A Novel Set of Nuclear Localization Signals Determine Distributions of the αCP RNA-Binding Proteins

Abstract

αCPs comprise a subfamily of KH-domain-containing RNA-binding proteins with specificity for C-rich pyrimidine tracts. These proteins play pivotal roles in a broad spectrum of posttranscriptional events. The five major αCP isoforms are encoded by four dispersed loci. Each isoform contains three repeats of the RNA-binding KH domain (KH1, KH2, and KH3) but lacks other identifiable motifs. To explore the complexity of their respective functions, we examined the subcellular localization of each αCP isoform. Immunofluorescence studies revealed three distinct distributions: αCP1 and αCP2 are predominantly nuclear with specific enrichment of αCP1 in nuclear speckles, αCP3 and αCP4 are restricted to the cytoplasm, and αCP2-KL, an αCP2 splice variant, is present at significant levels in both the nucleus and the cytoplasm. We mapped nuclear localization signals (NLSs) for αCP isoforms. αCP2 contains two functionally independent NLS. Both NLSs appear to be novel and were mapped to a 9-amino-acid segment between KH2 and KH3 (NLS I) and to a 12-amino-acid segment within KH3 (NLS II). NLS I is conserved in αCP1, whereas NLS II is inactivated by two amino acid substitutions. Neither NLS is present in αCP3 or αCP4. Consistent with mapping studies, deletion of NLS I from αCP1 blocks its nuclear accumulation, whereas NLS I and NLS II must both be inactivated to block nuclear accumulation of αCP2. These data demonstrate an unexpected complexity in the compartmentalization of αCP isoforms and identify two novel NLS that play roles in their respective distributions. This complexity of αCP distribution is likely to contribute to the diverse functions mediated by this group of abundant RNA-binding proteins.

ACKNOWLEDGMENTS

We thank R. Andino, G. Dreyfuss, and M. Malim for generously sharing reagents used in this study. We thank Julia Morales for early contributions to this project, Brian Calvi for advice on confocal microscopy, and Lili Wan for critical review of the manuscript.

This work was supported by NIH grant RO1 HL65449 and by the generosity of the Doris Duke Foundation.