Abstract
Identification of cell fate-controlling lncRNAs is essential to our understanding of molecular cell biology. Here we present a human genome-scale forward-genetics approach for the identification of lncRNAs based on gene function. This approach can identify genes that play a causal role, and immediately distinguish them from those that are differentially expressed but do not affect cell function. Our genome-scale library plus next-generation-sequencing and bioinformatic approach, radically upscales the breadth and rate of functional ncRNA discovery. Human gDNA was digested to produce a lentiviral expression library containing inserts in both sense and anti-sense orientation. The library was used to transduce human Jurkat T-leukaemic cells. Cell populations were selected using continuous culture ± anti-FAS IgM, and sequencing used to identify sequences controlling cell proliferation. This strategy resulted in the identification of thousands of new sequences based solely on their function including many ncRNAs previously identified as being able to modulate cell survival or to act as key cancer regulators such as AC084816.1*, AC097103.2, AC087473.1, CASC15*, DLEU1*, ENTPD1-AS1*, HULC*, MIRLET7BHG*, PCAT-1, SChLAP1, and TP53TG1. Independent validation confirmed 4 out of 5 sequences that were identified by this strategy, conferred a striking resistance to anti-FAS IgM-induced apoptosis.
Highlights
The first stage in this strategy was the preparation of a lentiviral library containing most of the human genome in both sense and antisense orientations and its transduction into the human T-leukaemic Jurkat clone JKM1, which is highly sensitive to apoptosis induced through ligation of the Fas receptor[66]
Considering selection at the gene level, rather than based upon the isolation of identical sequences contained within, we identified 11 long non-protein coding RNAs (lncRNAs) with known causal roles in cancer selected in both the d47, MFZ and d47 + anti-FAS, MF conditions including AC084816.1*1, AC097103.22, AC087473.11, CASC15*3–7, DLEU1*8–13, ENTPD1-AS1*1, HULC*14–22, MIRLET7BHG*1, PCAT123–33, SChLAP134–41, and TP53TG142–45
Investigation of long non-protein coding RNAs has become a key area in biological and biomedical research. The analysis of this vast number of transcripts is still at an early stage, it is evident that many lncRNAs play crucial roles in molecular cell biology
Summary
Non-protein-coding sequences in the genome play crucial functional roles in a range of different cellular processes[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47]. Demonstrations of the functional importance of individual RNAs are impressive and growing, the vast majority—many thousands—of lncRNAs are still entirely uncharacterised; some of these currently uncharacterised lncRNAs are likely to play critical roles in important control processes that have yet to be revealed This provides strong motivation for the development of high-throughput strategies for the identification of key lncRNAs by targeting their functional activity. We describe a genome-scale strategy that builds upon these observations and identifies critical sequences through their functional effects on cell proliferation and survival Our approach identifies those genes that play a causal role, and immediately distinguishes this group from those that are differentially expressed but do not affect cell function—this represents a key advantage over expression level studies. In addition to this new information on genes with clear potential in physiology and pathology, these observations provide proof of principle for the application of this novel strategy to the identification of lncRNAs that play rate-limiting roles in many different cellular activities
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