Studies on regulation of intronic polyadenylation by RNA helicase p68/DDX5 and U1snRNP in MCF7 cell line

Abstract

In eukaryotes, pre-mRNA has to undergo different processing steps; addition of cap at 5′ end, removal of introns to join functional exons, and addition of poly(A) tail at 3′ end to become fully functional mRNA. These processing events are linked to each other; one process effects efficiency of the other. Addition of polyA tail is not only limited at 3` end but poly A sequences are also found in genes (within exons and introns). The use of one of these intronic PAS results in primary transcript with different 3`UTR. DDX5 (p68) and DDX17 (p72) are RNA helicases performing different cellular functions; mi-RNA processing, transcription, mRNA processing, cell proliferation/transformation, cellular development and cancer. Four human genes MET, BCCIP, TGM2, and SMAD2 were selected to determine the relationship between p68, U1 snRNP, and activation of intronic polyadenylation. Genes were cloned into pGL3-TK vector having Firefly luciferase reporter gene. Mutation was introduced in 5`splice site to block U1. MCF7 cells were transfected with si-RNAp68/p72. After 24 hours cells were co-transfected with WT and Mut plasmids and pRL-SV40 control vector. Expression level of short isoform was determined by Dual Luciferase Reporter assay. The results suggest the role of p68 in IPA activation. Quantitative PCR was performed on uncleaved/total mRNA that confirmed the role of p68 RNA helicase in IPA acting through U1snRNP. To exclude the possibility that IPA is activated by splicing inhibition, si-RNAs against two splicing factors were used. If competitive inhibition of splicing result in IPA activation then should get the same expression level of short isoform with both si-RNAs, but it was not the case. IPA activation was seen only after si-U1 70k treatment while no or little short isoform was observed after si-U2AF65. These results clearly prove that IPA activation is not related to splicing inhibition. Overexpression of p68 and p72 enhanced the IPA event, again confirming the role of p68 in IPA. All experimental results prove that p68 activates intronic polyadenylation by removing U1 from 5`ss. Influence of p68 on IPA is not direct but it is acting through U1.