RNA Decay Research Articles

Light-induced cryptochrome 2 liquid-liquid phase separation and mRNA methylation.

Light is essential not only for photosynthesis but also for the regulation of various physiological and developmental processes in plants. While the mechanisms by which light regulates transcription and protein stability are well established, the effects of light on RNA methylation and their subsequent impact on plant growth and development are less understood. Upon exposure to blue light, the photoreceptor cryptochromes form nuclear speckles or nuclear bodies, termed CRY photobodies. The CRY2 photobodies undergo light-induced homo-oligomerization and liquid-liquid phase separation (LLPS), which are crucial for their physiological activity. Recent studies have proposed that blue light-induced CRY2 LLPS increases the local concentration or directly enhances the biochemical activities of RNA N6-methyladenosine (m6A) methyltransferases, thus, to regulate circadian clock and maintain Chl homeostasis through processes of RNA decay or translation. This review aimed to elucidate the functions of CRY2 and LLPS in RNA methylation, focusing on the light-controlled reversible phase transitions regulon and the outstanding questions that remain in RNA methylation.

Disrupted Post-Transcriptional Regulation of Gene Expression as a Hallmark of Fatty Liver Progression

It is known that both transcriptional and post-transcriptional mechanisms control messenger RNA (mRNA) levels. Compared to transcriptional regulations, our understanding of how post-transcriptional regulations adapt during fatty liver progression at the whole-transcriptome level is unclear. While traditional RNA-seq analysis uses only reads mapped to exons to determine gene expression, recent studies support the idea that intron-mapped reads can be reliably used to estimate gene transcription. In this study, we analyzed differential gene expression at both the exon and intron levels using two liver RNA-seq datasets from mice that were fed a high-fat diet for seven weeks (mild fatty liver) or thirty weeks (severe fatty liver). We found that the correlation between gene transcription and mature mRNA levels was much lower in mice with mild fatty liver as compared with mice with severe fatty liver. This result indicates broad post-transcriptional regulations for early fatty liver and such regulations are compromised for severe fatty liver. Specifically, gene ontology analysis revealed that genes involved in synapse organization and cell adhesion were transcriptionally upregulated, while their mature mRNAs were unaffected in mild fatty liver. Further characterization of post-transcriptionally suppressed genes in early fatty liver revealed that their mRNAs harbor a significantly longer 3′ UTR, one of the major features that may subject RNA transcripts to nonsense-mediated RNA decay (NMD). We further show that the expression of representative genes that were post-transcriptionally suppressed were upregulated in mice with a hepatocyte-specific defect of NMD. Finally, we provide data supporting a time-dependent decrease in NMD activity in the liver of a diet-induced metabolic-dysfunction-associated fatty liver disease mouse model. In summary, our study supports the conclusion that NMD is essential in preventing unwanted/harmful gene expression at the early stage of fatty liver and such a mechanism is lost due to decreased NMD activity in mice with severe fatty liver.

Regulation of maize growth and immunity by ZmSKI3-mediated RNA decay and post-transcriptional gene silencing.

Disease resistance is often associated with compromised plant growth and yield due to defense-growth tradeoffs. However, key components and mechanisms underlying the defense-growth tradeoffs are rarely explored in maize. In this study, we find that ZmSKI3, a putative subunit of the SUPERKILLER (SKI) complex that mediates the 3'-5' degradation of RNA, regulates both plant development and disease resistance in maize. The Zmski3 mutants showed retarded plant growth and constitutively activated defense responses, while the ZmSKI3 overexpression lines are more susceptible to Curvularia lunata and Bipolaris maydis. Consistently, the expression of defense-related genes was generally up-regulated, while expressions of growth-related genes were mostly down-regulated in leaves of the Zmski3-1 mutant compared to that of wild type. In addition, 223 differentially expressed genes that are up-regulated in Zmski3-1 mutant but down-regulated in the ZmSKI3 overexpression line are identified as potential target genes of ZmSKI3. Moreover, small interfering RNAs targeting the transcripts of the defense- and growth-related genes are differentially accumulated, likely to combat the increase of defense-related transcripts but decrease of growth-related transcripts in Zmski3-1 mutant. Taken together, our study indicates that plant growth and immunity could be regulated by both ZmSKI3-mediated RNA decay and post-transcriptional gene silencing in maize.

EDC-3 and EDC-4 regulate embryonic mRNA clearance and biomolecular condensate specialization

Animal development is dictated by the selective and timely decay of mRNAs in developmental transitions, but the impact of mRNA decapping scaffold proteins in development is unclear. This study unveils the roles and interactions of the DCAP-2 decapping scaffolds EDC-3 and EDC-4 in the embryonic development of C.elegans. EDC-3 facilitates the timely removal of specific embryonic mRNAs, including cgh-1, car-1, and ifet-1 by reducing their expression and preventing excessive accumulation of DCAP-2 condensates in somatic cells. We further uncover a role for EDC-3 in defining the boundaries between P bodies, germ granules, and stress granules. Finally, we show that EDC-4 counteracts EDC-3 and engenders the assembly of DCAP-2 with the GID (CTLH) complex, a ubiquitin ligase involved in maternal-to-zygotic transition (MZT). Our findings support a model where multiple RNA decay mechanisms temporally clear maternal and zygotic mRNAs throughout embryonic development.

Disruption of cotranscriptional splicing suggests RBM39 is a therapeutic target in acute lymphoblastic leukemia

AbstractThere are only a few options for patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL), thus, this is a major area of unmet medical need. In this study, we reveal that the inclusion of a poison exon in RBM39, which could be induced by both CDK9 or CDK9 independent cyclin-dependent kinases, mitogen-activated protein kinases, glycogen synthase kinases, CDC-like kinases (CMGC) kinase inhibition, is recognized by the nonsense-mediated messenger RNA decay pathway for degradation. Targeting this poison exon in RBM39 with CMGC inhibitors led to protein downregulation and the inhibition of ALL growth, particularly in relapsed/refractory B-ALL. Mechanistically, disruption of cotranscriptional splicing by the inhibition of CMGC kinases, including DYRK1A, or inhibition of CDK9, which phosphorylate the C-terminal domain of RNA polymerase II (Pol II), led to alteration in the SF3B1 and Pol II association. Disruption of SF3B1 and the transcriptional elongation complex altered Pol II pausing, which promoted the inclusion of a poison exon in RBM39. Moreover, RBM39 ablation suppressed the growth of human B-ALL, and targeting RBM39 with sulfonamides, which degrade RBM39 protein, showed strong antitumor activity in preclinical models. Our data reveal that relapsed/refractory B-ALL is susceptible to pharmacologic and genetic inhibition of RBM39 and provide 2 potential strategies to target this axis.

NMDtxDB: data-driven identification and annotation of human NMD target transcripts.

The nonsense-mediated RNA decay (NMD) pathway is a crucial mechanism of mRNA quality control. Current annotations of NMD substrate RNAs are rarely data-driven, but use generally established rules. We present a data set with four cell lines and combinations for SMG5, SMG6, and SMG7 knockdowns or SMG7 knockout. Based on this data set, we implemented a workflow that combines Nanopore and Illumina sequencing to assemble a transcriptome, which is enriched for NMD target transcripts. Moreover, we use coding sequence information (CDS) from Ensembl, Gencode consensus Ribo-seq ORFs, and OpenProt to enhance the CDS annotation of novel transcript isoforms. In summary, 302,889 transcripts were obtained from the transcriptome assembly process, out of which 24% are absent from Ensembl database annotations, 48,213 contain a premature stop codon, and 6433 are significantly upregulated in three or more comparisons of NMD active versus deficient cell lines. We present an in-depth view of these results through the NMDtxDB database, which is available at https://shiny.dieterichlab.org/app/NMDtxDB, and supports the study of NMD-sensitive transcripts. We open sourced our implementation of the respective web-application and analysis workflow at https://github.com/dieterich-lab/NMDtxDB and https://github.com/dieterich-lab/nmd-wf.

MYC Induces Oncogenic Stress through RNA Decay and Ribonucleotide Catabolism in Breast Cancer.

Upregulation of MYC is a hallmark of cancer, wherein MYC drives oncogenic gene expression and elevates total RNA synthesis across cancer cell transcriptomes. Although this transcriptional anabolism fuels cancer growth and survival, the consequences and metabolic stresses induced by excess cellular RNA are poorly understood. Herein, we discover that RNA degradation and downstream ribonucleotide catabolism is a novel mechanism of MYC-induced cancer cell death. Combining genetics and metabolomics, we find that MYC increases RNA decay through the cytoplasmic exosome, resulting in the accumulation of cytotoxic RNA catabolites and reactive oxygen species. Notably, tumor-derived exosome mutations abrogate MYC-induced cell death, suggesting excess RNA decay may be toxic to human cancers. In agreement, purine salvage acts as a compensatory pathway that mitigates MYC-induced ribonucleotide catabolism, and inhibitors of purine salvage impair MYC+ tumor progression. Together, these data suggest that MYC-induced RNA decay is an oncogenic stress that can be exploited therapeutically. Significance: MYC is the most common oncogenic driver of poor-prognosis cancers but has been recalcitrant to therapeutic inhibition. We discovered a new vulnerability in MYC+ cancer where MYC induces cell death through excess RNA decay. Therapeutics that exacerbate downstream ribonucleotide catabolism provide a therapeutically tractable approach to TNBC (Triple-negative Breast Cancer) and other MYC-driven cancers.

Sequestration of DBR1 to stress granules promotes lariat intronic RNAs accumulation for heat-stress tolerance

Heat stress (HS) poses a significant challenge to plant survival, necessitating sophisticated molecular mechanisms to maintain cellular homeostasis. Here, we identify SICKLE (SIC) as a key modulator of HS responses in Arabidopsis (Arabidopsis thaliana). SIC is required for the sequestration of RNA DEBRANCHING ENZYME 1 (DBR1), a rate-limiting enzyme of lariat intronic RNA (lariRNA) decay, into stress granules (SGs). The sequestration of DBR1 by SIC enhances the accumulation of lariRNAs, branched circular RNAs derived from excised introns during pre-mRNA splicing, which in turn promote the transcription of their parental genes. Our findings further demonstrate that SIC-mediated DBR1 sequestration in SGs is crucial for plant HS tolerance, as deletion of the N-terminus of SIC (SIC1–244) impairs DBR1 sequestration and compromises plant response to HS. Overall, our study unveils a mechanism of transcriptional regulation in the HS response, where lariRNAs are enriched through DBR1 sequestration, ultimately promoting the transcription of heat stress tolerance genes.

Adenosine Deaminase-Like Gene-Carried Lentivirus Toolkit for Identification of DNA N6-Methyladenine Origins.

Post-replicative DNA N6-methyladenine (pr6mdA) can form via bona fide methylase-catalyzed adenine methylation, playing a pivotal role in embryonic development and other biological processes. Surprisingly, pre-methylated adenine can be erroneously incorporated into DNA as misincorporated N6-methyladenine (i6mdA) via DNA polymerase-mediated replication. Despite pr6mdA and i6mdA sharing identical chemical structures, their biological functions diverge significantly, presenting a substantial challenge in distinguishing between the two. Here, for the first-time, it is exploited that the adenosine deaminase-like (Adal) protein and a corresponding activity-null mutant to construct an Adal lentivirus toolkit. With this newly designed toolkit, both pr6mdA and i6mdA can be identified and quantified simultaneously. The presence of 6mdA in the bone marrow cells of mice is shown, with its levels serving as indicators for growth with age, probably reflecting the cellular stress-caused changes in RNA decay, nucleotide pool sanitation, and transcription. Collectively, a powerful toolkit to advance understanding of both pr6mdA and i6mdA is demonstrated.

Genetic regulation of nascent RNA maturation revealed by direct RNA nanopore sequencing.

Quantitative trait loci analyses have revealed an important role for genetic variants in regulating alternative splicing (AS) and alternative cleavage and polyadenylation (APA) in humans. Yet, these studies are generally performed with mature mRNA, so they report on the outcome rather than the processes of RNA maturation and thus may overlook how variants directly modulate pre-mRNA processing. The order in which the many introns of a human gene are removed can substantially influence AS, while nascent RNA polyadenylation can affect RNA stability and decay. However, how splicing order and poly(A) tail length are regulated by genetic variation has never been explored. Here, we used direct RNA nanopore sequencing to investigate allele-specific pre-mRNA maturation in 12 human lymphoblastoid cell lines. We found frequent splicing order differences between alleles and uncovered significant single nucleotide polymorphism (SNP)-splicing order associations in 17 genes. This included SNPs located in or near splice sites as well as more distal intronic and exonic SNPs. Moreover, several genes showed allele-specific poly(A) tail lengths, many of which also had a skewed allelic abundance ratio. HLA class I transcripts, which encode proteins that play an essential role in antigen presentation, showed the most allele-specific splicing orders, which frequently co-occurred with allele-specific AS, APA or poly(A) tail length differences. Together, our results expose new layers of genetic regulation of pre-mRNA maturation and highlight the power of long-read RNA sequencing for allele-specific analyses.

RNase L-induced bodies sequester subgenomic flavivirus RNAs to promote viral RNA decay

Subgenomic flavivirus RNAs (sfRNAs) are structured RNAs encoded by flaviviruses that promote viral infection by inhibiting cellular RNA decay machinery. Herein, we analyze sfRNA production and localization using single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) throughout West Nile virus, Zika virus, or dengue virus serotype 2 infection. We observe that sfRNAs are generated during the RNA replication phase of viral infection in the cytosol and accumulate in processing bodies (P-bodies), which contain RNA decay machinery such as XRN1 and Dcp1b. However, upon activation of the host antiviral endoribonuclease, ribonuclease L (RNase L), sfRNAs re-localize to ribonucleoprotein complexes known as RNase L-induced bodies (RLBs). RLB-mediated sequestration of sfRNAs reduces sfRNA association with RNA decay machinery inP-bodies, which coincides with increased viral RNA decay. These findings establish a functional role forRLBs in enhancing the cell-mediated decay of viral RNA by sequestering functional viral RNA decay products.

Full-length direct RNA sequencing uncovers stress-granule dependent RNA decay upon cellular stress.

Cells react to stress by triggering response pathways, leading to extensive alterations in the transcriptome to restore cellular homeostasis. The role of RNA metabolism in shaping the cellular response to stress is vital, yet the global changes in RNA stability under these conditions remain unclear. In this work, we employ direct RNA sequencing with nanopores, enhanced by 5' end adaptor ligation, to comprehensively interrogate the human transcriptome at single-molecule and nucleotide resolution. By developing a statistical framework to identify robust RNA length variations in nanopore data, we find that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Unlike typical RNA decay models in normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but does not depend on deadenylation or decapping. We observed that RNAs undergoing decay are predominantly enriched in the stress granule transcriptome while inhibition of stress granule formation via genetic ablation of G3BP1 and G3BP2 rescues RNA length. Our findings reveal RNA decay as a key determinant of RNA metabolism upon cellular stress and dependent on stress-granule formation.

The Effect of Diurnal Temperature Fluctuations on the Decay of Japanese Encephalitis and Murray Valley Encephalitis Virus RNA Seeded in Piggery Wastewater

The Effect of Diurnal Temperature Fluctuations on the Decay of Japanese Encephalitis and Murray Valley Encephalitis Virus RNA Seeded in Piggery Wastewater

The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites.

RNA molecules are localized to subcellular regions through interactions between localization-regulatory cis-elements and trans-acting RNA binding proteins (RBPs). However, the identities of RNAs whose localization is regulated by a specific RBP as well as the impacts of that RNA localization on cell function have generally remained unknown. Here, we demonstrate that the RBP HNRNPA2B1 acts to keep specific RNAs out of neuronal projections. Using subcellular fractionation, high-throughput sequencing, and single molecule RNA FISH, we find that hundreds of RNAs demonstrate markedly increased abundance in neurites in HNRNPA2B1 knockout cells. These RNAs often encode motor proteins and are enriched for known HNRNPA2B1 binding sites and motifs in their 3' UTRs. The speed and processivity of microtubule-based transport is impaired in these cells, specifically in their neurites. HNRNPA2B1 point mutations that increase its cytoplasmic abundance relative to wildtype lead to stronger suppression of RNA mislocalization defects than seen with wildtype HNRNPA2B1. We further find that the subcellular localizations of HNRNPA2B1 target RNAs are sensitive to perturbations of RNA decay machinery, suggesting that it is HNRNPA2B1's known role in regulating RNA stability that may explain these observations. These findings establish HNRNPA2B1 as a negative regulator of neurite RNA abundance and link the subcellular activities of motor proteins with the subcellular abundance of the RNAs that encode them.

Crop antiviral defense: Past and future perspective.

Viral pathogens not only threaten the health and life of humans and animals but also cause enormous crop yield losses and contribute to global food insecurity. To defend against viral pathogens, plants have evolved an intricate immune system to perceive and cope with such attacks. Although most of the fundamental studies were carried out in model plants, more recent research in crops has provided new insights into the antiviral strategies employed by crop plants. We summarize recent advances in understanding the biological roles of cellular receptors, RNA silencing, RNA decay, hormone signaling, autophagy, and ubiquitination in manipulating crop host-mediated antiviral responses. The potential functions of circular RNAs, the rhizosphere microbiome, and the foliar microbiome of crops in plant-virus interactions will be fascinating research directions in the future. These findings will be beneficial for the development of modern crop improvement strategies.

The Story of RNA Unfolded: The Molecular Function of DEAD- and DExH-Box ATPases and Their Complex Relationship with Membraneless Organelles.

DEAD- and DExH-box ATPases (DDX/DHXs) are abundant and highly conserved cellular enzymes ubiquitously involved in RNA processing. By remodeling RNA-RNA and RNA-protein interactions, they often function as gatekeepers that control the progression of diverse RNA maturation steps. Intriguingly, most DDX/DHXs localize to membraneless organelles (MLOs) such as nucleoli, nuclear speckles, stress granules, or processing bodies. Recent findings suggest not only that localization to MLOs can promote interaction between DDX/DHXs and their targets but also that DDX/DHXs are key regulators of MLO formation and turnover through their condensation and ATPase activity.In this review, we describe the molecular function of DDX/DHXs in ribosome biogenesis, messenger RNA splicing, export, translation, and storage or decay as well as their association with prominent MLOs. We discuss how the enzymatic function of DDX/DHXs in RNA processing is linked to DDX/DHX condensation, the accumulation of ribonucleoprotein particles and MLO dynamics. Future research will reveal how these processes orchestrate the RNA life cycle in MLO space and DDX/DHX time.

Identification of nonsense variants in the genomes of 15 Murciano-Granadina bucks and analysis of their segregation in parent-offspring trios

Nonsense variants can inactivate gene function by causing the synthesis of truncated proteins or by inducing nonsense mediated decay of messenger RNAs. The occurrence of such variants in the genomes of livestock species is modulated by multiple demographic and selective factors. Even though nonsense variants can have causal effects on embryo lethality, abortions, and disease, their genomic distribution and segregation in domestic goats have not been characterized in depth yet. In this work, we have sequenced the genomes of 15 Murciano-Granadina bucks with an average coverage of 32.92 × ± 1.45 × . Bioinformatic analysis revealed 947 nonsense variants consistently detected with SnpEff and Ensembl-VEP. These variants were especially abundant in the 3'end of the protein-coding regions. Genes related to olfactory perception, ATPase activity coupled to transmembrane movement of substances, defense to virus, hormonal response, and sensory perception of taste were particularly enriched in nonsense variants. Seventeen nonsense variants expected to have harmful effects on fitness were genotyped in parent-offspring trios. We observed that several nonsense variants predicted to be lethal based on mouse knockout data did not have such effect, a finding that could be explained by the existence of multiple mechanisms counteracting lethality. These findings demonstrate that predicting the effects of putative nonsense variants on fitness is extremely challenging. As a matter of fact, such a goal could only be achieved by generating a high quality telomere-to-telomere goat reference genome combined with carefully curated annotation and functional testing of promising candidate variants.

A novel compound heterozygous mutation of UFC1 in a patient with neurodevelopmental disorder.

Neurodevelopmental disorders (NDDs) encompass a diverse group of disorders characterized by impaired cognition, behavior, and motor skills. Genetic factor is the leading cause in about 35% of NDDs patients. Mutations of UFC1, an E2 enzyme participating in the post-translational modification of proteins through attachment of ubiquitin-like proteins, were recently reported to be associated with NDDs. However, the UFC1 associated NDDs are rare and the data are scarce, thus making it difficult to identify this disease. This study reported a novel compound heterozygous mutation of UFC1 in a Chinese patient with NDD. Detailed clinical data were recorded. Whole exome sequencing (WES) was performed to determine the genetic cause of the patient. The candidate mutation was verified using Sanger sequencing. WES analysis identified a novel compound heterozygous mutation of UFC1 (c.19C > T, p.Arg7* and c.164G > A, p.Arg55Gln). The nonsense mutation c.19C > T (p.Arg7*) led to a premature truncation of UFC1 and nonsense-mediated RNA decay. Arg55 is highly conserved among orthologues. Molecular modeling predicted that mutation c.164G > A (p.Arg55Gln) may influence the correct folding of UFC1. These two mutations were evaluated as likely pathogenic based on the ACMG guideline. Moreover, neurodevelopmental delay, microcephaly, and epilepsy were confirmed as major phenotypes of UFC1 mutation. This study expands the mutational spectrum of NDDs. We reported the nonsense mutation of UFC1 for the first time. We also confirmed the major phenotypes that may guide clinical identification of UFC1 mutation. Ubiquitination mechanism is highlighted in NDDs pathogenesis.

A systematic search for RNA structural switches across the human transcriptome

RNA structural switches are key regulators of gene expression in bacteria, but their characterization in Metazoa remains limited. Here, we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3ʹ untranslated region of the RORC (RAR-related orphan receptor C) transcript. In vivo dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated messenger RNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.

A biallelic variant of the RNA exosome gene, EXOSC4, associated with neurodevelopmental defects impairs RNA exosome function and translation

The RNA exosome is an evolutionarily conserved complex required for both precise RNA processing and decay. Pathogenic variants in EXOSC genes, which encode structural subunits of this complex, are linked to several autosomal recessive disorders. Here, we describe a missense allele of the EXOSC4 gene that causes a collection of clinical features in two affected siblings. This missense variant (NM_019037.3: exon3:c.560T>C) changes a leucine residue within a conserved region of EXOSC4 to proline (p.Leu187Pro). The two affected individuals show prenatal growth restriction, failure to thrive, global developmental delay, intracerebral and basal ganglia calcifications, and kidney failure. Homozygosity for the damaging variant was identified by exome sequencing with Sanger sequencing to confirm segregation. To explore the functional consequences of this amino acid change, we modeled EXOSC4-L187P in the corresponding budding yeast protein, Rrp41 (Rrp41-L187P). Cells that express Rrp41-L187P as the sole copy of the essential Rrp41 protein show growth defects. Steady-state levels of both Rrp41-L187P and EXOSC4-L187P are decreased compared to controls, and EXOSC4-L187P shows decreased copurification with other RNA exosome subunits. RNA exosome target transcripts accumulate in rrp41-L187P cells, including the 7S precursor of 5.8S rRNA. Polysome profiles show a decrease in actively translating ribosomes in rrp41-L187P cells as compared to control cells with the incorporation of 7S pre-rRNA into polysomes. This work adds EXOSC4 to the structural subunits of the RNA exosome that have been linked to human disease and defines foundational molecular defects that could contribute to the adverse phenotypes caused by EXOSC pathogenic variants.