Murine Long Noncoding RNA Morrbid Contributes in the Regulation of NRAS Splicing in Hepatocytes In Vitro.

Anna Fefilova,Tatiana Prikazchikova,Timofei S Zatsepin,Olga Sergeeva,Tatiana Abakumova,Pavel Melnikov,Rustam Ziganshin,Pavel V Mazin,Ilya Kurochkin

doi:10.3390/ijms21165605

Abstract

The coupling of alternative splicing with the nonsense-mediated decay (NMD) pathway maintains quality control of the transcriptome in eukaryotes by eliminating transcripts with premature termination codons (PTC) and fine-tunes gene expression. Long noncoding RNA (lncRNA) can regulate multiple cellular processes, including alternative splicing. Previously, murine Morrbid (myeloid RNA repressor of Bcl2l11 induced death) lncRNA was described as a locus-specific controller of the lifespan of short-living myeloid cells via transcription regulation of the apoptosis-related Bcl2l11 protein. Here, we report that murine Morrbid lncRNA in hepatocytes participates in the regulation of proto-oncogene NRAS (neuroblastoma RAS viral oncogene homolog) splicing, including the formation of the isoform with PTC. We observed a significant increase of the NRAS isoform with PTC in hepatocytes with depleted Morrbid lncRNA. We demonstrated that the NRAS isoform with PTC is degraded via the NMD pathway. This transcript is presented almost only in the nucleus and has a half-life ~four times lower than other NRAS transcripts. Additionally, in UPF1 knockdown hepatocytes (the key NMD factor), we observed a significant increase of the NRAS isoform with PTC. By a modified capture hybridization (CHART) analysis of the protein targets, we uncovered interactions of Morrbid lncRNA with the SFPQ (splicing factor proline and glutamine rich)-NONO (non-POU domain-containing octamer-binding protein) splicing complex. Finally, we propose the regulation mechanism of NRAS splicing in murine hepatocytes by alternative splicing coupled with the NMD pathway with the input of Morrbid lncRNA.

Highlights

During the last decades, the basic dogma of molecular biology—DNA↔RNA→protein—evolved into a complex network with back loops that regulate gene expression in eukaryotes in a cell- and tissue-specific manner
We studied the subcellular localization of Morrbid Long noncoding RNA (lncRNA) in hepatocytes by a fluorescence in situ hybridization analysis (FISH) and found that the lncRNA predominantly localizes in the cell nucleus (Figure 1B)
We demonstrated that transfection of the antisense oligonucleotides (ASO) mix results in an ~80% decrease of the Morrbid lncRNA expression in AML12 cells in comparison to control ASO (Supplementary Figure S1A,B)

Summary

Introduction

The basic dogma of molecular biology—DNA↔RNA→protein—evolved into a complex network with back loops that regulate gene expression in eukaryotes in a cell- and tissue-specific manner. Spliced exons can introduce premature translation termination codons (PTC) in transcripts Such PTC-containing splice variants are degraded through the nonsense-mediated decay (NMD) pathway [9]. Discovered as a quality control (QC) step that helps to remove aberrant splicing transcripts, the NMD pathway can contribute in the fine-tuning of gene expression via so-called regulated unproductive splicing and translation (RUST) [10]. Lewis et al [11] estimated that ~30% of alternatively spliced exons introduce PTC This fact demonstrates the widespread coupling of alternative splicing and NMD for the regulation of gene expression. NEAT1 and MALAT1 can influence the phosphorylation of splicing regulatory proteins or act as chromatin remodelers Another example is lncRNA 51A, a natural antisense transcript to the intron 1 of the sortilin-related receptor 1 (SORL1) gene. LncRNAs can modulate transcriptome reprogramming in eukaryotes using several distinct mechanisms

Methods

Results

Conclusion