Abstract
Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.
Highlights
) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear
Complex transcription interwoven between and within protein-coding genes produces many thousands of long non-coding RNAs that are greater than 200 nucleotides in length but that appear to lack protein-coding potential (Djebali et al, 2012)
The function of a long non-coding RNAs (lncRNAs) may be mediated by the gene's RNA product which can bind to proteins or to other nucleic acids thereby modulating their functions
Summary
(Petruk et al, 2006; Latos et al, 2012; Marquardt et al, 2014). In these latter cases, any technique intended to dissect mechanism must alter the act and extent of transcription rather than change RNA levels. The earliest studied lncRNAs were those associated with imprinting, such as Airn and H19, or X chromosome regulation, such as Xist or roX1/2 (Table 1) In these cases, lncRNA expression was initially linked genetically to a known phenotype, and cell line models accurately reflected the in vivo models (Hao et al, 1993; Keniry et al, 2012; Latos et al, 2012; Helwak et al, 2013; Huppertz et al, 2014). Whilst useful for studies of many trans-acting lncRNAs, RNAi-based knockdown acts post-transcriptionally, and does not block the act of transcription, precluding analyses of lncRNAs which may produce their effects via this mechanism Another experimental approach is to genetically manipulate the lncRNA locus.
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