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.
