Malfunction of pre-mRNA processing factors are linked to several human diseases including cancer and neurodegeneration. Here we report the identification of a de novo heterozygous missense mutation ...in the SNRPE gene (c.65T>C (p.Phe22Ser)) in a patient with non-syndromal primary (congenital) microcephaly and intellectual disability. SNRPE encodes SmE, a basal component of pre-mRNA processing U snRNPs. We show that the microcephaly-linked SmE variant is unable to interact with the SMN complex and as a consequence fails to assemble into U snRNPs. This results in widespread mRNA splicing alterations in fibroblast cells derived from this patient. Similar alterations were observed in HEK293 cells upon SmE depletion that could be rescued by the expression of wild type but not mutant SmE. Importantly, the depletion of SmE in zebrafish causes aberrant mRNA splicing alterations and reduced brain size, reminiscent of the patient microcephaly phenotype. We identify the EMX2 mRNA, which encodes a protein required for proper brain development, as a major mis-spliced down stream target. Together, our study links defects in the SNRPE gene to microcephaly and suggests that alterations of cellular splicing of specific mRNAs such as EMX2 results in the neurological phenotype of the disease.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Exogeneous messenger ribonucleic acid (mRNA) can be used as therapeutic and preventive medication. However, during the enzymatic production process, commonly called
transcription, by-products occur ...which can reduce the therapeutic efficacy of mRNA. One such by-product is double-stranded RNA (dsRNA). We therefore sought to limit the generation of dsRNA by-products during
transcription.
transcription was performed with a DNA template including a poly(A)-tail-encoding region, dinucleotide or trinucleotide cap analogs for cotranscriptional capping, and relevant nucleoside triphosphates. Concentrations of UTP or modified UTP (m1ΨTP) and GTP were reduced and fed over the course of the reaction. mRNA was analyzed for dsRNA contamination, yield of the reaction, RNA integrity, and capping efficiency before translational activity was assessed.
Limiting the steady-state level of UTP or m1ΨTP during the enzymatic reaction reduced dsRNA formation, while not affecting mRNA yield or RNA integrity. Capping efficiency was optimized with the use of a combined GTP and UTP or m1ΨTP feed, while still reducing dsRNA formation. Lower dsRNA levels led to higher protein expression from the corresponding mRNAs.
Low steady-state concentrations of UTP and GTP, fed in combination over the course of the
transcription reaction, produce mRNA with high capping and low levels of dsRNA formation, resulting in high levels of protein expression. This novel approach may render laborious purification steps to remove dsRNA unnecessary.
Microglia are the resident phagocytes of the brain that are responsible for the clearance of injured neurons, an essential step in subsequent tissue regeneration. How death signals are controlled ...both in space and time to attract these cells toward the site of injury is a topic of great interest. To this aim, we have used the optically transparent zebrafish larval brain and identified rapidly propagating Ca2+ waves that determine the range of microglial responses to neuronal cell death. We show that while Ca2+-mediated microglial responses require ATP, the spreading of intercellular Ca2+ waves is ATP independent. Finally, we identify glutamate as a potent inducer of Ca2+-transmitted microglial attraction. Thus, this real-time analysis reveals the existence of a mechanism controlling microglial targeted migration to neuronal injuries that is initiated by glutamate and proceeds across the brain in the form of a Ca2+ wave.
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► Death signals travel across the brain in the form of Ca2+ waves ► Ca2+ waves define which microglia are targeted to neuronal injuries ► Ca2+ waves provide positional information via ATP to P2Y12-expressing microglia ► Ca2+ wave formation and propagation depends on glutamate acting via NMDA receptors
Using real-time analysis of the developing zebrafish brain, Sieger et al. identify Ca2+ waves that travel across the brain to guide microglia, the resident phagocytes, to sites of neuronal cell death. Neuronal glutamate/NMDA-receptor signaling generates and propagates these waves, establishing the extracellular ATP guidance cue needed for microglia migration.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The repressor element silencing transcription factor (REST) plays a crucial role in the differentiation of neural progenitor cells (NPCs). Effector proteins of REST are C-terminal domain small ...phosphatases (CTDSPs), which reduce polymerase II activity on genes required for neurogenesis. miR-26b regulates neurogenesis in zebrafish by targeting ctdsp2 mRNA, but the molecular events triggered by this microRNA remain unknown. Here we show in a murine embryonic stem cell differentiation paradigm that inactivation of miR-26 family members disrupts the formation of neurons and astroglia and arrests neurogenesis at the neural progenitor level. We further show that miR-26 directly targets Rest, thereby inducing the expression of a large set of REST complex-repressed neuronal genes including miRs required for the induction of the neuronal gene expression program. Our data identify the miR-26 family as the trigger of a self-amplifying system required for neural differentiation that acts upstream of REST-controlled miRs.
The assembly of spliceosomal U snRNPs depends on the coordinated action of PRMT5 and SMN complexes in vivo. These trans‐acting factors enable the faithful delivery of seven Sm proteins onto snRNA and ...the formation of the common core of snRNPs. To gain mechanistic insight into their mode of action, we reconstituted the assembly machinery from recombinant sources. We uncover a stepwise and ordered formation of distinct Sm protein complexes on the PRMT5 complex, which is facilitated by the assembly chaperone pICln. Upon completion, the formed pICln‐Sm units are displaced by new pICln‐Sm protein substrates and transferred onto the SMN complex. The latter acts as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to prevent mis‐assembly and to ensure the transfer of Sm proteins to cognate RNA. Investigation of mutant SMN complexes provided insight into the contribution of individual proteins to these activities. The biochemical reconstitution presented here provides a basis for a detailed molecular dissection of the U snRNP assembly reaction.
Synopsis
By reconstituting the assembly of human U snRNPs in vitro, this study establishes the sequential action of distinct Sm protein complexes and provides a basis for understanding defects in Spinal Muscular Atrophy.
Reconstitution of the human snRNP assembly machinery from recombinant proteins.
The PRMT5 complex acts as a scaffold for the stepwise organization of Sm proteins by the assembly chaperone pICln.
Release of pICln‐Sm protein intermediates from the PRMT5 complex scaffold is feed‐forward‐driven by new pICln‐Sm protein precursors.
The SMN complex acts as a Brownian machine, which enables Sm proteins to proofread cognate snRNAs and catalyzes faithful snRNP assembly.
Spinal muscular atrophy‐causing mutations differentially affect snRNP assembly.
By reconstituting the assembly of human U snRNPs in vitro, this study establishes the sequential action of distinct Sm protein complexes and provides a basis for understanding the defects in spinal muscular atrophy.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
T cell responses play an important role in protection against beta-coronavirus infections, including SARS-CoV-2, where they associate with decreased COVID-19 disease severity and duration. To enhance ...T cell immunity across epitopes infrequently altered in SARS-CoV-2 variants, we designed BNT162b4, an mRNA vaccine component that is intended to be combined with BNT162b2, the spike-protein-encoding vaccine. BNT162b4 encodes variant-conserved, immunogenic segments of the SARS-CoV-2 nucleocapsid, membrane, and ORF1ab proteins, targeting diverse HLA alleles. BNT162b4 elicits polyfunctional CD4+ and CD8+ T cell responses to diverse epitopes in animal models, alone or when co-administered with BNT162b2 while preserving spike-specific immunity. Importantly, we demonstrate that BNT162b4 protects hamsters from severe disease and reduces viral titers following challenge with viral variants. These data suggest that a combination of BNT162b2 and BNT162b4 could reduce COVID-19 disease severity and duration caused by circulating or future variants. BNT162b4 is currently being clinically evaluated in combination with the BA.4/BA.5 Omicron-updated bivalent BNT162b2 (NCT05541861).
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•BNT162b4 encodes conserved, immunogenic segments of the SARS-CoV-2 N, M, and ORF1ab antigens•Mass spectrometry detects BNT162b4 encoded peptides bound to diverse HLA-I alleles•BNT162b4 elicits non-spike T cell responses in mice while maintaining spike immunity•BNT162b4 protects animals from severe disease and enhances viral clearance by BNT162b2
Adding non-spike targeting components to mRNA vaccination elicits promising T cell responses against SARS-CoV-2 variant strains in rodent models of COVID-19.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Repressor element 1-silencing transcription factor (REST) plays a crucial role in the differentiation of neural progenitor cells (NPCs). C-terminal domain small phosphatases (CTDSPs) are REST ...effector proteins that reduce RNA polymerase II activity on genes required for neurogenesis. miR-26b regulates neurogenesis in zebrafish by targeting ctdsp2 mRNA, but the molecular events triggered by this microRNA (miR) remain unknown. Here, we show in a murine embryonic stem cell differentiation paradigm that inactivation of miR-26 family members disrupts the formation of neurons and astroglia and arrests neurogenesis at the neural progenitor level. Furthermore, we show that miR-26 directly targets Rest, thereby inducing the expression of a large set of REST complex-repressed neuronal genes, including miRs required for induction of the neuronal gene expression program. Our data identify the miR-26 family as the trigger of a self-amplifying system required for neural differentiation that acts upstream of REST-controlled miRs.
The SARS-CoV-2 Omicron variant and its sublineages show pronounced viral escape from neutralizing antibodies elicited by vaccination or prior SARS-CoV-2 variant infection owing to over 30-amino acid ...alterations within the spike (S) glycoprotein. Breakthrough infection of vaccinated individuals with Omicron sublineages BA.1 and BA.2 is associated with distinct patterns of cross-neutralizing activity against SARS-CoV-2 variants of concern (VOCs). In continuation of our previous work, we characterized the effect of Omicron BA.4/BA.5 S glycoprotein exposure on the neutralizing antibody response upon breakthrough infection in vaccinated individuals and upon variant-adapted booster vaccination in mice. We found that immune sera from triple mRNA-vaccinated individuals with subsequent breakthrough infection during the Omicron BA.4/BA.5 wave showed cross-neutralizing activity against previous Omicron variants BA.1, BA.2, BA.2.12.1, and BA.4/BA.5 itself. Administration of a prototypic BA.4/BA.5-adapted mRNA booster vaccine to mice after SARS-CoV-2 wild-type strain-based primary immunization is associated with broader cross-neutralizing activity than a BA.1-adapted booster. Whereas the Omicron BA.1-adapted mRNA vaccine in a bivalent format (wild-type + BA.1) broadens cross-neutralizing activity relative to the BA.1 monovalent booster, cross-neutralization of BA.2 and descendants is more effective in mice boosted with a bivalent wild-type + BA.4/BA.5 vaccine. In naïve mice, primary immunization with the bivalent wild-type + Omicron BA.4/BA.5 vaccine induces strong cross-neutralizing activity against Omicron VOCs and previous variants. These findings suggest that, when administered as boosters, mono- and bivalent Omicron BA.4/BA.5-adapted vaccines enhance neutralization breadth and that the bivalent version also has the potential to confer protection to individuals with no preexisting immunity against SARS-CoV-2.
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