Proximity mediated effects of RNA regulatory proteins (mRNA as a proxisome).
Extensive RNA-protein interactions occur during the lifecycle of mRNA. Proteins DCP2, ZFP36L2, and CNOT7, which induce mRNA degradation, are normally targeted to the 5' cap, 3' untranslated region (UTR), and 3' poly(A) tail, respectively. We directed these proteins to unconventional sites spanning the coding and non-coding regions of a reporter mRNA using an MS2 coat proteinRNA hairpin interaction. DCP2 reduced expression only when targeted to the 5'-UTR, as expected. ZFP36L2 reduced expression when targeted to either the 3'-UTR or the 3' half of the coding sequence, the latter a region outside its cognate site. Strikingly, CNOT7 reduced expression when targeted anywhere in the mRNA, most strongly at both "ends" of the RNA, in the 3'- and 5'-UTRs. Induction of mRNA degradation by CNOT7 when bound far from the the poly(A) tail supports a model in which global folding juxtaposes many mRNA regions, creating a functional proxisome. Overall design: To determine the RNA structure of exogenous mRNAs (expressed from a plasmid) encoding NanoLuc and containing a single MS2 stem-loop (C varient) HEK293 cells were chemically probed with dimethyl sulfate (DMS) and exogenous RNAs were subjected to mutational profiling. The resulting DNA libraries were sequenced and analyzed for DMS-MaP experiments.