The RNA Degradation Pathway Regulates the Function of GAS5 a Non-Coding RNA in Mammalian Cells

<div><p>Studies of various mRNAs have revealed that changes in the abundance of transcripts, through mRNA degradation, act as a critical step in the control of various biological pathways. Similarly, the regulation of non-coding RNA (ncRNA) levels is also considered to be important for their biological functions; however, far less is known about the mechanisms and biological importance of ncRNA turnover for the regulation of ncRNA functions. The growth arrest-specific 5 (GAS5) ncRNA accumulates during growth arrest induced by serum starvation and its transcript is degraded by the well characterized nonsense-mediated RNA decay (NMD) pathway. Historically, NMD was discovered as a RNA quality control system to eliminate aberrant transcripts; however, accumulating evidence shows that NMD also regulates the abundance of physiological transcripts. Interestingly, the GAS5 transcript has the ability to bind the glucocorticoid receptor (GR), resulting in the inhibition of its ligand-dependent association with DNA. The GR binds the promoters of various glucocorticoid-responsive genes, including apoptosis-related genes. In this study, we examined whether the RNA degradation pathway can regulate this function of GAS5. We measured the steady-state abundance and the decay rate of GAS5 in UPF1-depleted human cells using the 5′-bromo-uridine immunoprecipitation chase (BRIC) method, an inhibitor-free method for directly measuring RNA stability. We found that levels of the GAS5 transcript were elevated owing to prolonged decay rates in response to UPF1 depletion, and consequently the apoptosis-related genes, cIAP2 and SGK1, were down-regulated. In addition, serum starvation also increased the transcript levels of GAS5 because of prolonged decay rates, and conversely decreased levels of cIAP2 and SGK1 mRNA. Taken together, we found that the RNA degradation pathway can regulate the function of the GAS5 ncRNA in mammalian cells.</p> </div

Decay rate of GAS5.

<p>(A) Schematic representation of 5′-Bromo-uridine immunoprecipitation chase (BRIC). BrU is incorporated into newly transcribed RNA in place of uridine. Cells are collected and total RNA extracted at multiple time points after BrU washout. BrU-labeled RNAs are immunopurified using anti-BrdU beads. Non-labeled RNAs are discarded. Then, BrU-labeled RNAs are quantified by RT-qPCR. (B) The decay rate of GAS5 as determined by BRIC in HEK293T cells. Relative quantitative values at time 0 h were set to 100%. Values represent mean ± errors obtained from two independent experiments.</p

Serum starvation increases the expression level and prolongs the decay rate of GAS5, and decreases the expression of glucocorticoid-responsive genes.

<p>HEK293 cells were cultured with 0, 0.5, and 10% serum as indicated. (A) The relative expression levels of GAS5 in cells with 0 and 0.5% serum relative to control cells (cultured with 10% serum) were determined by RT-qPCR. (B) The decay rates of GAS5 were determined using the Click-iT Nascent RNA Capture Kit (Invitrogen) in control cells (solid circle and black bar) and in serum-starved cells (open circle and grey bar). Relative quantitative values at 0 h were set to 100%. (C) The relative expression levels of glucocorticoid-responsive genes, cIAP2 and SGK1, and the PPARδ-responsive gene, ADRP, in cells with 0 and 0.5% serum relative to control cells (cultured with 10% serum) were determined by RT-qPCR. (A–C) The levels of GAPDH and ACTB were used for normalization. Values represent mean±SD obtained from two (in B) or three (in A and C) independent experiments (*<i>P</i><0.05, **<i>P</i><0.01, Student's t test).</p

Knockdown of GAS5 suppresses the effects of serum withdrawal on the expression of glucocorticoid-responsive genes.

<p>HEK293 cells were treated with a control siRNA or with a siRNA targeting GAS5, and cultured either with or without serum for 48 h. (A, B) The relative expression levels of the indicated RNAs in cells with serum (black bar) or without serum (grey bar) relative to control (cells cultured at 0 h) were determined by RT-qPCR. The levels of GAPDH and ACTB were used for normalization. Values represent mean±SD obtained from three independent experiments (*<i>P</i><0.05, **<i>P</i><0.01, Student's t test).</p

Knockdown of UPF1 increases the expression level and prolongs the decay rate of GAS5, and decreases the expression of glucocorticoid-responsive genes.

<p>HEK293T cells were treated with a control siRNA or with a siRNA targeting UPF1. (A) The expression levels of UPF1 in control cells (black bar) and in UPF1-depleted cells (grey bar) were determined by RT-qPCR. (B) The expression levels of GAS5 and MALAT1 in control cells (black bar) and in UPF1-depleted cells (grey bar) were determined by RT-qPCR. (C) The decay rates of GAS5 and MALAT1 were determined by BRIC in control cells (solid circle and black bar) and in UPF1-depleted cells (open circle and grey bar). Relative quantitative values at 0 h were set to 100%. (D) The expression levels of glucocorticoid-responsive genes, cIAP2 and SGK1, and the PPARδ-responsive gene, ADRP, in control cells (black bar) and in UPF1-depleted cells (grey bar) were determined by RT-qPCR. (A–D) The levels of GAPDH were used for normalization. Values represent mean±SD obtained from three independent experiments (*<i>P</i><0.05, **<i>P</i><0.01, Student's t test).</p

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<p>The MALAT1 long noncoding RNA is strongly linked to cancer progression. Here we report a MALAT1 function in repressing the promoter of p53 (TP53) tumor suppressor gene. p21 and FAS, well-known p53 targets, were upregulated by MALAT1 knockdown in A549 human lung adenocarcinoma cells. We found that these upregulations were mediated by transcriptional activation of p53 through MALAT1 depletion. In addition, we identified a minimal MALAT1-responsive region in the P1 promoter of p53 gene. Flow cytometry analysis revealed that MALAT1-depleted cells exhibited G1 cell cycle arrest. These results suggest that MALAT1 affects the expression of p53 target genes through repressing p53 promoter activity, leading to influence the cell cycle progression.</p

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<p>The MALAT1 long noncoding RNA is strongly linked to cancer progression. Here we report a MALAT1 function in repressing the promoter of p53 (TP53) tumor suppressor gene. p21 and FAS, well-known p53 targets, were upregulated by MALAT1 knockdown in A549 human lung adenocarcinoma cells. We found that these upregulations were mediated by transcriptional activation of p53 through MALAT1 depletion. In addition, we identified a minimal MALAT1-responsive region in the P1 promoter of p53 gene. Flow cytometry analysis revealed that MALAT1-depleted cells exhibited G1 cell cycle arrest. These results suggest that MALAT1 affects the expression of p53 target genes through repressing p53 promoter activity, leading to influence the cell cycle progression.</p

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<p>The MALAT1 long noncoding RNA is strongly linked to cancer progression. Here we report a MALAT1 function in repressing the promoter of p53 (TP53) tumor suppressor gene. p21 and FAS, well-known p53 targets, were upregulated by MALAT1 knockdown in A549 human lung adenocarcinoma cells. We found that these upregulations were mediated by transcriptional activation of p53 through MALAT1 depletion. In addition, we identified a minimal MALAT1-responsive region in the P1 promoter of p53 gene. Flow cytometry analysis revealed that MALAT1-depleted cells exhibited G1 cell cycle arrest. These results suggest that MALAT1 affects the expression of p53 target genes through repressing p53 promoter activity, leading to influence the cell cycle progression.</p

Image4.tif

<p>The MALAT1 long noncoding RNA is strongly linked to cancer progression. Here we report a MALAT1 function in repressing the promoter of p53 (TP53) tumor suppressor gene. p21 and FAS, well-known p53 targets, were upregulated by MALAT1 knockdown in A549 human lung adenocarcinoma cells. We found that these upregulations were mediated by transcriptional activation of p53 through MALAT1 depletion. In addition, we identified a minimal MALAT1-responsive region in the P1 promoter of p53 gene. Flow cytometry analysis revealed that MALAT1-depleted cells exhibited G1 cell cycle arrest. These results suggest that MALAT1 affects the expression of p53 target genes through repressing p53 promoter activity, leading to influence the cell cycle progression.</p

Novel noncoding RNA candidates.

<p>Novel noncoding RNA candidates.</p