Summary

Liu et al. attempt to identify functional long non coding RNA(lncRNAs) using a technique known as CRISPRi. lncRNAs are non-protein-coding RNAs which are greater than 200 b.p in length. The function if any of the majority of these lncRNAs is yet to be determined and this paper addresses this question.

CRISPRi utilizes a guide RNA to direct a transcriptional repressor protein KRAB towards a specific lncRNA whose function is to be determined. The presence of the KRAB repressor protein turns off the transcription of the lncRNA and hence the effects of the missing lncRNA can be studied.

Liu et al. studied the effects of 16,401 lncRNAs in seven cell-lines. The functions of lncRNAs that can be tested using this approach include enhancer like function, cis and trans regulation of mRNA transcript production and cis-mediated regulation of the lncRNA transcription. The lncRNAs studied were prioritized based on their expression in the cancer and nontransformed cell lines used in the study.

499 lncRNA hits were found to increase or decrease cell-growth in the 7 cell-lines, this was determined by using a Mann Whitney U test on the growth phenotype. An increased number of hits were observed in the iPSCs, the authors verified that this was not a technical artefact. These cells appear to be more susceptible to lncRNAs targeting growth. The authors validated a representative subset of the lncRNAs by repeating the CRISPRi experiment and also verifying the knockdown using qPCR. The authors report a lncRNA PVT1 which has a pro-growth phenotype. The authors also analyzed the transcriptional response to knocking down the lncRNAs by performing RNAseq. The authors identify clusters of genes that are either upregulated or downregulated when lncRNAs that affect cell-growth are repressed. Most of the lncRNA hits were unique to one cell type. The authors suggest that the cell-type specificity could lend itself to lncRNAs being targets of cell type specific therapy.

LINC00263 is a lncRNA which the authors found to be expressed in multiple cell lines but causes a negative growth phenotype in just one of the cell lines U87. Shutting down LINC00263 in U87 resulted in upregulation of transcriptional programs related to apoptosis and ER stress but not in K562 cells and HELA cells. lncRNAs whose effects were similar amongst cell types resulted in similar transcriptional changes. The authors attribute the specificity of lncRNA function to the different transcriptional programs and not due to the differences in CRISPRi activity.

Identifying features of lncRNAs that affect growth phenotypes could help predict other lncRNAs which might have a similar function. Expression level of the lncRNA, distance from a FANTOM enhancer, distance from a cancer associated SNP and number of exons were identified by the authors as significant predictors of lncRNA hits.

To conclude, this study identifies 499 lncRNAs that modify growth phenotype in the cell lines tested, most of these are unique to just one cell line. lncRNAs that target other cellular phenotypes are yet to be identified.

Critical Evaluation

The cell type specificity of these growth affecting lncRNAs is an interesting observation that remains to be explained especially in the case of lncRNAs such as LINC00263 which is expressed in multiple cell types but only affects the growth phenotype in U87 cells. The target genes of the lncRNA hits identified in this study need to be followed up on to identify how they affect the growth phenotypes.