3′ terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing

Katherine C Goldfarb,Thomas R Cech

doi:10.1186/1471-2199-14-23

Katherine C Goldfarb, Thomas R Cech

Open Access

https://doi.org/10.1186/1471-2199-14-23

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Abstract

BackgroundPost-transcriptional 3′ end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3′ RACE coupled with high-throughput sequencing to characterize the 3′ terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes.ResultsThe 3′ terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3′ terminus of an in vitro transcribed MRP RNA control and the differing 3′ terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR).Conclusions3′ RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3′ terminal sequences of noncoding RNAs.

Highlights

Post-transcriptional 3′ end processing is a key component of RNA regulation
To comprehensively define the 3′-hydroxyl ends of endogenous RNA molecules in HEK293T cells, we adapted an RNA ligase-mediated 3′ RACE strategy coupled to deep sequencing (Figure 1)
Precise 3′ terminal nucleotides were demarcated by ligation of whole cell RNA preparations

Summary

Introduction

Post-transcriptional 3′ end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and may utilize this mode of regulation. Beyond the familiar long poly(A) tails of messenger RNAs, short 3′ tailing of uridine [1,2,3,4] and adenosine [5,6,7] nucleotides to noncoding RNAs is gathering increasing appreciation These simple oligonucleotide additions (one to ~20 identical bases) can alter the stability, binding partners or activity of the enzymatic reactions in which these RNAs participate. Many of these 3′ terminally extended noncoding RNAs, including U6 spliceosomal RNA [8], tRNAs [4] and several snoRNAs [9,10], have annotated termini flanked by a stretch of genomically encoded U’s. RNase MRP requires tight regulation to discern its proper RNA subunit and cleave its many substrates

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