A cell-based screen for splicing regulators identifies hnRNP LL as a distinct signal-induced repressor of CD45 variable exon 4.
The human CD45 gene encodes a protein-tyrosine phosphatase that exhibits differential isoform expression in resting and activated T cells due to alternative splicing of three variable exons. Previously, we have used biochemical methods to identify two regulatory proteins, hnRNP L and PSF, which contribute to the activation-induced skipping of CD45 via the ESS1 regulatory element in variable exon 4. Here we report the identification of a third CD45 regulatory factor, hnRNP L-like (hnRNP LL), via a cell-based screen for clonal variants that exhibit an activation-like phenotype of CD45 splicing even under resting conditions. Microarray analysis of two splicing-altered clones revealed increased expression of hnRNP LL relative to wild-type cells. We further demonstrate that both the expression of hnRNP LL protein and its binding to ESS1 are up-regulated in wild-type cells upon activation. Forced overexpression of hnRNP LL in wild-type cells results in an increase in exon repression, while knock-down of hnRNP LL eliminates activation-induced exon skipping. Interestingly, analysis of the binding of hnRNP L and hnRNP LL to mutants of ESS1 reveals that these proteins have overlapping, but distinct binding requirements. Together, these data establish that hnRNP LL plays a critical and unique role in the signal-induced regulation of CD45 and demonstrate the utility of cell-based screens for the identification of novel splicing regulatory factors.
Topp
Justin D
JD
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA.
Jackson
Jason
J
Melton
Alexis A
AA
Lynch
Kristen W
KW
D005091:Exons; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D014644:Genetic Variation; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000235:genetics*; D008213:Lymphocyte Activation; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D012326:RNA Splicing; D050436:Regulatory Elements, Transcriptional; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology; D015854:Up-Regulation
C491423:HNRNPLL protein, human; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012097:Repressor Proteins; D017493:Leukocyte Common Antigens