SRPK3 regulates alternative pre-mRNA splicing required for B lymphocyte development and humoral responsiveness

Abstract

One Sentence Summary: SRPK3 regulates alternative splicing of pre-mRNA that is crucial for B cell development, activation and antibody responses. Abstract:Alternative splicing (AS) of pre-mRNA is a critical component of transcriptional regulation that diversifies the cellular proteome. The Serine-Arginine Protein Kinases (SRPK) initiate early events in AS. Using conditional knockout mice (cKO), we demonstrated the importance of the X-linked gene Srpk3 in B lymphocyte development and in response to immunization in vivo.Significantly decreased numbers of immature and mature B cells were observed in Srpk3-cKO BM relative to wild-type (WT). Immunization of Srpk3-cKO mice with a T lymphocyteindependent type-2 antigen elicited greatly reduced amounts of specific IgG3. Srpk3 deletion resulted in hundreds of differentially spliced mRNAs in B cells, including mRNAs encoding proteins associated with signaling pathways and mitochondrial function. Several alternative splicing outcomes in Srpk3-cKO cells are due to altered splicing regulation of SR proteins. We conclude that Srpk3 is an immunomodulatory kinase that controls humoral immunity via its regulation of pre-mRNA splicing, antibody production, and metabolism in B cells. Introduction:Co-transcriptional mechanisms of gene regulation are emerging as important contributors to immune cell differentiation and function. One such mechanism is alternative pre-mRNA splicing (AS), which increases informational diversity, functional capacity, and the efficiency of protein expression (1). Recent studies have confirmed that AS influences immunity and tolerance through its roles in lymphocyte homeostasis (2). Additionally, B cell differentiation, activation, and maturation to antigen secreting cells (ASCs) are dependent on unique splicing patterns that modulate B cell responsiveness and function (3,4). Therefore, understanding how pre-mRNA splicing is regulated in these cells is of critical importance.The functional spliceosome is a dynamic machine comprised of approximately 20 protein and RNA components that regulate splice site selection in pre-mRNAs concurrently with transcription (5, 6). Upstream regulators of splicing include the Serine-Arginine Protein Kinase (SRPK) family, a group of highly conserved serine-threonine kinases that phosphorylate Serine-Arginine repeat (SR)-rich proteins in response to extracellular signals (7-10). SR proteins include splicing factors (SRSF1-12) that promote splice site selection by bridging between initial splice site selection in the pre-spliceosome and assembly of the mature spliceosome.In mammals, SRPK proteins (SRPK1 and SRPK2) have been extensively studied as regulators of AS and mRNA maturation (11-13), transduction of growth signaling (14), chromatin reorganization (15), cell cycle (7) and metabolic signaling (16). However, the function of SRPK3 has not been elucidated. SRPK3 was identified first as a transcriptional target of Myocyte Enhancer Factor 2C (MEF2C) in skeletal muscle and the heart (17). Later studies identified the X-linked ...