Cellular mRNA Research Articles

Developmental expression patterns of genes mutated in patients with neurodevelopmental disorders

Neurodevelopmental disorders (NDDs) comprise a heterogeneous spectrum of disorders with diverse manifestations, such as microcephaly, structural brain abnormalities, epilepsy, developmental delay, intellectual disability, and autism spectrum disorders [1]. Although relatively rare, each type of NDDs represents a significant population of neurological patients. The global prevalence of NDDs exceeds 15% [2]. NDDs typically arise from molecular cascades that are highly regulated and disrupted by either gene mutations or environmental factors. The genetic basis of a substantial proportion of such disorders is hard to discern given that not all are inherited according to Mendelian principles and involve allelic variations from multiple genes. However, roughly 40% of NDDs are believed to be caused by the disruption of a single gene, indicating monogenic conditions. [3] Understanding and predicting the physiological function of a protein encoded by a specific gene, determining its interactions with other proteins, and investigating the role of the gene in organ and tissue development requires a close examination of its expression. Thus, a crucial initial step in researching genes associated with neurodevelopmental disorders is to investigate the expression patterns of these genes in the mouse brain at various embryonic developmental stages. In situ RNA hybridization was used to analyze gene expression patterns in slices of mouse brain tissue. Fixed in 4% paraformaldehyde/phosphate-buffered saline/diethylpyrocarbonate mouse brain samples at embryonic (E12.5, E15.5, E18.5) and postnatal (P1, P21) developmental stages were sectioned using a Leica CM1520 cryostat with 15 µm slice thickness. Next, we conducted in situ hybridization of cellular mRNA using DIG-dUTP-labeled RNA probes that were previously synthesized by PCR with cDNA and gene-specific primers. 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) was used, which produces an insoluble dark blue or purple sediment visible under a light microscope by reaction with alkaline phosphatase, to visualize the localization of mRNA expression in tissues. The study examined the expression of a member of the CCDC gene family which encodes proteins involved in intercellular transmembrane signal transduction. In situ hybridization was performed on mouse brain slices, revealing significant mRNA expression of the gene in the cerebral cortex. Additionally, mouse knockout experiments are planned to investigate the gene’s role in brain development.

Vitrified turkey ovarian tissue cultured and assessed through gene expression patterns: A potential screening tool

Biobanking of turkey ovarian tissue has the potential to play a crucial part in preserving female genetics. To date, ovarian tissue has only been vitrified using a standard protocol, with immediate analyses after warming, therefore, long-term cryoinjury is unknown. Long-term cryoinjury was investigated here by in-ovo culturing, fresh (non-vitrified), a purposefully suboptimal poor vitrification (PV), and the standard vitrified (StV) protocol. Assessments were performed via cellular morphological changes and mRNA gene expression differences, immediately (day 0) or after 2, 4, or 6 days of in-ovo culturing. On day 0, the mRNA levels of heat-shock protein A2 (HSPA2) were lowest in the fresh tissue, and increased 5-fold in the StV treatment, and 18-fold in the PV treatment. Whereas, by day 6, growth determining factor 9 (GDF9) mRNA levels within the fresh tissue were over 3-fold and 21-fold higher than StV and PV treatments, respectively. After 6 days of in-ovo culture the follicle density was highest in the fresh ovarian tissue (4701 ± 950 #/mm3), followed by the StV (1601 ± 300 #/mm3), with PV having the lowest density (172 ± 145 #/mm3). This shows that although the density of follicles was higher in StV versus PV, a considerable number (∼65 %) were lost compared to the fresh treatment. Additionally, the HSPA2 expression could be an early screening tool, whereas GDF9 expression could be a late screening tool, used to assess turkey ovarian tissue vitrification protocols. We conclude that the StV protocol should be further optimized to try and improve follicle numbers post-warming.

Human papillomavirus testing using HPV APTIMA® assay and an external cellularity control in self-collected samples.

In cervical cancer screening programs, the detection of high-risk human papillomavirus (HR-HPV)is now widely implemented on physician-collected samples and has expanded to include self-collected samples. The use of a cellularity control (CC)is needed to reduce false-negative HPV results. An external mRNA CC forthe HPV APTIMA®assay was assessed for its analytical performance and the results were compared with both cervix cytobrush samples taken by physicians and self-collected vaginal samples from 148 women. The performance of the CC was adjusted to control for the presence of cellular mRNA in the ThinPrep® and Multitest® transport media. This CC is user-friendly but implies to perform two independent assays on PANTHER®automate. Self-collected vaginal sampling gives a lower median CC results (13.2 vs. 16.9 min) but a higher risk of negative CC results (3.3 vs. 0%). The usefulness of the CC for the HR-HPVassay may be optimized by the definition of a threshold for a minimum cell number to be tested to increase confidence in HPV-negative results. The systematic use of an RNA CC increases confidence for HPV RNA assays on self-collected vaginal samples.

Abstract A163: Exoribonuclease XRN1 is a therapeutic vulnerability in tumors with intrinsically elevated type I interferon signaling

Abstract Background: 5′→3′ exoribonuclease 1 (XRN1) degrades single stranded mRNA from the 5′→3′ direction and plays a key role in endogenous cellular mRNA turnover. In addition, XRN1 can degrade double-stranded RNA (dsRNA), and as such plays a role in innate immunity by prevention of dsRNA accumulation and pPKR induction in virus-infected cells. We identified XRN1 as a putative selective vulnerability in tumor cells with intrinsic elevation of a Type I Interferon Stimulated Gene (TISG) signature through analysis of publicly available CRISPR screen data across 483 tumor cell lines. We further explored the role of XRN1 in a set of lung cancer cell lines with differential TISG expression in order to validate XRN1 as a compelling target for oncology drug development. Materials and Methods: NCI-H1650, NCI-H1703, NCI-H1944, and NCI-H838 lung adenocarcinoma cell lines were infected with lentiviruses containing Cas9 and sgRNAs targeting XRN1. Non-targeting sgRNAs were used as negative controls. XRN1 knockout (KO) cells and controls were evaluated in proliferation, apoptosis, qPCR and western blot assays. Results: Consistent with a selective dependency on XRN1 in cells with high intrinsic interferon signaling, XRN1 KO via CRISPR leads to robust anti-proliferative effects in TISG high NCI-H1650 and NCI-H1703 cells, but not in TISG low NCI-H838 and NCI-H1944 cells. Annexin V staining of NCI-H1650 cells upon XRN1 KO demonstrates that apoptosis is induced in TISG high cells. XRN1 KO in the TISG high setting leads to robust upregulation of Interferon-β mRNA consistent with activation of the MDA5 pathway by elevation of cytoplasmic dsRNA. Similarly, XRN1 loss induces pPKR activation in TISG high, but not TISG low cells. As pPKR activation triggers translational shutdown, TISG high selective pPKR elevation is consistent with the cell death observed. Exogenous treatment with Interferon-β or dsRNA mimetic Poly I:C to induce elevated Type I Interferon expression sensitizes NCI-H838 cells to XRN1 KO, with effects on cell proliferation. Conclusions: Taken together, these results demonstrate a critical role of XRN1 in cancer cells with intrinsically elevated Type I Interferon signaling. Cell death is triggered via accumulation of dsRNA and downstream activation of the MDA5 and PKR innate immune pathways. These data suggest that XRN1, through its regulation of dsRNA burden, is a compelling oncology target for TISG high tumors. Conflict of Interests: All authors are current or former employees and shareholders of Accent Therapeutics, Inc. Citation Format: Maureen M Lynes, Sophie A Shen, Sunaina P Nayak, Brian A Sparling, Scott Ribich, Stephen J Blakemore, Serena J Silver. Exoribonuclease XRN1 is a therapeutic vulnerability in tumors with intrinsically elevated type I interferon signaling [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A163.

Directed evolution of biomass intensive CHO cells by adaptation to sub-physiological temperature

We report a simple and effective means to increase the biosynthetic capacity of host CHO cells. Lonza proprietary CHOK1SV® cells were evolved by serial sub-culture for over 150 generations at 32 °C. During this period the specific proliferation rate of hypothermic cells gradually recovered to become comparable to that of cells routinely maintained at 37 °C. Cold-adapted cell populations exhibited (1) a significantly increased volume and biomass content (exemplified by total RNA and protein), (2) increased mitochondrial function, (3) an increased antioxidant capacity, (4) altered central metabolism, (5) increased transient and stable productivity of a model IgG4 monoclonal antibody and Fc-fusion protein, and (6) unaffected recombinant protein N-glycan processing. This phenotypic transformation was associated with significant genome-scale changes in both karyotype and the relative abundance of thousands of cellular mRNAs across numerous functional groups. Taken together, these observations provide evidence of coordinated cellular adaptations to sub-physiological temperature. These data reveal the extreme genomic/functional plasticity of CHO cells, and that directed evolution is a viable genome-scale cell engineering strategy that can be exploited to create host cells with an increased cellular capacity for recombinant protein production.

Transcriptome-wide profiling of acute stress induced changes in ribosome occupancy level using external standards.

Acute cellular stress is known to induce a global reduction in mRNA translation through suppression of cap dependent translation. Selective translation in response to acute stress has been shown to play important roles in regulating the stress response. However, accurately profiling translational changes transcriptome-wide in response to acute cellular stress has been challenging. Commonly used data normalization methods operate on the assumption that any systematic shifts are experimental artifacts. Consequently, if applied to profiling acute cellular stress-induced mRNA translation changes, these methods are expected to produce biased estimates. To address this issue, we designed, produced, and evaluated a panel of 16 oligomers to serve as external standards for ribosome profiling studies. Using Sodium Arsenite treatment-induced oxidative stress in lymphoblastoid cell lines as a model system, we applied spike-in oligomers as external standards. We found our spike-in oligomers to display a strong linear correlation between the observed and the expected quantification, with small ratio compression at the lower concentration range. Using the expected fold changes constructed from spike-in controls, we found in our dataset that TMM normalization, a popular global scaling normalization approach, produced 87.5% false positives at a significant cutoff that is expected to produce only 10% false positive discoveries. In addition, TMM normalization produced a systematic shift of fold change by 3.25 fold. These results highlight the consequences of applying global scaling approaches to conditions that clearly violate their key assumptions. In contrast, we found RUVg normalization using spike-in oligomers as control genes recapitulated the expected stress induced global reduction of translation and resulted in little, if any, systematic shifts in the expected fold change. Our results clearly demonstrated the utility of our spike-in oligomers, both for constructing expected results as controls and for data normalization.

Ceg1 depletion reveals mechanisms governing degradation of non-capped RNAs in Saccharomyces cerevisiae

Most functional eukaryotic mRNAs contain a 5′ 7-methylguanosine (m7G) cap. Although capping is essential for many biological processes including mRNA processing, export and translation, the fate of uncapped transcripts has not been studied extensively. Here, we employed fast nuclear depletion of the capping enzymes in Saccharomyces cerevisiae to uncover the turnover of the transcripts that failed to be capped. We show that although the degradation of cap-deficient mRNA is dominant, the levels of hundreds of non-capped mRNAs increase upon depletion of the capping enzymes. Overall, the abundance of non-capped mRNAs is inversely correlated to the expression levels, altogether resembling the effects observed in cells lacking the cytoplasmic 5′−3′ exonuclease Xrn1 and indicating differential degradation fates of non-capped mRNAs. The inactivation of the nuclear 5′−3′ exonuclease Rat1 does not rescue the non-capped mRNA levels indicating that Rat1 is not involved in their degradation and consequently, the lack of the capping does not affect the distribution of RNA Polymerase II on the chromatin. Our data indicate that the cap presence is essential to initiate the Xrn1-dependent degradation of mRNAs underpinning the role of 5′ cap in the Xrn1-dependent buffering of the cellular mRNA levels.

Chikungunya virus nonstructural protein 1 is a versatile RNA capping and decapping enzyme

Chikungunya virus (CHIKV) non-structural protein 1 (nsP1) contains both the N7-guanine methyltransferase and guanylyltransferase activities and catalyzes the 5’ end cap formation of viral RNAs. To further understand its catalytic activity and role in virus-host interaction, we demonstrate that purified recombinant CHIKV nsP1 can reverse the guanylyl transfer reaction and remove the m7GMP from a variety of capped RNA substrates including host mRNAs. We then provide the structural basis of this function with a high-resolution cryogenic-electron microscopy structure of nsP1 in complex with the unconventional cap-1 substrate RNA m7GpppAmU. We show that the 5’ppRNA species generated by decapping can trigger RIG-I-mediated interferon (IFN) response. We further demonstrate that the decapping activity is conserved among the alphaviral nsP1s. To our knowledge, this is a new mechanism through which alphaviruses activate the antiviral immune response. This decapping activity could promote cellular mRNA degradation and facilitate viral gene expression which is functionally analogous to the cap-snatching mechanism by influenza virus.

CCR4-NOT differentially controls host versus virus poly(a)-tail length and regulates HCMV infection.

Unlike most RNA and DNA viruses that broadly stimulate mRNA decay and interfere with host gene expression, human cytomegalovirus (HCMV) extensively remodels the host translatome without producing an mRNA decay enzyme. By performing a targeted loss-of-function screen in primary human fibroblasts, we here identify the host CCR4-NOT deadenylase complex members CNOT1 and CNOT3 as unexpected pro-viral host factors that selectively regulate HCMV reproduction. We find that the scaffold subunit CNOT1 is specifically required for late viral gene expression and genome-wide host responses in CCR4-NOT-disrupted cells. By profiling poly(A)-tail lengths of individual HCMV and host mRNAs using nanopore direct RNA sequencing, we reveal poly(A)-tails of viral messages to be markedly longer than those of cellular mRNAs and significantly less sensitive to CCR4-NOT disruption. Our data establish that mRNA deadenylation by host CCR4-NOT is critical for productive HCMV replication and define a new mechanism whereby herpesvirus infection subverts cellular mRNA metabolism to remodel the gene expression landscape of the infected cell. Moreover, we expose an unanticipated host factor with potential to become a therapeutic anti-HCMV target.

Archaeological approaches to RNA virus evolution.

Studies with RNA enzymes (ribozymes) and protein enzymes have identified certain structural elements that are present in some cellular mRNAs and viral RNAs. These elements do not share a primary structure and, thus, are not phylogenetically related. However, they have common (secondary/tertiary) structural folds that, according to some lines of evidence, may have an ancient and common origin. The term 'mRNA archaeology' has been coined to refer to the search for such structural/functional relics that may be informative of early evolutionary developments in the cellular and viral worlds and have lasted to the present day. Such identified RNA elements may have developed as biological signals with structural and functional relevance (as if they were buried objects with archaeological value), and coexist with the standard linear information of nucleic acid molecules that is translated into proteins. However, there is a key difference between the methods that extract information from either the primary structure of mRNA or the signals provided by secondary and tertiary structures. The former (sequence comparison and phylogenetic analysis) requires strict continuity of the material vehicle of information during evolution, whereas the archaeological method does not require such continuity. The tools of RNA archaeology (including the use of ribozymes and enzymes to investigate the reactivity of the RNA elements) establish links between the concepts of communication and language theories that have not been incorporated into knowledge of virology, as well as experimental studies on the search for functionally relevant RNA structures.

In situ visualization of m6A sites in cellular mRNAs.

N 6-methyladenosine (m6A) is an abundant RNA modification which plays critical roles in RNA function and cellular physiology. However, our understanding of how m6A is spatially regulated remains limited due to a lack of methods for visualizing methylated transcripts of interest in cells. Here, we develop DART-FISH, a method for in situ visualization of specific m6A sites in target RNAs which enables simultaneous detection of both m6A-modified and unmodified transcript copies. We demonstrate the ability of DART-FISH to visualize m6A in a variety of mRNAs across diverse cell types and to provide information on the location and stoichiometry of m6A sites at single-cell resolution. Finally, we use DART-FISH to reveal that m6A is not sufficient for mRNA localization to stress granules during oxidative stress. This technique provides a powerful tool for examining m6A-modified transcript dynamics and investigating methylated RNA localization in individual cells.

Viruses as archaeological tools for uncovering ancient molecular relationships.

The entry of a virus into the host cell always implies the alteration of certain intracellular molecular relationships, some of which may involve the recovery of ancient cellular activities. In this sense, viruses are archaeological tools for identifying unexpressed activities in noninfected cells. Among these, activities that hinder virus propagation may represent cellular defense mechanisms, for example, activities that mutagenize the viral genome such as ADAR-1 or APOBEC activities. Instead, those that facilitate virus propagation can be interpreted as the result of viral adaptation to-or mimicking-cellular structures, enabling the virus to perform anthropomorphic activities, including hijacking, manipulating, and reorganizing cellular factors for their own benefit. The alternative we consider here is that some of these second set of cellular activities were already in the uninfected cell but silenced, under the negative control of the cell or lineage, and that they represent a necessary precondition for viral infection. For example, specifically loading an amino acid at the 3'-end of the mRNA of some plant viruses by aminoacyl-tRNA synthetases has proved essential for virus infection despite this reaction not occurring with cellular mRNAs. Other activities of this type are discussed here, together with the biological context in which they acquire a coherent meaning, that is, genetic latency and molecular conflict.

FRI025 Succinate Receptor Activation Increases Respiration In Human But Not Murine Brown Adipocytes

Abstract Disclosure: B.T. McNeill: None. A. Kelman: None. L.E. Ramage: None. K.J. Suchacki: None. S.J. Wakelin: None. N.M. Morton: None. R.H. Stimson: None. Brown adipose tissue (BAT) is a thermogenic tissue that generates heat to maintain body temperature. BAT activation is a potential therapeutic target for treating obesity-related cardiometabolic disease. We performed RNA sequencing of human primary brown and white adipocytes and identified SUCNR1, which encodes the succinate receptor, as highly differentially expressed (20-fold) in brown versus white adipocytes. Succinate is known to increase BAT thermogenesis, while SUCNR1 activation inhibits lipolysis in white adipose tissue (WAT). We hypothesized that SUCNR1 activation increases BAT thermogenesis in humans and in mice. To test this, we investigated the effect of succinate and the selective SUCNR1 agonist cis-epoxysuccinic acid (C-ESA) in human and murine primary brown/ beige adipocytes. In addition, we determined the metabolic effects of disruption of Sucnr1 in mice either fed control or a high fat (HFD) for 12 weeks and measured energy expenditure when housed at standard room temperature (21°C) and during cold exposure (4°C). In human brown and white adipocytes, succinate increased basal and noradrenaline-stimulated respiration, while C-ESA increased basal respiration only in human brown adipocytes without altering UCP1 mRNA levels. SUCNR1 activation reduced glycerol release only in human white adipocytes. Male (but not female) Sucnr1-/- mice exhibited impaired glucose tolerance versus Sucnr1+/+ littermates after 2 weeks of HFD, and greater fat mass was also observed in the Sucnr1-/- mice following chronic HFD. However, energy expenditure, locomotor activity and tail vein temperature were unchanged in male Sucnr1-/- mice either at 21°C or 4°C. In addition, expression of Ucp1 and Pgc1α in BAT and inguinal WAT were similar between genotypes. Sucnr1 deletion had no effect on fat mass or glucose tolerance in mice fed a control diet. In vitro, differentiated brown adipocytes from Sucnr1-/- mice demonstrated comparable cellular respiration and Ucp1 mRNA levels both basally and during noradrenaline stimulation to Sucnr1+/+ adipocytes. Succinate and C-ESA did not stimulate respiration in murine brown or white adipocytes. These data reveal that SUCNR1 activation enhances brown adipocyte respiration in human but not murine brown adipocytes, highlighting a novel pathway regulating BAT function in addition to differences between species. These data also reveal novel sex-specific differences in the metabolic effects of SUCNR1, although the mechanisms through which female mice are protected from the adverse metabolic effects of Sucnr1 deletion are unclear. SUCNR1 activation may be a novel therapeutic target to treat obesity and associated metabolic disease, but requires further research in humans to determine the specific mechanisms through which SUCNR1 regulates BAT thermogenesis. Presentation: Friday, June 16, 2023

SARS-CoV-2 protein NSP2 enhances microRNA-mediated translational repression.

Viruses use microRNAs (miRNAs) to impair the host antiviral response and facilitate viral infection by expressing their own miRNAs or co-opting cellular miRNAs. miRNAs inhibit translation initiation of their target mRNAs by recruiting the GIGYF2/4EHP translation repressor complex to the mRNA 5´-cap structure. We recently reported that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encoded non-structural protein 2 (NSP2) interacts with GIGYF2. This interaction is critical for blocking translation of the Ifn1-b mRNA that encodes the cytokine Interferon-ß, and thereby impairs the host antiviral response. However, it is not known whether NSP2 also affects miRNA-mediated silencing. Here, we demonstrate the pervasive augmentation of the miRNA-mediated translational repression of cellular mRNAs by NSP2. We show that NSP2 interacts with Argonaute 2, the core component of the miRNA-Induced Silencing Complex (miRISC) via GIGYF2 and enhances the translational repression mediated by natural miRNA binding sites in the 3´ UTR of cellular mRNAs. Our data reveal an additional layer of the complex mechanism by which SARS-CoV-2 and likely other coronaviruses manipulate the host gene expression program through co-opting the host miRNA-mediated silencing machinery.

The Helix-Loop-Helix motif of human EIF3A regulates translation of proliferative cellular mRNAs.

Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5' UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.

Profiling the polyadenylated transcriptome of extracellular vesicles with long-read nanopore sequencing

BackgroundWhile numerous studies have described the transcriptomes of extracellular vesicles (EVs) in different cellular contexts, these efforts have typically relied on sequencing methods requiring RNA fragmentation, which limits interpretations on the integrity and isoform diversity of EV-targeted RNA populations. It has been assumed that mRNA signatures in EVs are likely to be fragmentation products of the cellular mRNA material, and the extent to which full-length mRNAs are present within EVs remains to be clarified.ResultsUsing long-read nanopore RNA sequencing, we sought to characterize the full-length polyadenylated (poly-A) transcriptome of EVs released by human chronic myelogenous leukemia K562 cells. We detected 443 and 280 RNAs that were respectively enriched or depleted in EVs. EV-enriched poly-A transcripts consist of a variety of biotypes, including mRNAs, long non-coding RNAs, and pseudogenes. Our analysis revealed that 10.58% of all EV reads, and 18.67% of all cellular (WC) reads, corresponded to known full-length transcripts, with mRNAs representing the largest biotype for each group (EV = 58.13%, WC = 43.93%). We also observed that for many well-represented coding and non-coding genes, diverse full-length transcript isoforms were present in EV specimens, and these isoforms were reflective-of but often in different ratio compared to cellular samples.ConclusionThis work provides novel insights into the compositional diversity of poly-A transcript isoforms enriched within EVs, while also underscoring the potential usefulness of nanopore sequencing to interrogate secreted RNA transcriptomes.

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2.

Comparison of miRNA transcriptome of exosomes in three categories of somatic cells with derived iPSCs

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) through epigenetic manipulation. While the essential role of miRNA in reprogramming and maintaining pluripotency is well studied, little is known about the functions of miRNA from exosomes in this context. To fill this research gap,we comprehensively obtained the 17 sets of cellular mRNA transcriptomic data with 3.93 × 1010 bp raw reads and 18 sets of exosomal miRNA transcriptomic data with 2.83 × 107 bp raw reads from three categories of human somatic cells: peripheral blood mononuclear cells (PBMCs), skin fibroblasts(SFs) and urine cells (UCs), along with their derived iPSCs. Additionally, differentially expressed molecules of each category were identified and used to perform gene set enrichment analysis. Our study provides sets of comparative transcriptomic data of cellular mRNA and exosomal miRNA from three categories of human tissue with three individual biological controls in studies of iPSCs generation, which will contribute to a better understanding of donor cell variation in functional epigenetic regulation and differentiation bias in iPSCs.

In vitro reconstitution of SARS-CoV-2 Nsp1-induced mRNA cleavage reveals the key roles of the N-terminal domain of Nsp1 and the RRM domain of eIF3g.

SARS CoV-2 nonstructural protein 1 (Nsp1) is the major pathogenesis factor that inhibits host translation using a dual strategy of impairing initiation and inducing endonucleolytic cleavage of cellular mRNAs. To investigate the mechanism of cleavage, we reconstituted it in vitro on β-globin, EMCV IRES, and CrPV IRES mRNAs that use unrelated initiation mechanisms. In all instances, cleavage required Nsp1 and only canonical translational components (40S subunits and initiation factors), arguing against involvement of a putative cellular RNA endonuclease. Requirements for initiation factors differed for these mRNAs, reflecting their requirements for ribosomal attachment. Cleavage of CrPV IRES mRNA was supported by a minimal set of components consisting of 40S subunits and eIF3g's RRM domain. The cleavage site was located in the coding region 18 nt downstream from the mRNA entrance, indicating that cleavage occurs on the solvent side of the 40S subunit. Mutational analysis identified a positively charged surface on Nsp1's N-terminal domain (NTD) and a surface above the mRNA-binding channel on eIF3g's RRM domain that contain residues essential for cleavage. These residues were required for cleavage on all three mRNAs, highlighting general roles of the Nsp1 NTD and eIF3g's RRM domain in cleavage per se, irrespective of the mode of ribosomal attachment.

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.