Whole-genome-scale identification of novel non-protein-coding RNAs controlling cell proliferation and survival through a functional forward genetics strategy

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.

Figure 1

Experimental overview—(A) Human DNA was digested and cloned into pCDH-CMV4. (B) Jurkat clone JKM1 cells were transduced with our lentiviral library (CL3c) and harvested immediately following transduction (d0, Group = JCPZ), following 47 days of continuous culture (d47, Group = MFZ), or following 47 days of continuous culture followed by anti-FAS IgM selection for 96 h (d47 + anti-FAS, Group = MF). Four independent replicates were prepared for each condition (d0, d47 and d47 + anti-FAS).

Figure 2

Bioinformatics overview—Insert sequences were amplified by PCR and sequenced using the Illumina HiSeq system with a 300 bp paired end read metric. A custom bioinformatic analysis workflow was developed to enable appraisal of the inserts within cells prior to (d0, JCPZ) and (in subsequent experiments) following selection (d47, labelled MFZ, and d47 + anti-FAS, labelled). This workflow validated inserts as “library-derived” through the presence of flanking vector sequence (recalling that the library was human in origin and transfected into human cells), confirmed the presence of an appropriate restriction site (DraI or AanI), and provided an indication of insert frequency, in addition to reporting the likely direction of insertion (and thus transcription). The presence of selected inserts across multiple experimental replicates was used to prioritise sequences for further validation.

Figure 3

Jurkat proliferation rate—Continuous culture of transduced Jurkat cultures revealed a gradual increase in their proliferation rate (ergo decrease in doubling time). Mean viable cell counts from all 4 replicates at the end of each 2–3 days culture period are shown for each time point.

Figure 4

BAMFingerPrint analysis—Proportion of the entire human genome covered by sequence data is shown. Considering reads contained within 97% of all genomic bins (inset panel) revealed that > 85% (84.5–85.8) and 72.6% (71.4–74.3) of the total read count was reached for the CL3c samples (library before transduction) and JCPZ, d0 samples suggesting a broad distribution of reads from across the entire genome. In contrast, just 24.6% of the total read count was reached for those cells subjected to continuous culture (d47, MFZ) consistent with marked selection.

Figure 5

Frequency of inserts at defined coverage levels—Initial library (CL3c) and d0 transduced (JCPZ) samples presented relatively homogenously with the vast majority of inserts having a coverage of 20 or less reads per million. In contrast, the selected cell populations (d47 MFZ and d47 + anti-FAS MF) presented with a large increase in focused presence, and a very large increase in the maximal presence values noted. Maximal presence was increased dramatically in the selected sample sets; CL3c (1466), d0 (433), increasing to d47, MFZ (32,555), d47 + anti-FAS (28,055) [d47 + anti-FAS, MF_NoD (36,988)], suggesting the existence of large cell sub-populations that harbour a specific insert conferring a proliferation/survival advantage. The frequency of inserts at each coverage level in the CL3c library is indicated on all panels by way of a blue line to enable comparison between the initial library and the selected samples. The Y axis terminates at a frequency of ≥ 100 for clarity of data presentation—complete data presented in Supplementary Fig. S5.

Figure 6

Independent validation—Jurkat cells (5 × 10⁵ cells/ml) were transfected with our candidate functional sequences (see Table 7) in custom pcDNA3.1 plasmids and challenged with 10–20 ng/ml anti-Fas IgM. Viable (Trypan blue-excluding) viable cell concentrations were determined 7 to 18 days after the addition of antibody (see “Materials and methods” section). The mean viable cells ± standard deviation of 3 replicate cultures are shown. In 4 out of 5 cases, transfected cultures were significantly increased over empty vector transfected controls run in parallel (*p < 0.05).