Non-templated 3'-end oligouridylation is a poorly characterized RNA modification found in many species including humans. Recent studies have reported non-templated 3'-end oligouridylation of small non-coding RNAs and long mRNAs. To determine the prevalence of oligouridylation in higher eukaryotes, we used strand-specific paired-end RNA-Seq to deeply survey the population of small RNAs in human cells. We used custom sequence analysis programs to identify non-templated addition to RNA. Our data revealed widespread non-templated nucleotide addition to the 3'-ends of many classes of RNA, with short stretches of uridine being the most frequently added nucleotide.

To expand the search for RNAs subject to non-templated oligouridylation beyond those identified in the small RNA population, several changes to the RNA-Seq library preparation method were tested. A new RNA-Seq library was prepared based on these optimizations using total RNA purified from human embryonic stem cells. Analysis shows that RNA generated from repetitive elements are subject to non-templated oligouridylation.