Strategies for genetic inactivation of long noncoding RNAs in zebrafish.

Perrine Lavalou,Louise Damy,Helene Eckert,Antoine Graindorge,Florian Constanty,Angelo Bitetti,Alena Shkumatava,Sara Majello

doi:10.1261/rna.069484.118

Abstract

The number of annotated long noncoding RNAs (lncRNAs) continues to grow; however, their functional characterization in model organisms has been hampered by the lack of reliable genetic inactivation strategies. While partial or full deletions of lncRNA loci disrupt lncRNA expression, they do not permit the formal association of a phenotype with the encoded transcript. Here, we examined several alternative strategies for generating lncRNA null alleles in zebrafish and found that they often resulted in unpredicted changes to lncRNA expression. Removal of the transcription start sites (TSSs) of lncRNA genes resulted in hypomorphic mutants, due to the usage of either constitutive or tissue-specific alternative TSSs. Deletions of short, highly conserved lncRNA regions can also lead to overexpression of truncated transcripts. In contrast, knock-in of a polyadenylation signal enabled complete inactivation of malat1, the most abundant vertebrate lncRNA. In summary, lncRNA null alleles require extensive in vivo validation, and we propose insertion of transcription termination sequences as the most reliable approach to generate lncRNA-deficient zebrafish.

Highlights

Thousands of long noncoding RNAs (lncRNAs) have been identified in multiple vertebrate species (Necsulea et al 2014; Hezroni et al 2015), but their biological functions remain mostly unknown
A small fraction of zebrafish lncRNAs are conserved in mammals, representing a promising set of candidates for functional interrogation (Ulitsky et al 2011; Hezroni et al 2015)
To examine the effect of this strategy on lncRNA expression, we chose the deeply conserved lncRNA cyrano (Ulitsky et al 2011) for genetic interlncRNA transcription start sites (TSSs) removal leads to tissue-specific alternative TSS usage, maintaining lncRNA expression

Summary

INTRODUCTION

Thousands of lncRNAs have been identified in multiple vertebrate species (Necsulea et al 2014; Hezroni et al 2015), but their biological functions remain mostly unknown. Tion, including full or partial deletion of the lncRNA locus, deletion and subsequent replacement of the lncRNA locus by a reporter gene (Nakagawa et al 2012; Sauvageau et al 2013), deletion of the lncRNA transcription start site (TSS) and upstream regulatory regions (Fitzpatrick et al 2002; Zhang et al 2012) and sequence inversions (Fig.1; Bitetti et al 2018) Commonly used, these lncRNA inactivation strategies have several caveats and limitations. TSS deletion of the cyrano locus results in hypomorphic zebrafish mutants we tested if deleting the sequences surrounding and containing lncRNA TSS elements is a reliable alternative strategy for zebrafish lncRNA genetic inactivation To this end, we generated a minimally invasive cyranoΔTSS mutant allele by removing sequences containing the cyrano TSS (0 to +84) (Fig. 2E). This observation is consistent with recent zebrafish and mouse studies (Kleaveland et al 2018; Goudarzi et al 2019) and is in contrast to previous studies that used a morpholino-based knockdown approach to inactivate cyrano (Ulitsky et al 2011; Sarangdhar et al 2018)

RESULTS

DISCUSSION

MATERIALS AND METHODS