UPA-seq: prediction of functional lncRNAs using differential sensitivity to UV crosslinking.

Taiwa Komatsu,Saori Yokoi,Shinichi Nakagawa,Koichi Fujii,Shintaro Iwasaki,Mari Mito,Yusuke Kimura

doi:10.1261/rna.067611.118

Taiwa Komatsu, Saori Yokoi + Show 5 more

Open Access

https://doi.org/10.1261/rna.067611.118

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Abstract

While a large number of long noncoding RNAs (lncRNAs) are transcribed from the genome of higher eukaryotes, systematic prediction of their functionality has been challenging due to the lack of conserved sequence motifs or structures. Assuming that some lncRNAs function as large ribonucleoprotein complexes and thus are easily crosslinked to proteins upon UV irradiation, we performed RNA-seq analyses of RNAs recovered from the aqueous phase after UV irradiation and phenol-chloroform extraction (UPA-seq). As expected, the numbers of UPA-seq reads mapped to known functional lncRNAs were remarkably reduced upon UV irradiation. Comparison with ENCODE eCLIP data revealed that lncRNAs that exhibited greater decreases upon UV irradiation preferentially associated with proteins containing prion-like domains (PrLDs). Fluorescent in situ hybridization (FISH) analyses revealed the nuclear localization of novel functional lncRNA candidates, including one that accumulated at the site of transcription. We propose that UPA-seq provides a useful tool for the selection of lncRNA candidates to be analyzed in depth in subsequent functional studies.

Highlights

A large number of long noncoding RNAs are transcribed from the genomes of higher eukaryotes, and accumulating evidence suggests that lncRNAs are involved in a variety of molecular processes, including the epigenetic regulation of gene expression, formation of nonmembranous cellular bodies, and sequestration of miRNAs or RNA-binding proteins (RBPs)
We found that known functional lncRNAs including Xist, Neat1, Malat1, and Gomafu/Miat were efficiently crosslinked to proteins and were largely depleted from the aqueous phase upon UV irradiation, leading to a dramatic decrease in the number of mapped reads obtained by UPA-seq
To investigate whether lncRNAs tightly associate with multiple RBPs, we focused on UV irradiation, which induces the formation of covalent bonds between RNAs and RBPs at low efficiency (Greenberg 1979; Wagenmakers et al 1980)

Summary

Introduction

A large number of long noncoding RNAs (lncRNAs) are transcribed from the genomes of higher eukaryotes, and accumulating evidence suggests that lncRNAs are involved in a variety of molecular processes, including the epigenetic regulation of gene expression, formation of nonmembranous cellular bodies, and sequestration of miRNAs or RNA-binding proteins (RBPs) (for reviews, see Quinn and Chang 2016; Wu et al 2017; Kopp and Mendell 2018). Considering that certain fractions of lncRNAs might be “transcriptional noise” produced via stochastic associations of RNA polymerase with open chromatin regions (for review, see Struhl 2007), it is necessary to distinguish physiologically relevant lncRNAs from “junk” RNAs. While recent studies identified nuclear-localizing elements in lncRNAs (Zhang et al 2014; Lubelsky and Ulitsky 2018), lncRNAs commonly lack conserved sequence motifs or secondary structures, making functional classification of novel lncRNAs rather challenging. While recent studies identified nuclear-localizing elements in lncRNAs (Zhang et al 2014; Lubelsky and Ulitsky 2018), lncRNAs commonly lack conserved sequence motifs or secondary structures, making functional classification of novel lncRNAs rather challenging This situation is largely different from the case of proteins, which can be systematically categorized into families of molecules according to their individual domain structures (for review, see Hirose and Nakagawa 2016)

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