Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood

Abstract

The mammalian genome contains thousands of loci that transcribe long noncoding RNAs (lncRNAs)1-3, some of which are known to play critical roles in diverse cellular processes4-7. LncRNA loci can contribute to cellular regulation through a variety of mechanisms: while some encode RNAs that act non-locally (in trans)6,8, emerging evidence indicates that many lncRNA loci act locally (in cis)—for example, through functions of the lncRNA promoter, the process of lncRNA transcription, or the lncRNA transcript itself in regulating the expression of nearby genes7,9-11. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. To address these challenges, we developed a genome-scale CRISPR-Cas9 activation screen targeting more than 10,000 lncRNA transcriptional start sites to identify noncoding loci that influence a phenotype of interest. We found 11 novel lncRNA loci that, upon recruitment of an activator, each mediate BRAF inhibitor resistance in melanoma. We investigated potential local and non-local mechanisms at these candidate loci and found that most appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation results in dosage-dependent activation of four neighboring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit to systematically discover functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function.

ALKBH5 Inhibits Pancreatic Cancer Motility by Decreasing Long Non-Coding RNA KCNK15-AS1 Methylation

“…Cytoplasmic and nuclear lncRNAs function differently according to the cellular distribution of lncRNAs [9][10][11]. Cytoplasmic lncRNAs often regulate protein biosynthesis [12], while nuclear lncRNAs tend to affect mRNA transcription and/or alternative splicing [13,14].…”

Long Noncoding RNA Discovery in Cardiovascular Disease

“…A CRISPR activation screen from the same group also found 11 lncRNAs that mediate resistance to BRAF (B-Raf proto-oncogene, serine/threonine kinase) inhibitors and that most of them seem to function by influencing their local transcriptional environment. 91 These results highlight the fact that neighboring gene regulation by lncRNAs may be more common and more complex than thought previously. It is clear that multiple approaches may be required to glean the mechanism of action for a given lncRNA and assess its potential contributions to local gene regulation.…”

High-Throughput Approaches to Pinpoint Function within the Noncoding Genome

“…A 170,262 gRNA library was synthesized to target the TSS of 16,401 lncRNAs to identify hits playing a role in cell growth (Liu et al, 2017b). With CRISPRa, a 95,958 gRNA library was synthesized to target the TSS of 10,504 lncRNAs to identify key lncRNAs whose expression conferred resistance to a cancer therapy, including one lncRNA shown to regulate four nearby genes (Joung et al, 2017b). Similar to lncRNAs, pooled screens have been performed to interrogate microRNAs (Chen et al, 2015; Sanjana et al, 2014; Wallace et al, 2016).…”