Human DHX16/PRP2 protein in splicing and cell death
Human DHX16/PRP2 protein in splicing and cell death
Spliceosome requires different DExD/H-box RNA helicases at assembly, activation, and disassembly steps. Mutations in these helicase genes of S. cerevisiae could abolish splicing and cell growth; however, their effect in humans is less known. S. cerevisiae Prp2 protein is a late spliceosomal helicase; prp2 mutants accumulate unspliced pre-mRNA in vivo and their extracts form an inactive pre-catalytic spliceosome in vitro (see project 2). We showed that an siRNA-mediated knockdown of human DHX16 gene expression caused a quick and drastic decrease of mRNAs from several intron-containing genes. Overproduction of the protein mutated at its helicase domain had a similar effect. In both cases, the unspliced pre-mRNA from a reporter was detected. Splicing in HeLa extracts was inhibited by an antibody against a Dhx16 peptide; however, spliceosomes were nonetheless formed. Thus, sequence and function similarities indicate that human Dhx16 is likely the orthologue of S. cerevisiae Prp2. Interestingly, the antibody immunoprecipitated from splicing reactions the spliceosomal snRNAs, unspliced pre-mRNA, and the splicing intermediates. Yeast two-hybrid screens revealed an interaction between hPrp2 and a second-step splicing factor further suggesting a potential role of hPrp2 in spliceosome transition. We have established a stable cell line having a controllable dominant negative hPRP2; upon induction, DNA fragmentation occurred and cells died of apoptosis. This provides an opportunity to examine how splicing, transcription, and cell growth respond to a stall of spliceosome in vivo.