Investigating long noncoding RNAs using animal models

Abstract

The number of long noncoding RNAs (lncRNAs) has grown rapidly; however, our understanding of their function remains limited. Although cultured cells have facilitated investigations of lncRNA function at the molecular level, the use of animal models provides a rich context in which to investigate the phenotypic impact of these molecules. Promising initial studies using animal models demonstrated that lncRNAs influence a diverse number of phenotypes, ranging from subtle dysmorphia to viability. Here, we highlight the diversity of animal models and their unique advantages, discuss the use of animal models to profile lncRNA expression, evaluate experimental strategies to manipulate lncRNA function in vivo, and review the phenotypes attributable to lncRNAs. Despite a limited number of studies leveraging animal models, lncRNAs are already recognized as a notable class of molecules with important implications for health and disease.

Figure 1. lncRNA biology is a burgeoning field.

(A) The number of genes designated as lncRNAs in humans has steadily increased over successive GENCODE releases (http://www.gencodegenes.org/releases/) to nearly equal the number of protein-coding genes. (B) The number of publications in PubMed returned by querying “lncRNAs” has rapidly increased in recent years. However, few publications have explored lncRNAs using animal models.

Figure 2. Techniques to investigate lncRNA properties and tissue expression.

(A) These techniques include quantitative PCR (qPCR) and RNA-seq for transcript expression; RNA fluorescence ISH (FISH) or MS2 tagging (128) for localization; icSHAPE (24) or SHAPE-seq (25) for secondary structures; RIP, CLIP (21, 22), or PAR-CLIP (23) for protein interactions; RIA-seq (20) for RNA interactions; and CHART (18) or ChIRP (19) for DNA interactions. (B) Dissociation of tissue into single cells, followed by single-cell–sequencing analysis, can improve the sensitivity of detection for lncRNA expressed in a minority of cells within a tissue. (C) Fluorescently labeled cells within a tissue can be dissociated, sorted on the basis of fluorescence detection, and assayed for lncRNA expression to improve the sensitivity of detection.

Figure 3. DNA- and RNA-targeted strategies and general workflow.

(A) RNA-targeted approaches to attenuate lncRNA expression include RNAi, which degrades the lncRNA complementary to the experimentally introduced RNA. Other RNA-targeted approaches sterically interfere with a lncRNA complementary to the experimentally introduced RNA. (B) DNA-targeted approaches to ablate lncRNA expression include introducing a disruptive transgene within the lncRNA loci, excising the lncRNA loci or its regulatory elements, or inverting the lncRNA loci. (C) DNA- or RNA-targeted approaches to interfere with lncRNA function can be used either during development or adulthood in animal models. Functional ablation of a lncRNA may be sufficient to produce a phenotype or may result from gene-environment interactions. For trans-acting lncRNA, rescue experiments that revert the observed phenotype control for possible nonspecific effects, such as off-target effects of morpholinos or manipulation of DNA regulatory elements.