Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells

Abstract

Development of transcriptional pulsing approaches using the c-fos and Tet-off promoter systems greatly facilitated studies of mRNA turnover in mammalian cells. However, optimal protocols for these approaches vary for different cell types and/or physiological conditions, limiting their widespread application. In this study, we have further optimized transcriptional pulsing systems for different cell lines and developed new protocols to facilitate investigation of various aspects of mRNA turnover. We apply the Tet-off transcriptional pulsing strategy to investigate ARE-mediated mRNA decay in human erythroleukemic K562 cells arrested at various phases of the cell cycle by pharmacological inhibitors. This application facilitates studies of the role of mRNA stability in control of cell-cycle dependent gene expression. To advance the investigation of factors involved in mRNA turnover and its regulation, we have also incorporated recently developed transfection and siRNA reagents into the transcriptional pulsing approach. Using these protocols, siRNA and DNA plasmids can be effectively cotransfected into mouse NIH3T3 cells to obtain high knockdown efficiency. Moreover, we have established a tTA-harboring stable line using human bronchial epithelial BEAS-2B cells and applied the transcriptional pulsing approach to monitor mRNA deadenylation and decay kinetics in this cell system. This broadens the application of the transcriptional pulsing system to investigate the regulation of mRNA turnover related to allergic inflammation. Critical factors that need to be considered when employing these approaches are characterized and discussed.

FIGURE 1.

FACS analysis of K562III-2 cells arrested using cell cycle blockage reagents. K562 III-2 cells were cultured in the absence (Panel A, proliferating) or in the presence of indicated cell cycle inhibitors for 24 h (Panel B, aphidicolin treatment; Panel C, nocodazole treatment; Panel D, colchicine treatment; see Materials and Methods for treatment detail). Cells were then harvested for fixation followed by flow cytometry to determine the degree of cell synchronization at specific phases. The brackets indicate the cell cycle parameters, with “I” being the population of apoptotic cells (fragmented DNA), “B” being the population of cells in G₁ phase, “C” being the population of cells in S phase, and “D” being the population of cells in G₂/M phases.

FIGURE 2.

The destabilization function of AREs can be monitored at various phases of the cell cycle. K562 III-2 cells were electroporated with pTetBBB+ARE bearing either (A) the c-fos ARE or (B) the GM-CSF ARE. Transfected cells were kept in medium containing 40 ng/mL tetracycline (Tet) and the indicated cell cycle inhibitor for 24 h to synchronize cells at the desired phase. Cells were then induced for transcriptional pulsing as described in Materials and Methods. Cytoplasmic RNA was isolated immediately for the zero time point or at various time points after Tet (500 ng/mL) was added as indicated. RNA samples were analyzed by Northern blotting. Poly(A)⁻ RNA was prepared in vitro by treating the zero time point sample with oligo(dT) and RNase H. (BBB+ARE) β-Globin mRNA bearing an ARE; (GAPDH) glyceraldehyde-3-phosphate dehydrogenase mRNA served as an internal control. Semi-log plots were presented in the bottom to show the decay kinetics for BBB+ARE mRNA. Quantification of RNA was obtained by scanning radioactive blots with an imager (Bio-Rad).

FIGURE 3.

DNA and siRNA can be cotransfected effectively without compromising the knockdown efficiency. (A) Western blots of whole-cell lysates showing knockdown of UNR protein expression by the specific (UNR) siRNA and not by the nonspecific (ns) siRNA. DNA transfection was carried out at 8 h after siRNA transfection (lanes 3,4) or at the same time (lane 5) as the siRNA was transfected. (B, Left and middle panels) Northern blots showing decay and deadenylation of BBBspc+cd87 mRNA isolated from cells cotransfected with the DNA encoding pBBBspc+cd87 and either nonspecific siRNA (left) or UNR-specific siRNA (middle). The plasmid pSV-BBB, from which β-globin was expressed constitutively (SV-BBB) and which served as an internal standard, was cotransfected at the same time. (Right panel) Western blots of whole-cell lysate showing knockdown of UNR protein expression by the specific (UNR) siRNA and not by the nonspecific (NS) siRNA.

FIGURE 4.

Test for the optimal induction time and reporter plasmid levels for the Tet-off promoter system using PolyFect reagent. (A) NIH3T3 B2A2 cells were transfected with 1 μg of pTetBBB-PTC and 1 μg of pSV α-globin/GAPDH in the presence of Tet (25 ng/mL). Cytoplasmic RNA was isolated at various times after removal of tetracycline as indicated. (B) NIH3T3 B2A2 cells transfected with different amounts of pTetBBB-PTC were transcriptionally pulsed as described in Materials and Methods, and transcription was shut down after 110 min by adding 500 ng/mL tetracycline to culture medium. The number above each lane corresponds to hours (hr) after tetracycline addition. α-Globin/GAPDH mRNA was expressed constitutively and served as an internal standard for transfection efficiency and sample handling. (C) Semi-log plot showing the decay kinetics for BBB-PTC mRNA. Quantification of RNA was obtained by scanning radioactive blots with an imager (Bio-Rad).

FIGURE 5.

A decay intermediate with full-length poly(A) tail can be detected using the “consecutive-knockdown” approach. (A) Western blot analysis showing the expression of HA-tagged mutant PAN2 and knockdown of DCP2 protein expression in NIH3T3 B2A2 cells (“+” indicates the DNA or siRNA was transfected once, and “++” indicates the siRNA was transfected twice). The blot was probed with antibodies against HA-tag, DCP2, and GAPDH. (B–F) Northern blots showing deadenylation and decay of nonsense codon containing β-globin (BBB-PTC) mRNA in the (B,C) absence or (D,F) presence of ectopically expressed mutant PAN2, or combined with siRNA-mediated (E) single or (F) consecutive knockdown of DCP2. α-Globin/GAPDH mRNA was expressed constitutively and served as an internal standard for transfection efficiency and sample handling.

FIGURE 6.

Expression kinetics of the Tet-regulated promoter in human bronchial epithelial BEAS-2B cells. (A) Regulation of expression by tetracycline in the human bronchial epithelial BEAS-2B stable line harboring tTA. The BEAS-2B stable clone 19 (BEAS-2B-19) was transfected with pTet-Flag-eGFP. After transfection, cells were split into four dishes and grown for 48 h in various concentrations of tetracycline (Tet) as indicated. Total cytoplasmic lysates were isolated, and Western blot analysis was performed. Blots were then probed with an anti-Flag antibody for the eGFP and an anti-tubulin antibody for tubulin. (B) Kinetics of transcription resumption when tetracycline is removed. BEAS-2B-19 cells were grown in the presence of tetracycline (30 ng/mL). At 48 h after transient cotransfection with plasmids pTet-BBB and pSVα1/GAPDH (control), expression of the Tet-regulated gene was induced by replacing tetracycline-containing medium with fresh medium without tetracycline. Total cytoplasmic RNA was isolated at the times indicated at the top and subjected to Northern blot analysis. (C) Transcriptional pulsing achieved by modulating tetracycline in the culture medium. Transient mRNA expression from the Tet-regulated Flag-eGFP gene was induced for 110 min by transferring the cells to fresh culture medium without tetracycline, followed by addition of 500 ng/mL tetracycline to the medium to block transcription for the indicated time intervals to monitor mRNA decay analyzed by Northern blotting. In this blot, β-globin mRNA (SV-BBB) constitutively transcribed from cotransfected plasmid pSVBBB served as an internal standard.