Values used in the current report.

<p>Values used in the current report.</p

Comparative transcriptional chase analyses of a long-lived test mRNA.

<p>(A) Aggregate analysis using the conventional method. Cell aliquots were amended with dox at t₀, and sacrificed at defined intervals. Levels of β^WT mRNA were determined by RT-qPCR relative to control dox-indifferent β-actin mRNA, using the ΔΔCt method. Normalized RT-qPCR values for β^WT mRNA were corrected for aliquot-specific cell numbers, then plotted. Points represent the mean ± S.D. from three replicate experiments. A t_1/2 value was calculated from the exponential decay constant corresponding to the best-fit curve. (B) Aggregate analysis using the reverse-chase method. Cell aliquots were amended with dox at defined intervals and sacrificed simultaneously at t = 80 h. Normalized values for β^WT mRNA were determined by RT-qPCR, then plotted. Points represent the mean ± S.D. from three replicate experiments. A t_1/2 value was calculated from the exponential decay constant corresponding to the best-fit curve, corrected for an expansion factor describing the growth rate of cultured HeLa cells. (C) Analyses of individual replicates using the conventional method. Normalized values for each of three biological replicates reported in panel A were corrected for the number of cells present in each aliquot at the time of sacrifice, and t_1/2 values calculated. (D) Analyses of individual replicates using the reverse-chase method. Normalized values for each of three biological replicates reported in panel B were directly plotted, and t_1/2 values calculated following correction for interval cell expansion.</p

Effects of cell doubling time on the apparent t_1/2 value of a study mRNA.

<p>(A) Hypothetical mRNA decay. Cell aliquots, each originally containing 1.0×10⁶ cells, are incubated for defined intervals between 0 and 24 hours. Cells in each aliquot double every 6 hours, in parallel with the total amount of β-actin mRNA. At T = 0, each aliquot contains an equal quantity [1 arbitrary unit (au)] of an infinitely stable β-globin mRNA, encoded by a gene that has been transcriptionally silenced. The decline in the ratio of the globin:actin mRNAs, which erroneously indicates a half-life value of 6 hours for the globin mRNA, fails to account for interval expansion in cell number. These same principles apply to mRNAs with finite stabilities (panel B). (B) True and uncorrected t_1/2 values in cells with different doubling times. Curves illustrate the uncorrected half-life for a test mRNA, following transcriptional silencing of its encoding gene, if the interval expansion in cell number is not considered. The examples utilize an mRNA with a true t_1/2 = 6 h, expressed in cells that are growth arrested (curve <i>a</i>) or display doubling times of 24, 8, 6, or 4 hours (curves <i>b</i> through <i>e</i>, respectively).</p

HeLa cell growth is not affected by doxycycline supplement.

<p>Values are the mean of three independent cultures; error bars indicate ±1 S.E.</p

Experimental schemata for transcriptional chase analyses of mRNA stability.

<p>(A) Conventional chase method. Identical aliquots cultured in doxycycline (dox)-supplemented media (thick line) are sacrificed at defined intervals (arrowheads). A hypothetical 80-hr chase experiment is illustrated. (B) Reverse-chase method. Identical aliquots cultured in dox-free media (thin line) are amended with dox at defined intervals, and sacrificed simultaneously at the conclusion of the experiment.</p

HeLa cell expansion under transcriptional chase conditions.

<p>Data from dox-supplementation experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040827#pone-0040827-g002" target="_blank">Fig. 2</a> (1 µg/mL) was regressed to an exponential function, and expansion factors defined [j = 0.0224 (h) or 0.0004 (min)].</p

Rbm38 activates Exon 16 in Epb41 minigenes.

<p>A) Schematic depicting a portion of the mouse protein 4.1R (Epb41) gene from exons 13 to 17 (Upper panel). In early erythroid differentiation, exons 14-16 are skipped, and in late erythroid differentiation exon 16 is included. Dashed lines indicate the position of exon 13 and 17 truncations in the minigenes described below. Closed and open arrows indicate primer binding sites that specifically amplify mRNA expressed from the minigenes and endogenous Epb41 locus, respectively. 4.1wt is a 1.2 kb minigene with indicated exonic and intronic sequences (Middle panel). Minigene 4.1Δhex lacks all three UGCAUG Rbfox2 binding motifs due to a 186-nt deletion within intron 16 (Lower panel). B) Co-transfection of minigenes and Empty vector (EV), Rbm38-FF, Rbfox2-FF, or both Rbm38-FF and Rbfox2-FF. <i>Upper </i><i>panel</i>, expression of Rbm38 and Rbfox2 promotes inclusion of Exon 16 in the 4.1wt minigene. When Rbm38 and Rbfox2 are expressed together, there is no further enhancement of exon 16 inclusion. <i>Lower </i><i>panel</i>, expression of Rbm38 promotes inclusion of Exon 16 in the 4.1 Δhex, while Rbfox2 has little effect. When Rbm38 and Rbfox2 are expressed together, activation of exon 16 splicing is similar to Rbm38 alone. We also noted a small band above the exon 16 inclusion product in Rbm38 lanes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078031#pone-0078031-g003" target="_blank">Figure 3B</a>, asterisk). This band was sequenced and found to contain exon 13, a retained intronic sequence downstream of exon 13, a region from intron 15, and exon 17. Exon 16 was not included. C) RT-PCR of endogenous EPB41 exon 16 inclusion in response to transfection of EV, Rbm38, Rbfox2, or both Rbm38 and Rbfox2. Percent exon inclusion is indicated below each lane. RT-PCR product sizes are provided to aid the reader in distinguishing minigene Epb41 from endogenous EPB41. D) Western blot analysis of Rbm38 and Rbfox2 protein expression levels. Actin was used as a loading control. </p

RBM38 is expressed during late erythroid differentiation and RT-PCR analysis of a subset of RBM38-regulated microarray targets in erythroid differentiated cells.

<p>A) Western blot detection of RBM38 in erythroid differentiated CD34⁺ cells on days 2, 5, 7, and 11. (upper panel). Actin is shown as a loading control (lower panel). B) Quantitative RT-PCR of RBM38 mRNA expression levels during erythroid differentiation on days 3 to 13. C) Western blot detection of RBM38 in erythroid or granulocyte/monocyte differentiated CD34⁺ cells on days 7 and 10. SuperSignal West Femto Chemiluminescent ECL reagent was used to detect lower levels of RBM38 in early erythroid cells. D) Semi-quantitative RT-PCR analysis of RBM38 microarray targets ZDHHC18, ISOC2, and GUSB in erythroid differentiated cells. Average percentages of exon inclusion and standard deviations from three experiments are indicated below a representative gel. For the middle time point, D7/8, average and standard deviation are calculated from one Day 7 and two Day 8 replicate samples. </p

Direct tethering of Rbm38 to an intronic position downstream of a regulated exon activates splicing.

<p>A) Schematic of the PKC-40b-2xBoxB FGFR2 reporter minigene used in the lambda N-Box B tethering system. The minigene has a weak splice site, which promotes basal 40b exon skipping. The RNA sequence of Nut R Box B is provided in Materials and Methods B) Map of C- and N-terminal λN protein expression vectors. The amino acid sequence of lambda N peptide is provided in Materials and Methods C) Activation of 40b exon was examined by RT-PCR using RNA extracted from 293T cells transiently co-transfected for 48 h with expression vector: empty vector control (EV), Rbm38-FF, Rbm38-FF N-λN, or Rbm38-FF C-λN and minigene: no Box B insert or 2x Box B. Percent exon inclusion is provided below each lane. D) Western blot analysis of Flag tagged Rbm38 proteins used in (C). </p

EPB41 exon 16 splicing increases during erythroid differentiation and exon 16 splicing is regulated by RBM38.

<p>A) Semi-quantiative RT-PCR detection of the activation of EPB41 exon 16 during late erythroid differentiation.).Average percentages of exon inclusion with standard deviations compiled from three experiments are indicated below a representative gel. For the middle time point, D7/8, average and standard deviation are calculated from one Day 7 and two Day 8 replicate samples. B) Semi-quantitative RT-PCR analysis of EPB41 exon 16 inclusion in MCF-7 cells after siRNA mediated knockdown of RBM38 from three biological replicate experiments. (C) RT-PCR analysis of EPB41 exon 16 splicing in response to knockdown of RBM38 in RL-7 cells RT-PCR products in RL-7 blood cell line were uncut or digested with BstE II (labeled as B) to detect presence of exon 14. D) Western blot detection of siRNA knockdown of RBM38 in RL-7 cells. Asterisk indicates a higher molecular weight RT-PCR product that is slightly enhanced in lanes with high RBM38 expression. </p