Regulation of HIV-1 TAT pre-mRNA splicing.

Abstract

Pre-mRNA splicing has been shown to be regulated in a cell- and tissue-specific as well as a developmentally specific manner. A substantial number of proteins have been identified that play a specific role in pre-mRNA processing. Included in these, are serine/arginine-rich (SR) proteins, which are thought to be involved in the regulation of splicing. SR protein activity is believed to be modulated by their state of phosphorylation. A family of autophosphorylating dual-specific nuclear kinases termed Clk (CDC2/CDC28-like kinases) have been shown to interact with, phosphorylate, and cause the nuclear redistribution of, SR proteins. Previous studies in our laboratory have shown that catalytically active Clks influence their own pre-mRNA splicing and that active Clks also influence the alternative splicing of an E1A adenovirus expression vector in vivo. Here, we describe the development of a triple primer RT-PCR assay to measure the spliced and unspliced RNA transcripts produced from the HIV-1 TAT expression vector pgTAT. The splicing of this vector has previously shown to be influenced by the SR protein ASF/SF2 in both in vitro and in vivo studies. Using the RT-PCR approach, we show that the over expression of any of the four known human full-length Clks (hClk1 to 4) does not alter the RNA ratio of spliced to unspliced TAT transiently or stably expressed in 293T cells. As well, by this approach, we show that the splicing of this same vector, stably transfected in P19 embryonal carcinoma cells, is altered upon neuronal and endodermal differentiation with retinoic acid or dimethyl sulfoxide respectively, whereby differentiation caused an increase in the unspliced transcript.