Long noncoding RNAs: fresh perspectives into the RNA world

Abstract

Large-scale mapping of transcriptomes has revealed significant levels of transcriptional activity within both unannotated and annotated regions of the genome. Interestingly, many of the novel transcripts demonstrate tissue-specific expression and some level of sequence conservation across species, but most have low protein-coding potential. Here, we describe progress in identifying and characterizing long noncoding RNAs (lncRNAs) and review how these transcripts interact with other biological molecules to regulate diverse cellular processes. We also preview emerging techniques that will help advance the discovery and characterization of novel transcripts. Finally, we discuss the role of lncRNAs in disease and therapeutics.

Keywords: Long noncoding RNA; epigenetics; next-generation sequencing; therapeutics.

Figure 1. Mechanisms for long noncoding RNA (lncRNA) function

Characterization of lncRNA function has revealed the ability of these transcripts to regulate gene expression through chromatin remodeling, control of transcription initiation and post-transcriptional processing. (a) lncRNAs such as Xist, Kcnq1ot1, Airn and HOTAIR have been found to interact with chromatin remodeling proteins such as polycomb repressive complex 2 and G9a (represented in green) to mediate deposition of repressive chromatin marks. (b) lncRNAs can directly regulate messenger RNA synthesis at genomic loci by interacting with transcription factors (see text) or components of the basal transcriptional machinery. In the case of DHFR regulation, an upstream lncRNA transcribed from the minor promoter has been shown to bind both the major DHFR promoter as well as TFIIB, leading to displacement of TFIIB from the major promoter. (c) lncRNAs can regulate co-transcriptional processes such as RNA splicing (see text) and translation. The Uchl1-AS RNA is transcribed in times of cellular stress and acts to speed up translation of Uchl1 mRNA by enhancing polysome loading onto the mRNA in the cytoplasm. The mechanisms for this activity is not well understood, although it has been proposed that the SINEB2 repeat element could play a crucial role.

Figure 2. Techniques for mapping RNA-protein interactions

In both chromatin crosslinking and immunoprecipitation (CLIP) and UV RNA immunoprecipitation (UV-RIP), cells are irradiated with 254nm UV light, which crosslinks proteins and nucleic acids that are in close proximity. For PAR-CLIP, cells are incubated with 4-thiouracil and crosslinked at 360nm UV. In CLIP, cellular lysate is treated with RNAse after crosslinking to fragment RNA before performing immunoprecipitation. A 3’ adaptor can be added to enriched RNA fragments (not necessary unless for downstream sequencing library preparation). RNA is end labeled with [γ³²] p, following which the reaction is subjected to SDS-PAGE. Following transfer to nitrocellulose membranes, the appropriate region of the membrane is excised (dependent on size of protein probed) and treated with proteinase K to release RNA fragments. Denaturing conditions of SDS-PAGE ensures enrichment for RNA fragments in direct interaction with protein of interest. In UV-RIP, cell lysate is subjected to immunoprecipitation to enrich for RNA bound either directly or indirectly to the protein of interest. A high stringency salt wash further enriches for RNA bound both directly and indirectly to the protein of interest by removing uncrosslinked fragments. Proteinase K treatment releases bound RNA. The RNA fraction obtained from both CLIP and UV-RIP can be analyzed by RT-qPCR, array hybridization as well as high throughput sequencing.