Alternative Splicing Controlled by Heterogeneous Nuclear Ribonucleoprotein L Regulates Development, Proliferation, and Migration of Thymic Pre-T Cells

Abstract

The regulation of posttranscriptional modifications of pre-mRNA by alternative splicing is important for cellular function, development, and immunity. The receptor tyrosine phosphatase CD45, which is expressed on all hematopoietic cells, is known for its role in the development and activation of T cells. CD45 is known to be alternatively spliced, a process that is partially regulated by heterogeneous nuclear ribonucleoprotein (hnRNP) L. To investigate the role of hnRNP L further, we have generated conditional hnRNP L knockout mice and found that LckCre-mediated deletion of hnRNP L results in a decreased thymic cellularity caused by a partial block at the transition stage between double-negative 4 and double-positive cells. In addition, hnRNP L−/− thymocytes express aberrant levels of the CD45RA splice isoforms and show high levels of phosphorylated Lck at the activator tyrosine Y394, but lack phosphorylation of the inhibitory tyrosine Y505. This indicated an increased basal Lck activity and correlated with higher proliferation rates of double-negative 4 cells in hnRNP L−/− mice. Deletion of hnRNP L also blocked the migration and egress of single-positive thymocytes to peripheral lymphoid organs in response to sphingosine-1-phosphate and the chemokines CCL21 and CXCL12 very likely as a result of aberrant splicing of genes encoding GTPase regulators and proteins affecting cytoskeletal organization. Our results indicate that hnRNP L regulates T cell differentiation and migration by regulating pre-TCR and chemokine receptor signaling.

Global analysis of physical and functional RNA targets of hnRNP L reveals distinct sequence and epigenetic features of repressed and enhanced exons

“…HnRNP L has a widespread impact on the inclusion of cassette exons, but not intron retention or transcript abundance HnRNP L is critical for proper T cell development and function (Gaudreau et al 2012); however, only a relatively few confirmed targets of hnRNP L-dependent splicing regulation have been identified. To determine the full breadth of hnRNP L regulated splicing in T cells, we used complementary knockdown and sequencing approaches to generate high depth-and breadth-analysis of hnRNP L-dependent mRNA expression in Jurkat T cells.…”

“…Previous studies have demonstrated pervasive association of hnRNP L with RNA in human T cells as well as a critical role for hnRNP L in proper T-cell development and function (Gaudreau et al 2012;Shankarling et al 2014). Here we expand the understanding of the biologic function of hnRNP L by identifying changes in transcriptome expression upon knockdown of hnRNP L in a human T-cell line.…”

“…A handful of additional targets of hnRNP L splicing-regulatory activity have been identified in both T cells and other cell types (Hung et al 2008;Gaudreau et al 2012;Shankarling et al 2014); however, validated targets of hnRNP L splicing activity remain relatively few. Moreover, a transcriptomewide comparison of the binding and function of hnRNP L has not been performed.…”

“…HnRNP L binds to, and represses, the three variable exons of the CD45 pre-mRNA, leading to the expression of the smallest isoform of CD45 essential for maintaining T-cell homeostasis (Rothrock et al 2005;Tong et al 2005;MottaMena et al 2010;Preussner et al 2012). Furthermore, lymphoid-specific knockout of hnRNP L in mice causes significant defects in thymic development and reduced migration of hnRNP L −/− T cells to the periphery (Gaudreau et al 2012). Importantly, hnRNP L is expressed at consistent levels throughout T-cell development and activation and CD45 misregulation is insufficient to account for all of the physiological defects observed in the hnRNP L −/− T cells (Gaudreau et al 2012).…”

“…Furthermore, lymphoid-specific knockout of hnRNP L in mice causes significant defects in thymic development and reduced migration of hnRNP L −/− T cells to the periphery (Gaudreau et al 2012). Importantly, hnRNP L is expressed at consistent levels throughout T-cell development and activation and CD45 misregulation is insufficient to account for all of the physiological defects observed in the hnRNP L −/− T cells (Gaudreau et al 2012). Together these data indicate that hnRNP L plays a critical role throughout T-cell maturation and function via more than one target or mechanism.…”