Using EMOTE to Map the Exact 5′-Ends of Processed RNA on a Transcriptome-Wide Scale

Abstract

The presence or absence of structure in an RNA is often crucial to its function. This is evident for highly structured RNAs such as rRNA, tRNA, or riboswitches, but it is also the case for many mRNAs, where secondary structures in the 5′ or 3′ UTR can determine the efficiency of translation or the half-life of the RNA. There are paths to modify such secondary structures, (1) by the action of a helicase that allows an alternative RNA structure to form, (2) by the formation of a duplex with another RNA, or (3) by cleavage of the RNA in a way that favors a different secondary structure. None of the three exclude the others, and in vivo it is common that two or all three work together to remodel an RNA to the desired form. However, while the first two solutions can be reversible, the cleavage of RNA is final, and there is no chance to go back. In this chapter, a method for tracking the 5′ end created by RNA processing on a transcriptome-wide scale is presented. The Exact Mapping Of Transcriptome Ends (EMOTE) allows the large-scale identification of mono-phosphorylated RNA 5′-ends and provides the exact processing sites.