Functional Classification and Experimental Dissection of Long Noncoding RNAs

Abstract

Over the last decade, it has been increasingly demonstrated that the genomes of many species are pervasively transcribed, resulting in the production of numerous long noncoding RNAs (lncRNAs). At the same time, it is now appreciated that many types of DNA regulatory elements, such as enhancers and promoters, regularly initiate bi-directional transcription. Thus, discerning functional noncoding transcripts from a vast transcriptome is a paramount priority, and challenge, for the lncRNA field. In this review, we aim to provide a conceptual and experimental framework for classifying and elucidating lncRNA function. We categorize lncRNA loci into those that regulate gene expression in cis versus those that perform functions in trans and propose an experimental approach to dissect lncRNA activity based on these classifications. These strategies to further understand lncRNAs promise to reveal new and unanticipated biology with great potential to advance our understanding of normal physiology and disease.

Keywords: lncRNA; noncoding RNA.

Figure 1. Functions of lncRNA loci in local gene regulation

The ability of a lncRNA locus to regulate the expression of nearby genes in cis may be attributable to sequence-specific functions of the mature lncRNA transcript (A), may require transcription or splicing of an RNA, but the lncRNA itself is not functional (B), or may be due to DNA elements within the lncRNA promoter or gene body that function independently of the transcribed RNA (C). Pol II, RNA polymerase II; TF, transcription factor.

Figure 2. Functions of lncRNAs in trans

(A) lncRNAs may regulate the expression of unlinked genes by interacting with promoters and enhancers, or other proteins bound to these sites, and influencing chromatin states and RNA polymerase activity. (B) Some lncRNAs are components of dynamic subcellular structures, such as MALAT1 in nuclear speckles and NEAT1 in paraspeckles, and may help to position these structures near actively transcribed genes to facilitate delivery of splicing and transcription factors. lncRNA FIRRE organizes interchromosomal interactions within the nucleus. C) Transacting lncRNAs may regulate the activity of interacting proteins and RNAs in a stoichiometric manner. For example, NORAD binds many molecules of PUMILIO (PUM) proteins, limiting the availability of these post-transcriptional regulators to interact with other targets. CDR1as binds and stabilizes AGO:miR-7 complexes, whereas AGO:miR-671 cleaves the lncRNA. Pol I, RNA polymerase I; X chr, X chromosome.

Figure 3. Experimental dissection of lncRNA loci

A proposed experimental approach for classifying lncRNAs into those that function in cis or trans, followed by investigation of the underlying mechanisms of action.