Noncoding AUG circRNAs constitute an abundant and conserved subclass of circles.

Lotte Vw Stagsted,Katrine M Nielsen,Thomas B Hansen,Iben Daugaard

doi:10.26508/lsa.201900398

Abstract

Circular RNAs (circRNAs) are a subset of noncoding RNAs previously considered as products of missplicing. Now, circRNAs are considered functional molecules, although to date, only few functions have been experimentally validated. Here, based on RNA sequencing from the ENCODE consortium, we identify and characterize a subset of circRNAs, coined AUG circRNAs, encompassing the annotated translational start codon from the protein-coding host genes. AUG circRNAs are more abundantly expressed and conserved than other groups of circRNAs, and they display flanking sequences that suggest an Alu-independent mechanism of biogenesis. The AUG circRNAs contain part of bona fide open reading frame, and in the recent years, several studies have reported cases of circRNA translation. However, using thorough cross-species analysis, extensive ribosome profiling, proteomics analyses, and experimental data on a selected panel of AUG circRNAs, we observe no indications of translation of AUG circRNAs or any other circRNAs. Our data provide a comprehensive classification of circRNAs and, collectively, the data suggest that the AUG circRNAs constitute an abundant subclass of circRNAs produced independently of primate-specific Alu elements.

Highlights

Noncoding RNAs constitute the vast majority of the human transcriptome as only a few percent of the produced transcripts are translated into proteins (ENCODE Project Consortium, 2012)
The notable fraction of circRNAs only predicted by one algorithm—the so-called exotic circRNAs—is in general lowly expressed (Fig 1B), which is reflected by a small subset of exotic circRNAs in the top 1,000 expressed circRNA candidates predicted by each algorithm (1–8%, data not shown)
The Alu-independent biogenesis is consolidated by analysis of RNAseq on DHX9 depleted cells and DHX9 HITS-CLIP, where AUG circRNAs generally are insensitive to DHX9 perturbation and exhibit reduced DHX9 binding in the flanking regions compared with other circRNAs

Summary

Introduction

Noncoding RNAs (ncRNAs) constitute the vast majority of the human transcriptome as only a few percent of the produced transcripts are translated into proteins (ENCODE Project Consortium, 2012). CircRNAs are typically derived from annotated protein-coding genes, but because of their relatively low abundance compared with their linear mRNA counterparts, circRNA molecules were first presumed to be missplicing events of the spliceosome with little to no relevance (Cocquerelle et al, 1993; Zaphiropoulos, 1997) This may be the case for a substantial subset of circRNAs, the identification and functional characterization of the highly conserved circRNA and miR-7-sponge, CDR1as/ciRS-7 (Hansen et al, 2013b; Memczak et al, 2013), and extensive profiling of differentially expressed circRNAs from RNA sequencing analyses (Salzman et al, 2012; Memczak et al, 2013; Rybak-Wolf et al, 2015; Veno et al, 2015) strongly support circRNAs as biologically relevant RNA species in eukaryotic cells. This is conventionally facilitated by inverted Alu elements (IAEs) (Jeck et al, 2013; Zhang et al, 2014); trans-acting RNA-binding factors have been implicated in circRNA formation (Ashwal-Fluss et al, 2014; Conn et al, 2015; Li et al, 2017)

Methods

Results

Conclusion