Isolated 3’UTRs:
deciphering the post-transcriptional cleavage of mRNA
2Department of Molecular Neuroscience, Weizmann Institute of Science, Israel
Messenger RNAs (mRNAs) undergo a lengthy series of processing and transport steps during their lifetime, which include splicing, polyadenylation, export and degradation. These processes are relatively well understood and have been subjected to high-throughput experimental screens. Other RNA-centric processes remain more obscure, particularly those that are thought to be restricted to specific cell types and and/or are tightly regulated. One such process is the post-transcriptional cleavage of mRNAs in their 3’ untranslated regions (3’ UTRs). Most mRNA molecules remain intact throughout their lifetime, however that is not always the case. The core of the accumulating evidence for this phenomenon comes from imaging approaches used to visualize different parts of mRNA molecules in intact cells. Those evidence suggest that many mRNAs begin their existence as a continuous molecule and then become processed and cleaved into units with distinct fates. Genomic approaches have also recently been used to characterize the phenomena, and found ample support for this trend, but where, when, and how these events occur remains unclear. To answer these questions, we are establishing an AAV delivered massively parallel bicistronic reporter assays which use high-throughput sequencing to read out the fates of reporter RNA molecules in cells. The experimental design monitors both production of reporter proteins as well as the cleavage of RNA, and will report on its precise position. Importantly, the same reporter system can be used both in cells in tissue culture and in intact brain tissue.