HNRNP-L Interacts with the Signal-Responsive Alternative Splicing Regulatory Element of CD45

Alternative splicing is emerging as a common mechanism for altering protein expression in response to extracellular cues. Of particular interest is how cell-signaling pathways modify mRNA isoform expression. The T cell tyrosine phosphatase CD45 provides a useful model system to explore the regulation of cell-signaling induced alternative splicing. CD45 has three variable exons that are differentially excluded in T cells upon antigen challenge or activation. An exonic splicing silencer element, named ESS1, is necessary for regulation of inclusion or exclusion of CD45 exon 4 in the mature mRNA. This sequence is also sufficient to confer T cell activation induced repression of a heterologous exon. ESS1 contains a sequence motif that is present in CD45 variable exons 5 and 6, as well as exons of other signal responsive genes. Point mutations in conserved nucleotides within this motif abolish T cell activation-induced skipping of CD45 variable exons 4, 5, and 6. Biochemical purification of RNA binding proteins from nuclear extract identifies a number of hnRNP family members that bind ESS1 RNA sequence in a specific manner. Mass spectrometry identifies three of these, including p65 as heterogeneous ribonucleoprotein L (hnRNP-L), p55 as Polypyrimidine Tract Binding Protein (PTB or hnRNP-I), and p40 as heterogeneous ribonucleoprotein E2 (hnRNP-E2). Recombinant hnRNP-L, PTB, and hnRNP-E2 bind specificity to wild type ESS1 RNA, as tested by mobility shift and UV crosslinking assays. Mutations in the ESS1 sequence, which prevent exon skipping in vivo, reduce recombinant hnRNP-L binding in vitro. Recombinant PTB and hnRNP-E2 do not appear to have reduced binding to mutant ESS1 sequence. HnRNP-L appears to be the main sequence-specific binding component of ESS1 RNA, and may be involved in repressing CD45 variable exon 4 in response to T cell activation.