Early mRNA Research Articles

Alternative splicing in the genome of HPV and its regulation.

Persistent infection with high-risk human papillomavirus (HR-HPV) is the main cause of cervical cancer. These chronic infections are characterized by high expression of the HPV E6 and E7 oncogenes and the absence of the L1 and L2 capsid proteins. The regulation of HPV gene expression plays a crucial role in both the viral life cycle and rare oncogenic events. Alternative splicing of HPV mRNA is a key mechanism in post-transcriptional regulation. Through alternative splicing, HPV mRNA is diversified into various splice isoforms with distinct coding potentials, encoding multiple proteins and influencing the expression of HPV genes. The spliced mRNAs derived from a donor splicing site within the E6 ORF and one of the different acceptor sites located in the early mRNA contain E6 truncated mRNAs, named E6*. E6* is one of the extensively studied splicing isoforms. However, the role of E6* proteins in cancer progression remains controversial. Here, we reviewed and compared the alternative splicing events occurring in the genomes of HR-HPV and LR-HPV. Recently, new HPV alternative splicing regulatory proteins have been continuously discovered, and we have updated the regulation of HPV alternative splicing. In addition, we summarized the functions of known splice isoforms from three aspects: anti-tumorigenic, tumorigenic, and other cancer-related functions, including not only E6*, but also E6^E7, E8^E2, and so on. Comprehending their contributions to cancer development enhances insights into the carcinogenic mechanisms of HPV and explores the potential utility of alternative splicing in the diagnosis and treatment of cervical cancer.

Deciphering autism heterogeneity: a molecular stratification approach in four mouse models

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by impairments in social interaction and communication, as well as restrained or stereotyped behaviors. The inherent heterogeneity within the autism spectrum poses challenges for developing effective pharmacological treatments targeting core features. Successful clinical trials require the identification of robust markers to enable patient stratification. In this study, we identified molecular markers within the oxytocin and immediate early gene families across five interconnected brain structures of the social circuit. We used wild-type and four heterogeneous mouse models, each exhibiting unique autism-like behaviors modeling the autism spectrum. While dysregulations in the oxytocin family were model-specific, immediate early genes displayed widespread alterations, reflecting global changes across the four models. Through integrative analysis, we identified Egr1, Foxp1, Homer1a, Oxt, and Oxtr as five robust and discriminant molecular markers that allowed the successful stratification of the four models. Importantly, our stratification demonstrated predictive values when challenged with a fifth mouse model or identifying subgroups of mice potentially responsive to oxytocin treatment. Beyond providing insights into oxytocin and immediate early gene mRNA dynamics, this proof-of-concept study represents a significant step toward the potential stratification of individuals with ASD. This work has implications for the success of clinical trials and the development of personalized medicine in autism.

A sperm-enriched 5'fragment of tRNA-Valine regulates preimplantation embryonic transcriptome and development.

Sperm small RNAs have been implicated in intergenerational epigenetic inheritance of paternal environmental effects; however, their biogenesis and functions remain poorly understood. We previously identified a 5' fragment of tRNA-Valine-CAC-2 (tRFValCAC) as one of the most abundant small RNA in mature sperm. tRFValCAC is specifically enriched in sperm during post-testicular maturation in the epididymis, and we found that it is delivered to sperm from epididymis epithelial cells via extracellular vesicles. Here, we investigated the mechanistic basis of tRFValCAC delivery to sperm and its functions in the early embryo. We show that tRFValCAC interacts with an RNA binding protein, heterogeneous nuclear ribonucleoprotein A/B (hnRNPAB), in the epididymis, and this interaction regulates the sorting and packing of tRFValCAC into extracellular vesicles. In the embryo, we found that tRFValCAC regulates early embryonic mRNA processing and splicing. Inhibition of tRFValCAC in preimplantation embryos altered the transcript abundance of genes involved in RNA splicing and mRNA processing. Importantly, tRFValCAC-inhibited embryos showed altered mRNA splicing, including alternative splicing of various splicing factors and genes important for proper preimplantation embryonic development. Finally, we find that inhibition of tRFValCAC in zygotes delayed preimplantation embryonic development. Together, our results reveal a novel function of a sperm-enriched tRF in regulating alternating splicing and preimplantation embryonic development and shed light on the mechanism of sperm small RNA-mediated epigenetic inheritance.

Visualizing lipid nanoparticle trafficking for mRNA vaccine delivery in non-human primates.

mRNA delivered using lipid nanoparticles (LNPs) has become an important subunit vaccine modality, but mechanisms of action for mRNA vaccines remain incompletely understood. Here, we synthesized a metal chelator-lipid conjugate enabling positron emission tomography (PET) tracer labeling of LNP/mRNA vaccines for quantitative visualization of vaccine trafficking in live non-human primates (NHPs). Following i.m. injection, we observed LNPs distributing through injected muscle tissue, simultaneous with rapid trafficking to draining lymph nodes (dLNs). Deltoid injection of LNPs mimicking human vaccine administration led to stochastic LNP delivery to 3 different sets of dLNs. LNP uptake in dLNs was confirmed by histology, and cellular analysis of tissues via flow cytometry identified antigen-presenting cells as the primary cell type responsible for early LNP uptake and mRNA translation. These results provide insights into the biodistribution of mRNA vaccines administered at clinically relevant doses, injection volumes, and injection sites in an important large animal model for vaccine development.

Dgcr8 functions in the secondary heart field for outflow tract and right ventricle development in mammals

The DGCR8 gene, encoding a critical miRNA processing protein, maps within the hemizygous region in patients with 22q11.2 deletion syndrome. Most patients have malformations of the cardiac outflow tract that is derived in part from the anterior second heart field (aSHF) mesoderm. To understand the function of Dgcr8 in the aSHF, we inactivated it in mice using Mef2c-AHF-Cre. Inactivation resulted in a fully penetrant persistent truncus arteriosus and a hypoplastic right ventricle leading to lethality by E14.5. To understand the molecular mechanism for this phenotype, we performed gene expression profiling of the aSHF and the cardiac outflow tract with right ventricle in conditional null versus normal mouse littermates at stage E9.5 prior to morphology changes. We identified dysregulation of mRNA gene expression, of which some are relevant to cardiogenesis. Many pri-miRNA genes were strongly increased in expression in mutant embryos along with reduced expression of mature miRNA genes. We further examined the individual, mature miRNAs that were decreased in expression along with pri-miRNAs that were accumulated that could be direct effects due to loss of Dgcr8. Among these genes, were miR-1a, miR-133a, miR-134, miR143 and miR145a, which have known functions in heart development. These early mRNA and miRNA changes may in part, explain the first steps that lead to the resulting phenotype in Dgcr8 aSHF conditional mutant embryos.

Highly dynamic inflammatory and excitability transcriptional profiles in hippocampal CA1 following status epilepticus

Transient brain insults including status epilepticus (SE) can initiate a process termed ‘epileptogenesis’ that results in chronic temporal lobe epilepsy. As a consequence, the entire tri-synaptic circuit of the hippocampus is fundamentally impaired. A key role in epileptogenesis has been attributed to the CA1 region as the last relay station in the hippocampal circuit and as site of aberrant plasticity, e.g. mediated by acquired channelopathies. The transcriptional profiles of the distinct hippocampal neurons are highly dynamic during epileptogenesis. Here, we aimed to elucidate the early SE-elicited mRNA signature changes and the respective upstream regulatory cascades in CA1. RNA sequencing of CA1 was performed in the mouse pilocarpine-induced SE model at multiple time points ranging from 6 to 72 h after the initial insult. Bioinformatics was used to decipher altered gene expression, signalling cascades and their corresponding cell type profiles. Robust transcriptomic changes were detected at 6 h after SE and at subsequent time points during early epileptogenesis. Major differentially expressed mRNAs encoded primarily immediate early and excitability-related gene products, as well as genes encoding immune signalling factors. Binding sites for the transcription factors Nfkb1, Spi1, Irf8, and two Runx family members, were enriched within promoters of differentially expressed genes related to major inflammatory processes, whereas the transcriptional repressors Suz12, Nfe2l2 and Rest were associated with hyperexcitability and GABA / glutamate receptor activity. CA1 quickly responds to SE by inducing transcription of genes linked to inflammation and excitation stress. Transcription factors mediating this transcriptomic switch represent targets for new highly selected, cell type and time window-specific anti-epileptogenic strategies.

Fatigue, Toll-Like Receptor 4, and Pro-Inflammatory Cytokines in Adults With Subarachnoid Hemorrhage: A 6-Month Longitudinal Study.

Fatigue is prevalent in subarachnoid hemorrhage (SAH) survivors. Biological mechanisms underlying fatigue post-SAH are not clear. Inflammation may contribute to the development of fatigue. This study aimed to examine the associations between inflammatory markers and fatigue during the first 6months post-SAH. Specific biomarkers examined included both early and concurrent expression of Toll-Like Receptor 4 (TLR4) messenger RNA (mRNA) and plasma concentrations of pro-inflammatory cytokines, Tumor Necrosis Factor-alpha (TNF-α), Interleukin (IL)1β, and IL6. We conducted a 6-month longitudinal study with a convenience sample of 43 SAH survivors. We collected blood samples on days 2, 3, and 7 and 2, 3, and 6months post-SAH to assess biomarkers. Fatigue was assessed by the PROMIS Fatigue Scale at 2, 3, and 6months. Linear mixed models were used to test the associations between early (days 2, 3, and 7) and concurrent (2, 3, and 6months) TLR4 mRNA expression (TagMan gene expression assays) and TNF-α, IL1β, and IL6 plasma concentrations (multiplex assays) and concurrent fatigue. 28% of SAH survivors experienced fatigue during the first 6months post-SAH. Fatigue levels in SAH survivors were higher than those of the U.S. population and consistent during the 6months. Experience of fatigue during the 6months post-SAH was associated with higher IL1β plasma concentrations on day 7 and IL1β, IL6, and TNF-α plasma concentrations during the 6months post-SAH. Inflammation appears to underlie the development of fatigue in SAH survivors.

Epstein‒Barr virus and human herpesvirus 6 infection in patients with systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is a complex autoimmune disease, and the etiology is still unclear. Some studies have indicated that viral infection might contribute to the development of SLE.MethodsA total of 105 individuals with SLE and 110 matched healthy controls were tested for EBV-specific DNA fragments in peripheral blood monocytes by PCR-Southern blotting. The expression of EBV-encoded genes was determined by RT-PCR and Southern blotting in EBV-positive patients. Serum EBV-specific IgM antibody was determined by ELISA. HHV-6 DNA in peripheral blood monocytes of those SLE patients and normal controls was tested by nested PCR.ResultsStatistical analysis showed that the EBV-positive rate of SLE patients was significantly higher than that of the control group (χ2 = 87.329, P = 0), while the difference in the HHV-6-positive rate between the two groups was not significant (P > 0.05). An association of EBV and HHV-6 positivity in SLE patients was found (P = 0, r = 0.38). The EBV IgM level was significantly higher in SLE patients than in healthy controls (χ2 = 25.184, P = 0). Forty-two of the 75 EBV DNA-positive specimens were positive for EBNA2 mRNA, and an association between EBV EBNA2 mRNA and anti-Sm antibody positivity was found (P = 0, r = 0.409). LMP1 mRNA was positive in 2 SLE patients with active phase, and no LMP2A mRNA expression was detected in EBV DNA-positive specimens. EBV early gene BARF1 mRNA was detected in 2 cases of EBV-positive SLE patients, and these 2 patients were also HHV-6 DNA positive. Thirty-eight patients were BcLF1 mRNA positive, and 33 of them were HHV-6 positive as well. These factors were associated (χ2 = 15.734, P = 0). The expression of the EBV immediate early gene BZLF1 was negative in all 75 EBV-positive SLE patients.ConclusionsThe results suggest that EBV infection might be related to the occurrence of SLE. Although there is no direct evidence that HHV-6 infection is associated with the development of SLE, EBV and HHV-6 infection may have a coacceleration effect in SLE patients. This study provides a new theoretical and experimental basis for the study of viral etiology and the prevention and treatment of SLE.

Clinical efficacy and Long-term Immunogenicity of early triple dose COVID-19 mRNA vaccine in cancer patients

Clinical efficacy and Long-term Immunogenicity of early triple dose COVID-19 mRNA vaccine in cancer patients

Lipid metabolism mRNA expression and cellularity of intramuscular adipocytes within the Longissimus muscle of Angus- and Wagyu-sired cattle fed for a similar days on feed or body weight endpoint

This study investigates intramuscular (IM) adipocyte development in the Longissimus muscle (LM) between Wagyu- and Angus-sired steers compared at a similar age and days on feed (D) endpoint or similar body weight (B) endpoint by measuring IM adipocyte cell area and lipid metabolism mRNA expression. Angus-sired steers (AN, n = 6) were compared with steers from two different Wagyu sires (WA), selected for either growth (G) or marbling (M), to be compared at a similar days on feed (DOF; 258 ± 26.7 d; WA-GD, n = 5 and WA-MD, n = 5) in Exp. 1 or body weight (BW; 613 ± 18.0 kg; WA-GB, n = 4 and WA-MB, n = 5) in Exp. 2, respectively. In Exp. 1, WA-MD steers had a greater (P ≤ 0.01) percentage of IM fat in the LM compared with AN and WA-GD steers. In Exp. 2, WA-MB steers had a greater (P ≤ 0.01) percentage of IM fat in the LM compared with AN and WA-GB steers. The distribution of IM adipocyte area was unimodal at all biopsy collections, with IM adipocyte area becoming progressively larger as cattle age (P ≤ 0.01) and BW increased (P ≤ 0.01). Peroxisome proliferator activated receptor delta (PPARd) was upregulated earlier for WA-MD and WA-MB cattle compared with other steers at a similar DOF and BW (P ≤ 0.02; treatment × biopsy interaction). Peroxisome proliferator activated receptor gamma was upregulated (PPARg) at a lesser BW for WA-MB steers (P = 0.09; treatment × biopsy interaction), while WA-MD steers had a greater (P ≤ 0.04) overall mean PPARg mRNA expression compared with other steers. Glycerol-3-phosphate acyltransferase, lipin 1, and hormone sensitive lipase demonstrated mRNA expression patterns similar to PPARg and PPARd or CCAAT enhancer binding protein beta, which emphasizes their importance in marbling development and growth. Additionally, WA-MD and WA-MB steers often had a greater early mRNA expression of fatty acid transporters (fatty acid transport protein 1; P < 0.02; treatment × biopsy interaction) and binding proteins (fatty acid binding protein 4) compared with other steers. Cattle with a greater marbling propensity appear to upregulate adipogenesis at a younger chronological and physiological maturity through PPARd, PPARg, and possibly adipogenic regulating compounds, lysophosphatidic acid, and diacylglycerol. These genes and compounds could be used as potential markers for marbling propensity of cattle in the future.

Global Transcriptome Analyses of Cellular and Viral mRNAs during HAdV-C5 Infection Highlight New Aspects of Viral mRNA Biogenesis and Cytoplasmic Viral mRNA Accumulations.

It is well established that human adenoviruses such as species C, types 2 and 5 (HAdV-C2 and HAdV-C5), induce a nearly complete shutoff of host-cell protein synthesis in the infected cell, simultaneously directing very efficient production of viral proteins. Such preferential expression of viral over cellular genes is thought to be controlled by selective nucleocytoplasmic export and translation of viral mRNA. While detailed knowledge of the regulatory mechanisms responsible for the translation of viral mRNA is available, the viral or cellular mechanisms of mRNA biogenesis are not completely understood. To identify parameters that control the differential export of viral and cellular mRNAs, we performed global transcriptome analyses (RNAseq) and monitored temporal nucleocytoplasmic partitioning of viral and cellular mRNAs during HAdV-C5 infection of A549 cells. Our analyses confirmed previously reported features of the viral mRNA expression program, as a clear shift in viral early to late mRNA accumulation was observed upon transition from the early to the late phase of viral replication. The progression into the late phase of infection, however, did not result in abrogation of cellular mRNA export; rather, viral late mRNAs outnumbered viral early and most cellular mRNAs by several orders of magnitude during the late phase, revealing that viral late mRNAs are not selectively exported but outcompete cellular mRNA biogenesis.

Adipocyte commitment of 3T3-L1 cells is required to support human adenovirus 36 productive replication concurrent with altered lipid and glucose metabolism.

Human adenovirus 36 (HAdV-D36) can cause obesity in animal models, induces an adipogenic effect and increased adipocyte differentiation in cell culture. HAdV-D36 infection alters gene expression and the metabolism of the infected cells resulting in increased glucose internalization and triglyceride accumulation. Although HAdV-D36 prevalence correlates with obesity in humans, whether human preadipocytes may be targeted in vivo has not been determined and metabolic reprogramming of preadipocytes has not been explored in the context of the viral replication cycle. HAdV-D36 infection of the mouse fibroblasts, 3T3-L1 cells, which can differentiate into adipocytes, promotes proliferation and differentiation, but replication of the virus in these cells is abortive as indicated by short-lived transient expression of viral mRNA and a progressive loss of viral DNA. Therefore, we have evaluated whether a productive viral replication cycle can be established in the 3T3-L1 preadipocyte model under conditions that drive the cell differentiation process. For this purpose, viral mRNA levels and viral DNA replication were measured by RT-qPCR and qPCR, respectively, and viral progeny production was determined by plaque assay. The lipogenic effect of infection was evaluated with Oil Red O (ORO) staining, and expression of genes that control lipid and glucose metabolism was measured by RT-qPCR. In the context of a viral productive cycle, HAdV-D36 modulated the expression of the adipogenic genes, C/EBPα, C/EBPβ and PPARγ, as well as intracellular lipid accumulation, and the infection was accompanied by altered expression of glucolytic genes. The results show that only adipocyte-committed 3T3-L1 cells are permissive for the expression of early and late viral mRNAs, as well as viral DNA replication and progeny production, supporting productive HAdV-D36 viral replication, indicating that a greater effect on adipogenesis occurs in adipocytes that support productive viral replication.

DExD/H Box Helicases DDX24 and DDX49 Inhibit Reactivation of Kaposi's Sarcoma Associated Herpesvirus by Interacting with Viral mRNAs.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that is the causative agent of primary effusion lymphoma and Kaposi’s sarcoma. In healthy carriers, KSHV remains latent, but a compromised immune system can lead to lytic viral replication that increases the probability of tumorigenesis. RIG-I-like receptors (RLRs) are members of the DExD/H box helicase family of RNA binding proteins that recognize KSHV to stimulate the immune system and prevent reactivation from latency. To determine if other DExD/H box helicases can affect KSHV lytic reactivation, we performed a knock-down screen that revealed DHX29-dependent activities appear to support viral replication but, in contrast, DDX24 and DDX49 have antiviral activity. When DDX24 or DDX49 are overexpressed in BCBL-1 cells, transcription of all lytic viral genes and genome replication were significantly reduced. RNA immunoprecipitation of tagged DDX24 and DDX49 followed by next-generation sequencing revealed that the helicases bind to mostly immediate-early and early KSHV mRNAs. Transfection of expression plasmids of candidate KSHV transcripts, identified from RNA pull-down, demonstrated that KSHV mRNAs stimulate type I interferon (alpha/beta) production and affect the expression of multiple interferon-stimulated genes. Our findings reveal that host DExD/H box helicases DDX24 and DDX49 recognize gammaherpesvirus transcripts and convey an antiviral effect in the context of lytic reactivation.

Control of immediate early gene expression by CPEB4-repressor complex-mediated mRNA degradation

BackgroundCytoplasmic polyadenylation element-binding protein 4 (CPEB4) is known to associate with cytoplasmic polyadenylation elements (CPEs) located in the 3′ untranslated region (UTR) of specific mRNAs and assemble an activator complex promoting the translation of target mRNAs through cytoplasmic polyadenylation.ResultsHere, we find that CPEB4 is part of an alternative repressor complex that mediates mRNA degradation by associating with the evolutionarily conserved CCR4-NOT deadenylase complex. We identify human CPEB4 as an RNA-binding protein (RBP) with enhanced association to poly(A) RNA upon inhibition of class I histone deacetylases (HDACs), a condition known to cause widespread degradation of poly(A)-containing mRNA. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis using endogenously tagged CPEB4 in HeLa cells reveals that CPEB4 preferentially binds to the 3′UTR of immediate early gene mRNAs, at G-containing variants of the canonical U- and A-rich CPE located in close proximity to poly(A) sites. By transcriptome-wide mRNA decay measurements, we find that the strength of CPEB4 binding correlates with short mRNA half-lives and that loss of CPEB4 expression leads to the stabilization of immediate early gene mRNAs. Akin to CPEB4, we demonstrate that CPEB1 and CPEB2 also confer mRNA instability by recruitment of the CCR4-NOT complex.ConclusionsWhile CPEB4 was previously known for its ability to stimulate cytoplasmic polyadenylation, our findings establish an additional function for CPEB4 as the RNA adaptor of a repressor complex that enhances the degradation of short-lived immediate early gene mRNAs.

Relative Expression of mRNA BARF1 Epstein-Barr Virus from Tumor Tissue Biopsy in Formalin-Fixed Paraffin-Embedded in Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from nasopharyngeal epithelial cells. NPC is endemic in some regions, especially in Southeast Asia countries. NPC is also a multifactorial disease involving environmental factors, genetic factors, and infection from Epstein-Barr virus (EBV). According to WHO classification, Undifferentiated NPC is histopathologically associated with EBV infection and categorized into WHO subtype 3. EBV has two phases in its infection cycle: the lytic and latent phases. The BARF1 gene is a mediator of the transition from the latent phase to the lytic phase. Previous studies suggest measurement of EBV mRNA activity at the primary tumor site in the nasopharyngeal reflects the pathogenesis of NPC rather than measuring circulating EBV DNA or serological diagnosis. This study aimed to determine the relative expression potential of BARF1 mRNA at different tumor stages in NPC patients as a predictor of NPC pathogenesis. This research design was a descriptive research method in the form of a cross-sectional study. The samples used were 22 patients diagnosed as NPC WHO class III at the Anatomical Pathology Section of Prof. Dr. Margono Soekarjo, Purwokerto, who met the inclusion criteria. The relative expression of BARF1 mRNA was carried out using the one-step real-time RT-PCR technique and then calculated using a formula of 2-ΔCt. The T-test was used to compare the relative expression of early and late-stage BARF1 mRNA. The relative expression of BARF1 mRNA in the late-stage advanced stage (n = 6; 0.708292-0.840177; med = 0.7164655) was increased compared to the early stage (n = 2; 0.708841-0.712423; med = 0. 710632).

B Cells Specific CpG Induces High IL-10 and IL-6 Expression In Vitro in Neuro-Behçet's Disease.

Remitting-RelapsingMultiple Sclerosis (RRMS) and Neuro-Behçet Disease (NBD) are two chronic neuroinflammatory disorders leading to neurological damage. Herein, we investigated in these patients the IL-10-producing cells during the early stages of these disorders. Cellular and molecular investigations were carried out on treatment naive patients suffering from RRMS and NBD recruited at the first episode of clinical relapse. Our findings demonstrate that CSF-B cells from NBD patients, but not RRMS, are the major source of intrathecal IL-10 as compared to T-CD4 cells. Moreover, we showed a lower expression of TGF-β and IL35, in the CSF cells of NBD patients as compared to the control group. Specific in vitro CpG stimulation of peripheral blood B cells from NBD patients resulted in a concomitant early mRNA expression of IL6 and IL10 but was limited to IL10 for RRMS patients. Furthermore, mRNA expression of IL-6 and IL-10 receptors was assessed and intriguingly IL6ST receptor subunit was significantly lower in NBD CSF, but not RRMS while IL10RB was increased in both. Deciphering the role of increased IL-10-producing B cells and IL10RB despite relapsing disease as well as the discordant expression of IL6 and IL6ST may pave the way for a better understanding of the pathophysiology of these neuro-inflammatory disorders.

Developing mRNA for Therapy.

Messenger RNA was discovered in 1961 and it took 60 years until the first mRNA became FDA-approved product in the form of COVID-19 mRNA vaccine. During those years a lot of progress has been made by hundreds of scientists. It was 1978 when the first-time isolated mRNA delivered into mammalian cells produced the encoded protein. In vitro transcription introduced in 1984 made it possible to generate any desired mRNA from the encoding plasmid using phage RNA polymerases. In the early 90s mRNA was used for therapy as well as vaccine against infectious diseases and cancer. Inflammatory nature of the mRNAs limited their in vivo use. Replacing uridine with pseudouridine made the mRNA non-immunogenic, more stable and highly translatable. Delivery of the lipid nanoparticle-formulated nucleoside-modified mRNA encoding viral antigens became a platform for effective vaccine. Labile nature of the mRNA is ideal for transient production of the viral antigen, to generate effective antibody and cellular immune response. The mRNA platform is revolutionizing the delivery of effective and safe vaccines, therapeutics and gene therapies.

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

Structural basis for the recognition of the S2, S5-phosphorylated RNA polymerase II CTD by the mRNA anti-terminator protein hSCAF4.

The C-terminal domain (CTD) of RNA polymerase II serves as a binding platform for numerous enzymes and transcription factors involved in nascent RNA processing and the transcription cycle. The S2, S5-phosphorylated CTD is recognized by the transcription factor SCAF4, which functions as a transcription anti-terminator by preventing early mRNA transcript cleavage and polyadenylation. Here, we measured the binding affinities of differently modified CTD peptides by hSCAF4 and solved the complex structure of the hSCAF4-CTD-interaction domain (CID) bound to a S2, S5-quadra-phosphorylated CTD peptide. Our results revealed that the S2, S5-quadra-phosphorylated CTD peptide adopts a trans conformation and is located in a positively charged binding groove of hSCAF4-CID. Although hSCAF4-CID has almost the same binding pattern to the CTD as other CID-containing proteins, it preferentially binds to the S2, S5-phosphorylated CTD. Our findings provide insight into the regulatory mechanism of hSCAF4 in transcription termination.

Single Cell Analysis Elucidates the Maturation of Human Stem and Progenitor Cell Function from Fetal through Adult Hematopoiesis

Hematopoiesis continually replenishes the supply of circulating blood cells from embryonic development through the entirety of human lifespan. Although all hematopoietic lineages are produced throughout life, biases in lineage output occur at various stages, including lymphoid bias in childhood and myeloid bias in adulthood. Furthermore, many blood disorders demonstrate marked biases in age of onset, such as bone marrow failure disorders, clonal hematopoiesis of indeterminate potential, and numerous hematologic malignancies. A lack of insight into the normal physiologic changes occurring in the hematopoietic stem and progenitor cell (HSPC) compartments during development and maturation fundamentally limits our understanding of how age biased blood disorders arise. Two major unresolved questions are: (1) what changes in the molecular regulation of hematopoietic lineage commitment occur over the course of human life? And (2) are certain HSPC states present only during specific ages of life, and if so, do age-specific HSPCs have distinct biology?To address these questions we performed single cell RNA sequencing (scRNAseq) on human HSPCs from first and second trimester fetal liver hematopoiesis, and bone marrow hematopoiesis spanning childhood into mature adulthood. In total, HSPC samples were obtained from 14 distinct human donors. Dimensionality reduction and marker gene analysis identified uncommitted hematopoietic stem cells (HSCs) and the developmental trajectories of each lineage emanating from multipotent HSCs. We then identified the genes activated upon commitment to each hematopoietic lineage during fetal, childhood, and mature adult hematopoiesis using the Population Balance Analysis and Stationary Optimal Transport algorithms, followed by Elastic Net gene regression. For each lineage we determined the putative transcription factor network that is consistently active in driving commitment to that lineage throughout life, but surprisingly also found the existence of adjunctive transcription factor networks that only drove lineage commitment at specific ages.We next used unbiased clustering of scRNAseq data to identify 21 distinct subtypes in the HSPC compartment across human life. Using marker gene analysis and singleCellNet algorithm comparisons to an existing human adult bone marrow scRNAseq data set, we hierarchically ordered and annotated these HSPC subtypes ranging from uncommitted HSCs to lineage committed progenitor cells. We found that cellular distribution within the HSPC compartment amongst these subtypes varied markedly throughout human lifetime, with higher representation of HSCs in fetal life, predominance of lymphoid progenitors in childhood, and higher representation of myeloid progenitors in adulthood. Focusing on the distribution of cells among HSC subtypes over human life, we identified an HSC subtype exclusive to mid-gestation that was not present in early fetal or postnatal timepoints. This HSC subtype had a characteristic immunophenotype and was enriched for expression of early response transcription factors and mRNA decay factors. We functionally validated that this mid-gestation-specific HSC subtype was phenotypically unique using colony formation assays and xenotransplantation. Mid-gestation-specific HSCs were more clonogenic with a greater number of multi-lineage outcomes, and also demonstrated increased multilineage engraftment capacity compared to other HSC subtypesOur findings reveal that the intrinsic biology of hematopoietic lineage commitment fundamentally changes over the course of the human lifetime, and define and validate age-specific HSPC subtypes. In particular, the biology of the mid-gestation-specific HSC we identified has potential applications for improving engraftment and multi-lineage reconstitution in hematopoietic cell transplantation. DisclosuresRegev: Genentech: Current Employment; Celsius Therapeutics: Current equity holder in publicly-traded company, Other: Co-founder; Immunitas: Current equity holder in publicly-traded company; ThermoFisher Scientific: Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Neogene Therapeutics: Membership on an entity's Board of Directors or advisory committees; Asimov: Membership on an entity's Board of Directors or advisory committees.