Growth arrest on inhibition of nonsense-mediated decay is mediated by noncoding RNA GAS5

Abstract

Nonsense-mediated decay is a key RNA surveillance mechanism responsible for the rapid degradation of mRNAs containing premature termination codons and hence prevents the synthesis of truncated proteins. More recently, it has been shown that nonsense-mediated decay also has broader significance in controlling the expression of a significant proportion of the transcriptome. The importance of this mechanism to the mammalian cell is demonstrated by the observation that its inhibition causes growth arrest. The noncoding RNA growth arrest specific transcript 5 (GAS5) has recently been shown to play a key role in growth arrest induced by several mechanisms, including serum withdrawal and treatment with the mTOR inhibitor rapamycin. Here we show that inhibition of nonsense-mediated decay in several human lymphocyte cell lines causes growth arrest, and siRNA-mediated downregulation of GAS5 in these cells significantly alleviates the inhibitory effects observed. These observations hold true for inhibition of nonsense-mediated decay both through RNA interference and through pharmacological inhibition by aminoglycoside antibiotics gentamycin and G418. These studies have important implications for ototoxicity and nephrotoxicity caused by gentamycin and for the proposed use of NMD inhibition in treating genetic disease. This report further demonstrates the critical role played by GAS5 in the growth arrest of mammalian cells.

Figure 1

UPF1-specific siRNA increases cell death and inhibits cell proliferation in the human T-cell lines Jurkat and CEM-C7 and in the B-lymphoblastoid cell line BJAB. (a) Cells were transfected with 3 different specific UPF1 siRNAs or negative control siRNA ((−)siRNA) and cultured at 37°C. After 72 h the expression of endogenous UPF1 in the siRNA transfected cells was determined by real-time RT-PCR (mean ± s.e.m. from three separate experiments) and expressed relative to untransfected controls. ((b)–(d)) Cells were transfected with UPF1-siRNA1 or negative control siRNA ((−)siRNA) and cultured at 37°C. Viable cell numbers were determined after 48 h (b) and 72 h (c) by the LIVE/DEAD assay (Section 2.2). Results are represented as mean ± s.e.m. from three independent experiments. (d) Cell proliferation was measured after 48 h using the BrdU colorimetric ELISA assay. Results are represented as mean ± s.e.m. from three independent experiments. *P < 0.01 compared with (−)siRNA.

Figure 2

Down-regulation of UPF1 increases GAS5 mRNA levels. The human T-cell lines Jurkat and CEM-C7 and in the B-lymphoblastoid cell line BJAB were transfected with specific UPF1siRNA1 or negative control siRNA ((−)siRNA) and cultured at 37°C. 48 h, 72 h, and 96 h after transfection, and the expression of endogenous GAS5 mRNA in the siRNA transfected BJAB (a), Jurkat (b), and CEM-C7 (c) was determined by real-time RT-PCR using the comparative C _T method, normalised with 18S RNA as an internal control. The results are represented as the mean ± s.e.m. from three separate experiments. *P < 0.01 compared with (–)siRNA.

Figure 3

Down-regulation of GAS5 alleviates the inhibitory effects of UPF1 siRNA on cell viability and proliferation. CEM-C7, Jurkat, and BJAB cells were transfected with either control (−)siRNA or UPF1-siRNA1. 24 h after transfection, control cells and cells transfected with UPF1 siRNA were transfected with (−)siRNA or GAS5 siRNAs. Viable cell number (a) and cell proliferation (b) were determined after 72 h. Results are calculated as percentage viable cell number and absorbance relative to controls transfected with (−)siRNA. Data represent means ± s.e.m. from three independent experiments. *P < 0.01 compared to UPF1 siRNA, (−)siRNA1-transfected cells.

Figure 4

Down-regulation of GAS5 alleviates the inhibitory effects of aminoglycoside antibiotics on the colony forming ability of the human T-cell lines Jurkat and CEM-C7 and the B-lymphoblastoid cell line BJAB. CEM-C7, Jurkat, and BJAB cells were transfected with either control (−)siRNA or GAS5 siRNA2. 48 h after transfection, transfected cells were treated with gentamycin (100 μg/mL) (a), G418 (0.5 μg/mL) (b), or G418 (0.3–0.5 μg/mL) on BJAB cells (c) for 72 h. Colony forming ability was then determined by plating in soft agar. Results are calculated as percentage colony numbers relative to controls incubated in the absence of aminoglycoside antibiotics. Means ± s.e.m. from four independent experiments are shown. *P < 0.01 compared to (−)siRNA.