Global analysis of expression, maturation and subcellular localization of mouse liver transcriptome identifies novel sex-biased and TCPOBOP-responsive long non-coding RNAs

Christine N Goldfarb,David J Waxman

doi:10.1186/s12864-021-07478-5

Abstract

BackgroundWhile nuclear transcription and RNA processing and localization are well established for protein coding genes (PCGs), these processes are poorly understood for long non-coding (lnc)RNAs. Here, we characterize global patterns of transcript expression, maturation and localization for mouse liver RNA, including more than 15,000 lncRNAs. PolyA-selected liver RNA was isolated and sequenced from four subcellular fractions (chromatin, nucleoplasm, total nucleus, and cytoplasm), and from the chromatin-bound fraction without polyA selection.ResultsTranscript processing, determined from normalized intronic to exonic sequence read density ratios, progressively increased for PCG transcripts in going from the chromatin-bound fraction to the nucleoplasm and then on to the cytoplasm. Transcript maturation was similar for lncRNAs in the chromatin fraction, but was significantly lower in the nucleoplasm and cytoplasm. LncRNA transcripts were 11-fold more likely to be significantly enriched in the nucleus than cytoplasm, and 100-fold more likely to be significantly chromatin-bound than nucleoplasmic. Sequencing chromatin-bound RNA greatly increased the sensitivity for detecting lowly expressed lncRNAs and enabled us to discover and localize hundreds of novel regulated liver lncRNAs, including lncRNAs showing sex-biased expression or responsiveness to TCPOBOP a xenobiotic agonist ligand of constitutive androstane receptor (Nr1i3).ConclusionsIntegration of our findings with prior studies and lncRNA annotations identified candidate regulatory lncRNAs for a variety of hepatic functions based on gene co-localization within topologically associating domains or transcription divergent or antisense to PCGs associated with pathways linked to hepatic physiology and disease.

Highlights

While nuclear transcription and RNA processing and localization are well established for protein coding genes (PCGs), these processes are poorly understood for long non-codingRNAs
We find a strong enrichment of thousands of liver-expressed long non-coding RNA (lncRNA) in the chromatin fraction, including lncRNAs that respond to endogenous hormonal factors or external chemical exposures, many expressed at too low a level for discovery by traditional RNA-seq analysis of whole liver tissue or even in purified liver nuclei
Liver tissue was homogenized under conditions expected to preserve nuclear membrane integrity, and cytoplasmic and nuclear RNA purified from the cytoplasmic lysate and nuclear pellet, respectively

Summary

Introduction

While nuclear transcription and RNA processing and localization are well established for protein coding genes (PCGs), these processes are poorly understood for long non-coding (lnc)RNAs. Since the discovery of more than a thousand novel, poly-adenylated long non-coding RNAs (lncRNAs) in mouse and human cells [1], lncRNAs have increasingly been shown to play key roles in gene regulation and disease states, including liver disease [2,3,4]. Many lncRNA genes display striking patterns of developmental regulation and tissuespecific expression, which enables them to serve as condition-specific regulators of diverse biological processes [6]. LncRNAs can regulate cellular functions at multiple levels, including epigenetic modification and chromatin remodeling, transcriptional regulation, alternative splicing and mRNA translation [7,8,9]. The biological functions and mechanisms of action of the vast majority of lncRNAs expressed in liver and other tissues are unknown

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