The mammalian cell cycle is a complex and tightly controlled event. Myriads of different control mechanisms are involved in its regulation. Long non-coding RNAs (lncRNA) have emerged as important regulators of many cellular processes including cellular proliferation. However, a more global and unbiased approach to identify lncRNAs with importance for cell proliferation is missing. Here, researchers from Hannover Medical School present a lentiviral shRNA library-based approach for functional lncRNA profiling. They validated their library approach in NIH3T3 (3T3) fibroblasts by identifying lncRNAs critically involved in cell proliferation. Using stringent selection criteria the researchers identified lncRNA NR_015491.1 out of 3842 different RNA targets represented in our library. They termed this transcript Ntep (non-coding transcript essential for proliferation), as a bona fide lncRNA essential for cell cycle progression. Inhibition of Ntep in 3T3 and primary fibroblasts prevented normal cell growth and expression of key fibroblast markers. Mechanistically, they discovered that Ntep is important to activate P53 concomitant with increased apoptosis and cell cycle blockade in late G2/M. Our findings suggest Ntep to serve as an important regulator of fibroblast proliferation and function. In summary, this study demonstrates the applicability of an innovative shRNA library approach to identify long non-coding RNA functions in a massive parallel approach.
Experimental design and selection strategy for the identification
of lncRNAs essential for proliferation
(a) Schematic workflow of proliferation-based lncRNA shRNA library screen in 3T3 cells. (b) Library infection quality control: Scatter plot of the log 2-transformed DNA sequencing read values from baseline sample (5 days post library infection) normalized to 20 million reads versus the sequencing reads of the input pooled plasmid library normalized to 20 million reads. (c) Sequencing reads of shRNA barcodes against lncRNA NR_015491.1 (Ntep) in CFSE-low and -high sub-populations. Data are fold-change (FC) relative to barcode reads of shRNAs in each sub-population against luciferase control. (d) Scheme of the selection strategy to narrow down initial lncRNA candidates that influence proliferation. UCE, ultraconserved elements