Three of the eight RNA segments encoded by the influenza A virus (IAV) undergo alternative splicing to generate distinct proteins. Previously, we found that host proteins hnRNP K and NS1-BP regulate IAV M segment splicing, but the mechanistic details were unknown. Here we show NS1-BP and hnRNP K bind M mRNA downstream of the M2 5′ splice site (5′ss). NS1-BP binds most proximal to the 5′ss, partially overlapping the U1 snRNP binding site, while hnRNP K binds further downstream and promotes U1 snRNP recruitment. Mutation of either or both the hnRNP K and NS1-BP-binding sites results in M segment mis-splicing and attenuated IAV replication. Additionally, we show that hnRNP K and NS1-BP regulate host splicing events and that viral infection causes mis-splicing of some of these transcripts. Therefore, our proposed mechanism of hnRNP K/NS1-BP mediated IAV M splicing provides potential targets of antiviral intervention and reveals novel host functions for these proteins.

Over 95% of human multi-exon genes undergo alternative splicing, a process important in normal development and often dysregulated in disease. We sought to analyze the global splicing regulatory network of CELF2 in human T cells, a well-studied splicing regulator critical to T cell development and function. By integrating high-throughput sequencing data for binding and splicing quantification with sequence features and probabilistic splicing code models, we find evidence of splicing antagonism between CELF2 and the RBFOX family of splicing factors. We validate this functional antagonism through knockdown and overexpression experiments in human cells and find CELF2 represses RBFOX2 mRNA and protein levels. Because both families of proteins have been implicated in the development and maintenance of neuronal, muscle, and heart tissues, we analyzed publicly available data in these systems. Our analysis suggests global, antagonistic coregulation of splicing by the CELF and RBFOX proteins in mouse muscle and heart in several physiologically relevant targets, including proteins involved in calcium signaling and members of the MEF2 family of transcription factors. Importantly, a number of these coregulated events are aberrantly spliced in mouse models and human patients with diseases that affect these tissues, including heart failure, diabetes, or myotonic dystrophy. Finally, analysis of exons regulated by ancient CELF family homologs in chicken, Drosophila, and Caenorhabditis elegans suggests this antagonism is conserved throughout evolution.

Over 95% of human multi-exon genes undergo alternative splicing, a process important in normal development and often dysregulated in disease. We sought to analyze the global splicing regulatory network of CELF2 in human T cells, a well-studied splicing regulator critical to T cell development and function. By integrating high-throughput sequencing data for binding and splicing quantification with sequence features and probabilistic splicing code models, we find evidence of splicing antagonism between CELF2 and the RBFOX family of splicing factors.We validate this functional antagonism through knockdown and overexpression experiments in human cells and find CELF2 represses RBFOX2 mRNA and protein levels. Because both families of proteins have been implicated in the development and maintenance of neuronal, muscle, and heart tissues, we analyzed publicly available data in these systems. Our analysis suggests global, antagonistic co-regulation of splicing by the CELF and RBFOX proteins in mouse muscle and heart in several physiologically relevant targets including proteins involved in calcium signaling and members of the MEF2 family of transcription factors. Importantly, a number of these co-regulated events are aberrantly spliced in mouse models and human patients with diseases that affect these tissues including heart failure, diabetes, or myotonic dystrophy. Finally, analysis of exons regulated by ancient CELF family homologs in chicken, and Drosophila suggests this antagonism is conserved through evolution.

Vaccine-induced immune thrombotic thrombocytopenia (VITT), also known as thrombosis with thrombocytopenia syndrome, is a catastrophic and life-threatening reaction to coronavirus disease 2019 (COVID-19) vaccines, which occurs disproportionately in response to vaccination with non-replicating adenovirus vector (AV) vaccines. The mechanism of VITT is not well defined and it has not been resolved why cases of VITT are predominated by vaccination with AV vaccines. However, virtually all VITT patients have positive platelet-activating anti-platelet factor 4 (PF4) antibody titers. Subsequently, platelets are activated and depleted in an Fcγ-receptor IIa (FcγRIIa or CD32a)-dependent manner, but it is not clear why or how the anti-PF4 response is mounted. This review describes the pathogenesis of VITT and provides insight into possible mechanisms that prompt the formation of a PF4/polyanion complex, which drives VITT pathology, as an amalgam of current experimental data or hypotheses.