated over 100 patient-derived xenotransplant models. We analyzed genome and transcriptome sequencing data of multiple tumors to interrogate the NMD target repertoire in SCLC. We determined tumor-specific mutations and predicted NeoAg based on the HLA-I-binding ability of mutant peptides. Upon NMD inhibiton, we validated the expression and presentation of NeoAg via transcriptomics, proteomics and HLA-I-immunopeptidomics. NMD-sensitive mutated transcripts included a wide range of somatic alterations beyond FS-mutations, suggesting an overall increase of potential tumor-specific NeoAg. We furthermore evaluated immunogenicity after NMD perturbation employing in vitro co-cultures as well as immunocompetent in vivo mouse models. Inhibition of NMD in tumor cells resulted in an HLA-I-dependent enhancement of T cell-mediated tumor killing in vitro, and in vivo experiments showed reduced tumor growth accompanied by increased immune infiltration upon controlled induction of NMD inhibition. Altogether, NMD inhibition in SCLC led to an upregulation of NeoAg expression in tumor cells accompanied by enhanced T cell responses in vitro and in vivo. Our strategy provides a novel approach for the treatment of low immunogenic tumors which could be combined with ICI to enhance the efficacy of immunotherapy. Citation Format: Lucia A. Torres Fernández, Volker Böhm, Lukas Maas, Anna Schöllhorn, Joel Kaufmann, Joshua D'Rozario, Laura Kaiser, Niels Gehring, Martin Peifer, Cécile Gouttefangeas, Roman K. Thomas, Julie George. Exploring nonsense-mediated decay inhibition as a novel approach to improve immunogenicity in small cell lung cancer [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 3986.

An adapted MS2-MCP system to visualize endogenous cytoplasmic mRNA with live imaging in Caenorhabditis elegans.

Live imaging of RNA molecules constitutes an invaluable means to track the dynamics of mRNAs, but live imaging in Caenorhabditis elegans has been difficult to achieve. Endogenous transcripts have been observed in nuclei, but endogenous mRNAs have not been detected in the cytoplasm, and functional mRNAs have not been generated. Here, we have adapted live imaging methods to visualize mRNA in embryonic cells. We have tagged endogenous transcripts with MS2 hairpins in the 3' untranslated region (UTR) and visualized them after adjusting MS2 Coat Protein (MCP) expression. A reduced number of these transcripts accumulates in the cytoplasm, leading to loss-of-function phenotypes. In addition, during epithelial morphogenesis, MS2-tagged mRNAs for dlg-1 fail to associate with the adherens junction, as observed for untagged, endogenous mRNAs. These defects are reversed by inactivating the nonsense-mediated decay pathway. RNA accumulates in the cytoplasm, mutant phenotypes are rescued, and dlg-1 RNA associates with the adherens junction. These data suggest that MS2 repeats can induce the degradation of endogenous RNAs and alter their cytoplasmic distribution. Although our focus is RNAs expressed in epithelial cells during morphogenesis, we find that this method can be applied to other cell types and stages.

Global deletome profile of Saccharomyces cerevisiae exposed to lithium.

The increasing use of lithium (Li) in new technologies raises the question of its impact on living microorganisms. In the present study, we aimed to identify putative Li targets and resistance mechanisms in the yeast model Saccharomyces cerevisiae using a deletomic approach based on the screening of a collection of 4733 knockout mutants under Li exposure. This screening highlighted 60 mutants resistant to Li and 124 mutants sensitive to Li. Through functional enrichment analyses, transport systems were identified as playing a central role in cell resistance to toxic concentrations of Li. In contrast, the AKT/protein kinase B family, signal transduction or cell communication were identified as potential toxic targets of Li. The majority of the mutants with a Li-sensitive phenotype were also sensitive to other alkali and alkaline earth metals, whereas the Li-resistance phenotype was mostly resistant to Na but poorly resistant to other metals. A comparison with the results of deletomics studies carried out in the presence of other metals highlighted Li-specific phenotypes. Three genes (NAM7, NMD2, UPF3) of the nonsense-mediated decay pathway were specifically involved in resistance to Li. In contrast, mutants with the NCA2, SPT20, GCN5, YOR376W, YPK3, and DCW1 genes deleted were specifically resistant to Li. These genes encode various functions from putative mannosidase to constitution of the Spt-Ada-Gcn5 acetyltransferase complex. This work provides a better understanding of potential specific resistance mechanisms and cellular targets of Li in yeast.

REM-422, a Potent, Selective, Oral Small Molecule mRNA Degrader of the MYB Oncogene, Demonstrates Anti-Tumor Activity in Mouse Xenograft Models of AML

The c-MYB (MYB) oncogenic transcription factor is a key regulator of hematopoietic cell differentiation and proliferation. Recurrent genetic lesions or dysregulation of MYB have been identified in a variety of cancers, including adenoid cystic carcinoma (ACC), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), pediatric low-grade glioma (pLGG), and others. AML is characterized by multiple oncogenic abnormalities and high MYB mRNA expression. Evidence from in vitro and in vivo animal studies suggests that MYB may be a key downstream effector of these oncogenic abnormalities through the direct regulation of genes such as BCL2, MYC and FLT3. Functional genomics studies reveal MYB dependencies in human leukemia cell lines and mouse models of leukemia. Together, these data suggest that MYB is an attractive therapeutic target for AML and other hematological malignancies. Therapeutic agents that directly target MYB have yet to be developed, presumably due to the challenges of targeting this intrinsically disordered transcription factor, which lacks tractable ligand binding pockets. Here, we describe the mechanism of action and biological activity of REM-422, a potent, selective, oral small molecule messenger RNA (mRNA) degrader of MYB. REM-422 was identified through screening and medicinal chemistry efforts directed towards identifying small molecules that induce the inclusion of a normally unused ‘poison exon’ (PE), which contains a premature termination codon, into the MYB pre-mRNA transcript. Poison exon inclusion activates the nonsense-mediated decay pathway and promotes the degradation of MYB mRNA. We demonstrate in a cell-free assay that REM-422 acts by selectively facilitating the binding of the U1 snRNP spliceosome complex, which governs exon usage by recognizing 5‘ splice sites, to oligonucleotides containing the MYB PE 5‘ splice site sequence. REM-422 treatment of human leukemia cell lines promotes poison exon inclusion, leading to degradation of MYB mRNA and subsequent reduction in MYB protein levels (Figure 1A). In a panel of AML cell lines, REM-422 demonstrated preferential anti-proliferative activity in cell lines with high MYB expression, and in those shown to be MYB-dependent through functional genomics studies. To understand the pharmacokinetic (PK) and pharmacodynamic (PD) effects of MYB in vivo, Kasumi-1 AML cells were implanted subcutaneously in CB17 scid immunocompromised mice and REM-422 was dosed by oral gavage after the formation of established tumors. QD PO administration of REM-422 demonstrated dose-dependent effects in tumors on MYB mRNA levels, with induction of the MYB poison exon leading to reductions of MYB functional mRNA. In a 21-day study, REM-422 demonstrated significant anti-tumor activity, including tumor regressions at well tolerated dose levels (Figure 1 left). To further investigate the activity of REM-422, AML patient cells were injected into NOG immunocompromised mice and allowed to engraft. After successful engraftment, mice were dosed by oral gavage once daily with REM-422 at 10 mg/kg or vehicle for 24 days. Flow cytometry analysis of bone marrow and peripheral blood samples demonstrated that REM-422 led to the eradication of human leukemia cells from both compartments (Figure 1 right). Together, these data demonstrate that REM-422 treatment leads to degradation of MYB mRNA in preclinical models of AML and supports the therapeutic potential for REM-422 in AML patients.

Roles for Bile Acid Signaling and Nonsense-Mediated Ribonucleic Acid Decay in Small Bowel Resection-Associated Liver Injury

IntroductionMassive intestinal loss resulting in short bowel syndrome has been linked to intestinal failure associated liver disease. Efforts to elucidate the driving force behind the observed hepatic injury have identified inflammatory mediators, alterations in the microbiome, extent of structural and functional intestinal adaptation, and toxic shifts in the bile acid pool. In the present study, we posit that ileocecal resection interrupts the delivery of these hepatotoxic substances to the liver by physically disrupting the enterohepatic circulation, thereby shielding the liver from exposure to the aforementioned noxious stimuli. MethodsMice underwent sham, 50% proximal, or 50% distal small bowel resection (SBR), with or without tauroursodeoxycolic acid supplementation. Enterohepatic signaling and nonsense-mediated ribonucleic acid (RNA) decay were evaluated and correlated with hepatic injury. ResultsWhen compared to 50% proximal SBR, mice that underwent ileocecal resection exhibited reduced hepatic oxidative stress and exhibited a more physiological bile acid profile with increased de novo bile acid synthesis, enhanced colonic bile acid signaling, and reduced hepatic proliferation. Distal intestinal resection promoted an adaptive response including via the nonsense-mediated RNA decay pathway to satisfactorily process injurious messenger RNA and successfully maintain homeostasis. By contrast, this adaptive response was not observed in the proximal SBR group and hepatic injury persisted. ConclusionsIn summary, interruption of enterohepatic circulation via ileocecal resection abrogates the liver's exposure to toxic and inflammatory mediators while promoting physiological adaptations in bile acid metabolism and maintaining existing homeostatic pathways.

Exposure to environmental airborne particulate matter caused wide-ranged transcriptional changes and accelerated Alzheimer's-related pathology: A mouse study

Air pollution poses a significant threat to human health, though a clear understanding of its mechanism remains elusive. In this study, we sought to better understand the effects of various sized particulate matter from polluted air on Alzheimer's disease (AD) development using an AD mouse model. We exposed transgenic Alzheimer's mice in their prodromic stage to different sized particulate matter (PM), with filtered clean air as control. After 3 or 6 months of exposure, mouse brains were harvested and analyzed. RNA-seq analysis showed that various PM have differential effects on the brain transcriptome, and these effects seemed to correlate with PM size. Many genes and pathways were affected after PM exposure. Among them, we found a strong activation in mRNA Nonsense Mediated Decay pathway, an inhibition in pathways related to transcription, neurogenesis and survival signaling as well as angiogenesis, and a dramatic downregulation of collagens. Although we did not detect any extracellular Aβ plaques, immunostaining revealed that both intracellular Aβ1–42 and phospho-Tau levels were increased in various PM exposure conditions compared to the clean air control. NanoString GeoMx analysis demonstrated a remarkable activation of immune responses in the PM exposed mouse brain. Surprisingly, our data also indicated a strong activation of various tumor suppressors including RB1, CDKN1A/p21 and CDKN2A/p16. Collectively, our data demonstrated that exposure to airborne PM caused a profound transcriptional dysregulation and accelerated Alzheimer's-related pathology.

Turnip crinkle virus-encoded suppressor of RNA silencing suppresses mRNA decay by interacting with Arabidopsis XRN4.

Plant cells employ intricate defense mechanisms, including mRNA decay pathways, to counter viral infections. Among the RNA quality control (RQC) mechanisms, nonsense-mediated decay (NMD), no-go decay (NGD), and nonstop decay (NSD) pathways play critical roles in recognizing and cleaving aberrant mRNA molecules. Turnip crinkle virus (TCV) is a plant virus that triggers mRNA decay pathways, but it has also evolved strategies to evade this antiviral defense. In this study, we investigated the activation of mRNA decay during TCV infection and its impact on TCV RNA accumulation. We found that TCV infection induced the upregulation of essential mRNA decay factors, indicating their involvement in antiviral defense and the capsid protein (CP) of TCV, a well-characterized viral suppressor of RNA silencing (VSR), also compromised the mRNA decay-based antiviral defense by targeting AtXRN4. This interference with mRNA decay was supported by the observation that TCV CP stabilized a reporter transcript with a long 3' untranslated region (UTR). Moreover, TCV CP suppressed the decay of known NMD target transcripts, further emphasizing its ability to modulate host RNA control mechanisms. Importantly, TCV CP physically interacted with AtXRN4, providing insight into the mechanism of viral interference with mRNA decay. Overall, our findings reveal an alternative strategy employed by TCV, wherein the viral coat protein suppresses the mRNA decay pathway to facilitate viral infection.

Stress decreases spermatozoa quality and induces molecular alterations in zebrafish progeny

BackgroundChronic stress can produce a severe negative impact on health not only in the exposed individuals but also in their offspring. Indeed, chronic stress may be contributing to the current worldwide scenario of increasing infertility and decreasing gamete quality in human populations. Here, we evaluate the effect of chronic stress on behavior and male reproductive parameters in zebrafish. Our goal is to provide information on the impact that chronic stress has at molecular, histological, and physiological level in a vertebrate model species.ResultsWe evaluated the effects of a 21-day chronic stress protocol covering around three full waves of spermatogenesis in Danio rerio adult males. The induction of chronic stress produced anxiety-like behavior in stressed males as assessed by a novel tank test. At a molecular level, the induction of chronic stress consistently resulted in the overexpression of two genes related to endoplasmic reticulum (ER) stress in the brain. Gene set enrichment analysis (GSEA) of testes suggested a dysregulation of the nonsense-mediated decay (NMD) pathway, which was also confirmed on qPCR analysis. Histological analysis of the testicle did not show significant differences in terms of the relative proportions of each germ-cell type; however, the quality of sperm from stressed males was compromised in terms of motility. RNA-seq analysis in stress-derived larval progenies revealed molecular alterations, including those predicted to affect translation initiation, DNA repair, cell cycle control, and response to stress.ConclusionsInduction of chronic stress during a few cycles of spermatogenesis in the vertebrate zebrafish model affects behavior, gonadal gene expression, final gamete quality, and progeny. The NMD surveillance pathway (a key cellular mechanism that regulates the stability of both normal and mutant transcripts) is severely affected in the testes by chronic stress and therefore the control and regulation of RNAs during spermatogenesis may be affected altering the molecular status in the progeny.Graphical

Tissue-specific regulation of gene expression via unproductive splicing.

Eukaryotic gene expression is regulated post-transcriptionally by a mechanism called unproductive splicing, in which mRNA is triggered to degrade by the nonsense-mediated decay (NMD) pathway as a result of regulated alternative splicing (AS). Only a few dozen unproductive splicing events (USEs) are currently documented, and many more remain to be identified. Here, we analyzed RNA-seq experiments from the Genotype-Tissue Expression (GTEx) Consortium to identify USEs, in which an increase in the NMD isoform splicing rate is accompanied by tissue-specific down-regulation of the host gene. To characterize RNA-binding proteins (RBPs) that regulate USEs, we superimposed these results with RBP footprinting data and experiments on the response of the transcriptome to the perturbation of expression of a large panel of RBPs. Concordant tissue-specific changes between the expression of RBP and USE splicing rate revealed a high-confidence regulatory network including 27 tissue-specific USEs with strong evidence of RBP binding. Among them, we found previously unknown PTBP1-controlled events in the DCLK2 and IQGAP1 genes, for which we confirmed the regulatory effect using small interfering RNA (siRNA) knockdown experiments in the A549 cell line. In sum, we present a transcriptomic pipeline that allows the identification of tissue-specific USEs, potentially many more than were reported here using stringent filters.

Targeting nonsense-mediated RNA decay does not increase progranulin levels in the Grn R493X mouse model of frontotemporal dementia.

A common cause of frontotemporal dementia (FTD) are nonsense mutations in the progranulin (GRN) gene. Because nonsense mutations activate the nonsense-mediated RNA decay (NMD) pathway, we sought to inhibit this RNA turnover pathway as a means to increase progranulin levels. Using a knock-in mouse model harboring a common patient mutation, we tested whether either pharmacological or genetic inhibition of NMD upregulates progranulin in these GrnR493X mice. We first examined antisense oligonucleotides (ASOs) targeting an exonic region in GrnR493X mRNA predicted to block its degradation by NMD. As we previously reported, these ASOs effectively increased GrnR493X mRNA levels in fibroblasts in vitro. However, following CNS delivery, we found that none of the 8 ASOs we tested increased Grn mRNA levels in the brains of GrnR493X mice. This result was obtained despite broad ASO distribution in the brain. An ASO targeting a different mRNA was effective when administered in parallel to wild-type mice. As an independent approach to inhibit NMD, we examined the effect of loss of an NMD factor not required for embryonic viability: UPF3b. We found that while Upf3b deletion effectively perturbed NMD, it did not increase Grn mRNA levels in Grn+/R493X mouse brains. Together, our results suggest that the NMD-inhibition approaches that we used are likely not viable for increasing progranulin levels in individuals with FTD caused by nonsense GRN mutations. Thus, alternative approaches should be pursued.

Recoding of Nonsense Mutation as a Pharmacological Strategy.

Approximately 11% of genetic human diseases are caused by nonsense mutations that introduce a premature termination codon (PTC) into the coding sequence. The PTC results in the production of a potentially harmful shortened polypeptide and activation of a nonsense-mediated decay (NMD) pathway. The NMD pathway reduces the burden of unproductive protein synthesis by lowering the level of PTC mRNA. There is an endogenous rescue mechanism that produces a full-length protein from a PTC mRNA. Nonsense suppression therapies aim to increase readthrough, suppress NMD, or are a combination of both strategies. Therefore, treatment with translational readthrough-inducing drugs (TRIDs) and NMD inhibitors may increase the effectiveness of PTC suppression. Here we discuss the mechanism of PTC readthrough and the development of novel approaches to PTC suppression. We also discuss the toxicity and bioavailability of therapeutics used to stimulate PTC readthrough.

Regulation of alternative splicing by retrograde and light signals converges to control chloroplast proteins.

Retrograde signals sent by chloroplasts control transcription in the nucleus. These signals antagonistically converge with light signals to coordinate the expression of genes involved in chloroplast functioning and seedling development. Although significant advances have been made in understanding the molecular interplay between light and retrograde signals at the transcriptional level, little is known about their interconnection at the post-transcriptional level. By using different publicly available datasets, this study addresses the influence of retrograde signaling on alternative splicing and defines the molecular and biological functions of this regulation. These analyses revealed that alternative splicing mimics transcriptional responses triggered by retrograde signals at different levels. First, both molecular processes similarly depend on the chloroplast-localized pentatricopeptide-repeat protein GUN1 to modulate the nuclear transcriptome. Secondly, as described for transcriptional regulation, alternative splicing coupled with the nonsense-mediated decay pathway effectively downregulates expression of chloroplast proteins in response to retrograde signals. Finally, light signals were found to antagonistically control retrograde signaling-regulated splicing isoforms, which consequently generates opposite splicing outcomes that likely contribute to the opposite roles these signals play in controlling chloroplast functioning and seedling development.

Modulation of NBAS-Related Functions in the Early Response to SARS-CoV-2 Infection.

Upon infection, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is predicted to interact with diverse cellular functions, such as the nonsense-mediated decay (NMD) pathway, as suggested by the identification of the core NMD factor upframeshift-1 (UPF1) in the SARS-CoV-2 interactome, and the retrograde transport from the Golgi to the endoplasmic reticulum (ER) through the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), where coronavirus assembly occurs. Here, we investigated the expression and localization of the neuroblastoma-amplified sequence (NBAS) protein, a UPF1 partner for the NMD at the ER, participating also in retrograde transport, and of its functional partners, at early time points after SARS-CoV-2 infection of the human lung epithelial cell line Calu3. We found a significant decrease of DExH-Box Helicase 34 (DHX34), suppressor with morphogenetic effect on genitalia 5 (SMG5), and SMG7 expression at 6 h post-infection, followed by a significant increase of these genes and also UPF1 and UPF2 at 9 h post-infection. Conversely, NBAS and other genes coding for NMD factors were not modulated. Known NMD substrates related to cell stress (Growth Arrest Specific 5, GAS5; transducin beta-like 2, TBL2; and DNA damage-inducible transcript 3, DDIT3) were increased in infected cells, possibly as a result of alterations in the NMD pathway and of a direct effect of the infection. We also found that the expression of unconventional SNARE in the ER 1, USE1 (p31) and Zeste White 10 homolog, ZW10, partners of NBAS in the retrograde transport function, significantly increased over time in infected cells. Co-localization of NBAS and UPF1 proteins did not change within 24 h of infection nor did it differ in infected versus non-infected cells at 1 and 24 h after infection; similarly, the co-localization of NBAS and p31 proteins was not altered by infection in this short time frame. Finally, both NBAS and UPF1 were found to co-localize with SARS-CoV-2 S and N proteins. Overall, these data are preliminary evidence of an interaction between NBAS and NBAS-related functions and SARS-CoV-2 in infected cells, deserving further investigation.

What Can RNA-Based Therapy Do for Monogenic Diseases?

The use of RNA-based approaches to treat monogenic diseases (i.e., hereditary disorders caused by mutations in single genes) has been developed on different fronts. One approach uses small antisense oligonucleotides (ASOs) to modulate RNA processing at various stages; namely, to enhance correct splicing, to stimulate exon skipping (to exclude premature termination codon variants), to avoid undesired messenger RNA (mRNA) transcript degradation via the nonsense-mediated decay (NMD) pathway, or to induce mRNA degradation where they encode toxic proteins (e.g., in dominant diseases). Another approach consists in administering mRNA, which, like gene therapy, is a mutation-agnostic approach with potential application to any recessive monogenic disease. This is simpler than gene therapy because instead of requiring targeting of the nucleus, the mRNA only needs to be delivered to the cytoplasm. Although very promising (as demonstrated by COVID-19 vaccines), these approaches still have potential for optimisation, namely regarding delivery efficiency, adverse drug reactions and toxicity.

Reversal of splicing infidelity is a pre-activation step in B cell differentiation.

B cell activation and differentiation is central to the adaptive immune response. Changes in exon usage can have major impacts on cellular signaling and differentiation but have not been systematically explored in differentiating B cells. We analyzed exon usage and intron retention in RNA-Seq data from subsets of human B cells at various stages of differentiation, and in an in vitro laboratory model of B cell activation and differentiation (Epstein Barr virus infection). Blood naïve B cells were found to have an unusual splicing profile, with unannotated splicing events in over 30% of expressed genes. Splicing changed substantially upon naïve B cell entry into secondary lymphoid tissue and before activation, involving significant increases in exon commitment and reductions in intron retention. These changes preferentially involved short introns with weak splice sites and were likely mediated by an overall increase in splicing efficiency induced by the lymphoid environment. The majority of transcripts affected by splicing changes showed restoration of encoded conserved protein domains and/or reduced targeting to the nonsense-mediated decay pathway. Affected genes were enriched in functionally important immune cell activation pathways such as antigen-mediated signaling, cell cycle control and mRNA processing and splicing. Functional observations from donor B cell subsets in progressive states of differentiation and from timecourse experiments using the in vitro model suggest that these widespread changes in mRNA splicing play a role in preparing naïve B cells for the decisive step of antigen-mediated activation and differentiation.

Proximally Biased V(D)J Recombination and Evolution of Non-Productive Clones in B-Cell Precursor Acute Lymphocytic Leukemia with KMT2A::AFF1 Fusion Genes

Background: B-cell precursor acute lymphocytic leukemia (BCP-ALL) carrying KMT2A::AFF1 rearrangements is an aggressive disease with unsatisfactory response to treatment and poor prognosis. KMT2A is a histone methyl transferase involved in the establishment of epigenetic memory. In the fusion protein encoded by the KMT2A::AFF1 fusion gene, the catalytic SET domain of KMT2A mediating H3K4 trimethylation is lost. Instead, the fusion protein interacts with DOT1L and mediates H3K79 methylation. The subversion of the catalytic activity of KMT2A arrests affected cells in an immature stage of development that may precede V(D)J recombination. Aim: Investigate the immune repertoire of KMT2A::AFF1 rearranged BCP-ALL and evaluate the feasibility of measurable residual disease (MRD) detection using immunoglobulin (Ig) gene rearrangements. Patients and Methods: The cohort includes 290 adult and 89 pediatric BCP-ALL patients. The diagnosis was established following WHO classification. WTS (5x107 reads, 2x101bp) data were generated on HiSeq and NovaSeq instruments. Ig gene rearrangements were analyzed using MiXCR and NCBI-IgBlast. Results: We analyzed Ig heavy-chain variable region (IGHV) gene rearrangements in BCP-ALL samples. We observed that the immune repertoire of leukemic cells carrying KMT2A::AFF1 is less diverse than that of other BCP-ALL samples. We investigated the expression levels of IGHV genes using WTS data. We found that IGHV genes located proximally to the IGHD/IGHJ cluster of genes on chr14 were more likely to be expressed in samples carrying KMT2A::AFF1 (Fig 1A). This difference was particularly significant (p<10-18) for IGHV6-1, the IGHV gene closest to the IGHD/IGHJ cluster (Fig 1B). Proximally biased V(D)J recombination is indicative of incomplete Ig locus contraction. Locus contraction is regulated by PAX5, which suppresses WAPL, a negative regulator of chromatin loop extrusion. Chromatin looping of IGHV genes ensures a balanced immune repertoire by bringing distant IGHV genes in proximity to the V(D)J recombination center. We found that in samples with KMT2A::AFF1 fusion genes WAPL expression levels are significantly (p<10-10) higher than in other subtypes of BCP-ALL. Analysis of the Ig gene rearrangements in KMT2A::AFF1 samples revealed abundant expression of alleles encoding non-productive V(D)J rearrangements or IGHV alleles in germline configuration. Only 35% of complementarity determining region 3 covering reads encoded productive rearrangements in KMT2A::AFF1 samples while in other types of BCP-ALL 67% of reads are productive (p<10-6). In samples with chronic lymphocytic leukemia, a tumor of post-germinal center B cells, this percentage is generally 90% or more. Among the non-productive V(D)J rearranged alleles, in-frame stop codons were equally overrepresented as out-of-frame V-J fusions when compared to their abundance in other types of BCP-ALL (p<10-15 in both cases). As light chain rearrangements require signaling from a productive heavy chain, the significantly (p<9x10-7) lower expression of light chain alleles detectable in KMT2A::AFF1 samples supports the notion that the abundant expression of non-productive alleles is not an artefact. Clonal expansion as measured by the Gini index for the number of reads per clonotype is distributed uniformly between 0 (polyclonality) and 1 (monoclonality). Monoclonality was observed in only 10% of cases. Furthermore, some DJ-rearrangements with identical non-templated linker sequences were found to be recombined with different IGHV genes, indicating ongoing V(D)J recombination and clonal evolution. Conclusions: Our data suggest that the KMT2A::AFF1 rearrangement locks the cell of origin at a point of ongoing V(D)J recombination. V(D)J recombination appears to be induced prematurely in these cells. Poor locus contraction could explain the proximally biased V(D)J recombination pattern. The non-sense mediated decay pathway involved in down regulating the expression of non-productive alleles seems to be poorly functional. Consequently, the cells express unusually high amounts of non-productive alleles or alleles in germline configuration that are clonally evolving and derived frequently from IGHV6-1. These features distinguish KMT2A::AFF1 BCP-ALL from other BCP-ALL subtypes and make MRD detection in KMT2A::AFF1 BCP-ALL based on specific Ig gene rearrangements highly error prone. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Defining the Functional Interactome of Spliceosome-Associated G-Patch Protein Gpl1 in the Fission Yeast Schizosaccharomyces pombe.

Pre-mRNA splicing plays a fundamental role in securing protein diversity by generating multiple transcript isoforms from a single gene. Recently, it has been shown that specific G-patch domain-containing proteins are critical cofactors involved in the regulation of splicing processes. In this study, using the knock-out strategy, affinity purification and the yeast-two-hybrid assay, we demonstrated that the spliceosome-associated G-patch protein Gpl1 of the fission yeast S. pombe mediates interactions between putative RNA helicase Gih35 (SPAC20H4.09) and WD repeat protein Wdr83, and ensures their binding to the spliceosome. Furthermore, RT-qPCR analysis of the splicing efficiency of deletion mutants indicated that the absence of any of the components of the Gpl1-Gih35-Wdr83 complex leads to defective splicing of fet5 and pwi1, the reference genes whose unspliced isoforms harboring premature stop codons are targeted for degradation by the nonsense-mediated decay (NMD) pathway. Together, our results shed more light on the functional interactome of G-patch protein Gpl1 and revealed that the Gpl1-Gih35-Wdr83 complex plays an important role in the regulation of pre-mRNA splicing in S. pombe.