Long noncoding RNAs (lncRNAs) are emerging as important regulators of diverse biological functions. Although mechanistic models are starting to emerge, it is also clear that the lncRNA field needs appropriate model systems in order to better elucidate the functions of lncRNAs and their roles in both physiological and pathological conditions. The field of lncRNAs is new, and the biochemical and genetic methods used to address function and mechanisms of lncRNAs have only recently been developed or adapted from techniques used to investigate protein-coding genes. In this review, the authors discuss the strengths and weaknesses of available techniques for the analysis of chromatin-associated lncRNAs and emerging models for the recruitment to specific genomic sites such as triple-helix, RNA-protein-DNA recognition and proximity-guided search models.
Strategies to analyze lncRNA loss of function
Strategies to alter lncRNA expression. The lncRNA locus is indicated in orange, neighbouring protein- coding gene in blue and the process of transcription by dotted lines. Knockdown of lncRNA can be achieved by RNA interference (RNAi), a process activated by dsRNA species delivered to the cytoplasm of cells. Downregulation of nuclear lncRNAs can be achieved using antisense oligonucleotides (LNA or chemically modified RNA aptamers), which bind to nascent transcripts forming a DNA/RNA hybrid, triggering RNase H-dependent degradation of the RNA in the nucleus. Examples how to alter lncRNA gene locus are also depicted. Another possibility is genome engineering using dCas9 and TALENs fused to a repressor (Rep), which can establish transcriptional silencing of lncRNA genes. Outcome and limitations of the method chosen to alter lncRNA expression are described.