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

Mammalian expression and purification of Rbm38, followed by SELEX-Seq analysis to identify an RBM38 binding motif.

<p>A) Schematic of mammalian expressed Rbm38-FF-SBP purification steps. Mammalian 293T cells were transiently transfected for 48 h and protein was extracted using RIPA buffer. RIPA extract was added to Streptavidin resin and washed as indicated. The SBP tag was cleaved by incubation with TEV protease. Purified Rbm38-FF was used for SELEX-Seq. B) Map of FF-SPB tagged mammalian protein expression vector used in this study. Coomassie stain (C) and western blot (D) analysis of samples collected during purification of Rbm38-FF-SBP. E) Schematic for SELEX-Seq protocol. SELEX-Seq 7-mer motifs identified after two and three rounds of selection. </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

Validation of microarray predicted RBM38-regulated splicing events.

<p>A) Semi-quantitative RT-PCR analysis of HJAY microarray targets in MCF-7 cells. Average percentages of exon inclusion with standard deviations compiled from three experiments are indicated below a representative gel. . For AP1G2, only two replicates were included in analyses. B) Western blot detection of siRNA knockdown of RBM38 in MCF-7 cell line (upper panel). Actin is shown as a loading control (lower panel). C) Bar graph representing data from panel A. siControl and siRBM38 treated cells are shown in light and dark gray bars, respectively (<i>P</i>-values of ≤ 0.05 are annotated with an asterisk).</p