Molecular characterization of Ct-hrp65: identification of two novel isoforms originated by alternative splicing

Hrp65, a protein with two conserved RNA-binding domains, has been identified in Chironomus tentans as a component of nuclear fibers associated with ribonucleoprotein particles in transit from the gene to the nuclear pore. We have cloned two novel hrp65 isoforms and characterized the structure of the hrp65 gene. Comparison of the hrp65 gene to the hrp65 cDNAs revealed that the multiple hrp65 isoforms, hrp65-1, hrp65-2 and hrp65-3, are generated by alternative splicing of a single pre-mRNA. The hrp65-3 mRNA is only detected in C. tentans tissue culture cells of embryonic origin, whereas hrp65-1 and hrp65-2 mRNAs appear to be constitutively expressed. The hrp65 mRNAs are generated by differential 3' splice site selection at the last exon of the gene. Thus, the three hrp65 transcripts contain different 3' UTRs and encode proteins that vary in their C-terminal ends. Interestingly, the variant C-terminal region determines the subcellular localization of the hrp65 proteins. In transient transfection assays, hrp65-1 is efficiently targetted to the nucleus, whereas hrp65-2 and hrp65-3 localize mainly to the cytoplasm. Moreover, hrp65-3 is associated with cytoplasmic actin fibers. All together, our findings suggest that the different hrp65 isoforms serve specialized roles related to mRNA localization/transport in the different cell compartments.