B52 promotes alternative splicing of Dscam in Chinese mitten crab, Eriocheir sinensis.

B52 is a member of the classical serine/arginine (SR)-rich proteins, which are phylogenetically conserved and play significant roles in mRNA maturation, including alternative splicing. In the present study, the docking site, selector sequences and locus control region of the Chinese mitten crab (Eriocheir sinensis) Down syndrome cell adhesion molecule (EsDscam) were identified. Alternative splicing of Dscam is essential to generate different isoforms. We also isolated and characterised the B52 gene from E. sinensis (EsB52). The 876 bp open reading frame of EsB52 encodes a 291 amino acid residue polypeptide, and EsB52 has two RNA recognition motifs (RRMs) at the N-terminus and an arginine/serine-rich domain at the C-terminus. Each RRM contains two degenerate short submotifs, RNP-1 and RNP2. Analysis of tissue distribution revealed that EsB52 mRNA expression was widespread in all tested tissues, and especially high in brain and hemocytes. In hemocytes, EsB52 was upregulated significantly after stimulation with pathogen-associated molecular patterns and bacteria. Furthermore, EsB52 RNAi decreased the number of Ig7 inclusion in mRNA rather than Ig2 or Ig3. Taken together, these findings suggest that EsB52 acts as an alternative splicing activator of EsDscam.

JC polyomavirus expression and bell-shaped regulation of its SF2/ASF suppressor during the follow-up of multiple sclerosis patients treated with natalizumab.

Natalizumab is effective against multiple sclerosis (MS), but is associated with progressive multifocal leukoencephalopathy (PML), fatal disease caused by the JCV polyomavirus. The SF2/ASF (splicing factor2/alternative splicing factor) inhibits JCV in glial cells. We wondered about SF2/ASF modulation in the blood of natalizumab-treated patients and if this could influence JCV reactivation. Therefore, we performed a longitudinal study of MS patients under natalizumab, in comparison to patients under fingolimod and to healthy blood donors. Blood samples were collected at time intervals. The expression of SF2/ASF and the presence and expression of JCV in PBMC were analyzed. A bell-shaped regulation of SF2/ASF was observed in patients treated with natalizumab, increased in the first year of therapy, and reduced in the second one, while slightly changed, if any, in patients under fingolimod. Notably, SF2/ASF was up-regulated, during the first year, only in JCV DNA-positive patients, or with high anti-JCV antibody response; the expression of the JCV T-Ag protein in circulating B cells was inversely related to SF2/ASF protein expression. The SF2/ASF reduction, parallel to JCV activation, during the second year of therapy with natalizumab, but not with fingolimod, may help explain the increased risk of PML after the second year of treatment with natalizumab, but not with fingolimod. We propose that SF2/ASF has a protective role against JCV reactivation in MS patients. This study suggests new markers of disease behavior and, possibly, help in re-evaluations of therapy protocols.

Immune suppression of JC virus gene expression is mediated by SRSF1.

Progressive multifocal leukoemcephalopathy (PML) is a fatal demyelinating disease caused by the human neurotropic JC virus (JCV). JCV infects the majority of the human population during childhood and establishes a latent/persistent life-long infection. The virus reactivates under immunosuppressive conditions by unknown mechanisms, resulting in productive infection of oligodendrocytes in the central nervous system (CNS). Given the fact that the natural occurrence of PML is strongly associated with immunosuppression, the functional and molecular interaction between glial cells and neuroimmune signaling mediated by soluble immune mediators is likely to play a major role in reactivation of JCV and the progression of the lytic viral life cycle leading to the development of PML. In order to explore the effect of soluble immune mediators secreted by peripheral blood mononuclear cells (PBMCs) on JCV transcription, primary human fetal glial (PHFG) cells were treated with conditioned media from PBMCs. We observed a strong suppression of JCV early as well as late gene transcription in cells treated with conditioned media from induced PBMCs. Using a variety of virological and molecular biological approaches, we demonstrate that immune mediators secreted by PBMCs induce the expression of SRSF1, a strong inhibitor of JCV gene expression, and inhibit the replication of JCV. Our results show that downregulation of SRSF1 in glial cells overcomes the suppression of JCV gene expression and its replication mediated by soluble immune mediators. These findings suggest the presence of a novel immune signaling pathway between glial cells and PBMCs that may control JCV gene expression during the course of viral reactivation.

Splicing factor SRSF1 is essential for CD8 T cell function and host antigen-specific viral immunity.

Cytotoxic CD8 T cells are crucial for the host antigen-specific immune response to viral pathogens. Here we report the identification of an essential role for the serine/arginine-rich splicing factor (SRSF) 1 in CD8 T cell homeostasis and function. Specifically, SRSF1 is necessary for the maintenance of normal CD8 T lymphocyte numbers in the lymphoid compartment, and for the proliferative capacity and cytotoxic function of CD8 T cells. Furthermore, SRSF1 is required for antigen-specific IFN-γ cytokine responses in a viral infection challenge in mice. Transcriptomics analyses of Srsf1-deficient T cells reveal that SRSF1 controls proliferation, MAP kinase signaling and IFN signaling pathways. Mechanistically, SRSF1 controls the expression and activity of the Mnk2/p38-MAPK axis at the molecular level. Our findings reveal previously unrecognized roles for SRSF1 in the physiology and function of cytotoxic CD8 T lymphocytes and a potential molecular mechanism in viral immunopathogenesis.

Splicing factor SRSF1 controls distinct molecular programs in regulatory and effector T cells implicated in systemic autoimmune disease.

Systemic autoimmune diseases are characterized by hyperactive effector T cells (Teffs), aberrant cytokines and chemokines, and dysfunctional regulatory T cells (Tregs). We previously uncovered new roles for serine/arginine-rich splicing factor 1 (SRSF1) in the control of genes involved in T cell signaling and cytokine production in human T cells. SRSF1 levels are decreased in T cells from patients with systemic lupus erythematosus (SLE), and low levels correlate with severe disease. Moreover, T cell-conditional Srsf1-deficient mice recapitulate the autoimmune phenotype, exhibiting CD4 T cell hyperactivity, dysfunctional Tregs, systemic autoimmunity, and tissue inflammation. However, the role of SRSF1 in controlling molecular programs in Teffs and Tregs and how these pathways are implicated in autoimmunity is not known. Here, by comparative bioinformatics analysis, we demonstrate that SRSF1 controls largely distinct gene programs in Tregs and Teffs in vivo. SRSF1 regulates 189 differentially expressed genes (DEGs) unique to Tregs, 582 DEGs unique to Teffs, and 29 DEGs shared between both. Shared genes included IL-17A, IL-17F, CSF1, CXCL10, and CXCR4, and were highly enriched for inflammatory response and cytokine-cytokine receptor interaction pathways. SRSF1 controls distinct pathways in Tregs, which include chemokine signaling and immune cell differentiation, compared with pathways in Teffs, which include cytokine production, T cell homeostasis, and activation. We identified putative mRNA binding targets of SRSF1 which include CSF1, CXCL10, and IL-17F. Finally, comparisons with transcriptomics profiles from lupus-prone MRL/lpr mice reveal that SRSF1 controls genes and pathways implicated in autoimmune disease. The target genes of SRSF1 and putative binding targets we discovered, have known roles in systemic autoimmunity. Our findings suggest that SRSF1 controls distinct molecular pathways in Tregs and Teffs and aberrant SRSF1 levels may contribute to their dysfunction and immunopathogenesis of systemic autoimmune disease.

Characterization of a Broadly Neutralizing Monoclonal Antibody against SARS-CoV-2 Variants.

The constant mutation of SARS-CoV-2 has led to the emergence of new variants, which call for urgent effective therapeutic interventions. The trimeric spike (S) protein of SARS-CoV-2 is highly immunogenic with the receptor-binding domain (RBD) that binds first to the cellular receptor angiotensin-converting enzyme 2 (ACE2) and is therefore the target of many neutralizing antibodies. In this study, we characterized a broadly neutralizing monoclonal antibody (mAb) 9G8, which shows potent neutralization against the authentic SARS-CoV-2 wild-type (WT), Alpha (B.1.1.7), and Delta (1.617.2) viruses. Furthermore, mAb 9G8 also displayed a prominent neutralizing efficacy in the SARS-CoV-2 surrogate virus neutralization test (sVNT) against the Epsilon (B.1.429/7), Kappa (B.1.617.1), Gamma (P.1), Beta (B.1.351), and Delta Plus (1.617.2.1) RBD variants in addition to the variants mentioned above. Based on our in vitro escape mutant studies, we proved that the mutations V483F and Y489H within the RBD were involved in ACE2 binding and caused the neutralizing evasion of the virus from mAb 9G8. The development of such a cross-reactive neutralizing antibody against majority of the SARS-CoV-2 variants provides an important insight into pursuing future therapeutic agents for the prevention and treatment of COVID-19.

Mycobacterium tuberculosis H37Rv infection regulates alternative splicing in Macrophages.

OBJECTIVE: The objective of this study was to evaluate the expression of genes encoding SR proteinsand alternative splicing of IL4 and TLR4 in Mycobacterium tuberculosis (M. tb) H37Rv-infected macrophages. MATERIALS AND METHODS: THP-1 cells were induced to differentiate into macrophages with 200 nM PMA, and H37Rv strains were used for macrophage infection. After RNA extraction, qRT-PCR was performed to evaluate the expression of many SR proteins as well as the alternative splicing of IL4 and TLR4. RESULTS: IL4 and TLR4 play significant roles in host immunity to tuberculosis. The level of IL-4 splice variants in THP-1 cells increased after M. tb H37Rv infection. Three splice variants of TLR4 were detected in M. tb-infected THP-1 cells, when compared with uninfected controls; the expression level of these splicing variants in M. tb-infected THP-1 cell was down-regulated. Since SR proteins are RNA-binding proteins that regulate RNA splicing, the expression of SR proteins was examined, and SRSF2 and SRSF3 were significantly down-regulated. In addition, splicing factors SRp75 and SF3a were also significantly down-regulated post M. tb infection. CONCLUSION: Our findings indicate that alternative splicing may be involved in host gene regulation post M. tb infection of macrophage cells.

Diverging mRNA and Protein Networks in Activated Microglia Reveal SRSF3 Suppresses Translation of Highly Upregulated Innate Immune Transcripts.

Uncontrolled microglial activation may lead to the development of inflammation-induced brain damage. Here, we uncover a ribosome-based mechanism/checkpoint involved in control of the innate immune response and microglial activation. Using an in vivo model system for analysis of the dynamic translational state of microglial ribosomes, with mRNAs as input and newly synthesized peptides as an output, we find a marked dissociation of microglia mRNA and protein networks following innate immune challenge. Highly upregulated and ribosome-associated mRNAs were not translated, resulting in two distinct microglial molecular signatures, a highly specialized pro-inflammatory mRNA signature and an immunomodulatory/homeostatic protein signature. We find that this is due to specific translational suppression of highly expressed mRNAs through a 3' UTR-mediated mechanism involving the RNA-binding protein SRSF3. This discovery suggests avenues for therapeutic modulation of innate immune response in resident microglia.