Cross-talk Between PRMT1-mediated Methylation and Ubiquitylation on RBM15 Controls RNA Splicing
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Cross-talk Between PRMT1-mediated Methylation and Ubiquitylation on RBM15 Controls RNA Splicing
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RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia.
CNOT4; PRMT1; RBM15; RNA metabolism; arginine methylation; biochemistry; human; human biology; medicine; ubiquitylation.
Conflict of interest statement
The authors declare that no competing interests exist.
Figure 1.. RBM15 is methylated by PRMT1 at R578 in mammalian cells.
A) Alignment of RBM15 sequences among different species shows R578 within a conserved protein region. ( B) RBM15-Flag and its mutant (R578K), affinity purified with anti-Flag antibody from transfected 293T cells, were detected by WB with anti-monomethyl arginine antibody. ( C) Flag-tagged RBM15 was affinity purified by Flag antibody for WB with two generic antibodies against mono-methyl arginine and dimethyl arginine. The 293T cells overexpressing wild type RBM15-Flag protein with PRMT1 V2 (lane 2) and V1 (lane 3) were treated with 20nM MG132 for 6 hr before harvesting. ( D) The differences between N terminal sequences of PRMT1 V1 and V2 isoforms. ( E) In vitro methylation assays. Affinity purified RBM15 protein was methylated by incubation with purified HA-PRMT1 and 0.15 mM of S-adenosyl-methionine at 30°C for 4 hours. The methylated RBM15 was detected by anti-dimethyl arginine antibody in WB. ( F) RBM15 was immunoprecipitated with a mouse monoclonal anti-RBM15 antibody from whole cell extract prepared from a MEG-01 stable cell line expressing inducible short hairpin RNA against PRMT1. Normal IgG was used as immunoprecipitation controls. The immunoprecipitated RBM15 was detected by WB with antibodies against mono-methyl arginine (mono-R100) and asymmetrical dimethyl arginine (D4H5). As controls, we did WB with anti-PRMT5 and anti-PRMT4 antibodies. ( G) Detection of methylated RBM15 in MEG-01-stable cell lines expressing Flag-RBM15 and R578K mutant proteins after a PRMT1 inhibitor (DB75) treatment for 24 hr. RBM15 is affinity purified by anti-Flag antibody and detected by WB with mono-methyl arginine antibody and dimethyl arginine antibody. PRMT, protein arginine methyltransferases; WB, western blot. DOI:
Figure 1—figure supplement 1.. PRMT1 is overexpressed in AMKL leukemia and associated with short survival rate in AML.
AML, acute myeloid leukemia; AMKL, acute megakaryoblastic leukemia; PRMT, protein arginine methyltransferases. (
A) PRMT1 is overexpressed in AMKL leukemia compared to other types of AML. Microarray data set (GSE4119) (Bourquin et.al. 2006) was analyzed by NCBI GEO2R tool. Expression values were exported from NCBI GEO2R tool and then re-plotted by GraphPad PRISM 5 software. ( B) PRMT1 high expression is correlated with low survival rate in acute myeloid leukemia. DOI:
Figure 1—figure supplement 2.. RBM15 is discovered as a PRMT1 substrate via BPPM.
A) Schematic description of substrate identification with PRMT1 Y39FM48G and Pob-SAM using BPPM technology. ( B) HEK293T cells were transfected with either empty vector or the PRMT1 Y39FM48G mutant, followed with treatment of Adox to induce hypomethylation. These cells were then lysed to release the PRMT1 substrates as previously described and the cell lysates were treated with Pob-SAM cofactor. The terminal alkyne-modified substrates were conjugated with the cleavable azido-azo-biotin probe, followed by streptavidin enrichment, sodium dithionite elution and western blotting detection. ( C) Western blotting analysis of the pull-down substrate of PRMT1. The eluted targets of PRMT1 were incubated with RBM15 antibody. The pull-down sample from 293T cells transfected with PRMT1 Y39FM48G variant showed significantly higher level of RBM15 compared to the control group. Cell lysates without streptavidin enrichment were assessed by anti-RBM15 western blotting as loading control (Input panel). BPPM, bio-orthogonal profiling of protein methylation; Pob-SAM, propargyloxy-but-2-enyl-S-adenosylmethionine; PRMTs, protein arginine methyltransferases DOI:
Figure 1—figure supplement 3.. Mapping the methylation site for RBM15.
A) Tandem mass spectrometry analysis for Flag-tagged RBM15 purified from 293T cells overexpressing RBM15-Flag. The arrow indicates the modified peptide. ( B) Schematic diagram shows domains on RBM15. ( C) Dot blot to test the antibody (Mono-R100 from Cell Signaling, Danvers, MA) recognizing monomethylated RBM15 peptide based on mass spectrometry analysis. Nitrocellulose membrane was spotted with peptides with no modification, with arginine mono-methylated or with arginine asymmetrically dimethylated. The peptide sequences were listed on the right side of the dot blot gel. ( D) Dot blot to test the antibody (D4H5 from Cell Signaling) which recognizes asymmetrically dimethylated RBM15 peptide. Peptides were spotted in the same order as in panel C. ( E) IP-WBwestern blot for RBM15 protein in Meg-01 cell lines expressing Flag-tagged RBM15 and Flag-tagged RBM15 R578K. Anti-monomethyl arginine antibody was used for immunoprecipitation and the ectopically expressed RBM15 proteins were detected by Flag antibody. ( F) IP-western blotting assays for detecting methylated RBM15 and R3K mutant expressed in 293T cells. RBM15-R3K stands for RBM15 protein with all the arginines in the LYRDRDRD sequence mutated to lysines. ( G) The in vitro methylation reaction with peptide (LYRDRDRDLY) incubated with purified PRMT1. H4 peptide (20 mer) was used as a positive control. The methylated peptide was detected by the D4H5 dimethyl arginine antibody not by the mono-methyl antibody. IP, immunoprecipitation protocol; PRMTs, protein arginine methyltransferases; SPOC, spen paralog and ortholog C-terminal. DOI:
Figure 1—figure supplement 4.. RBM15 methylation status is further confirmed by a methyl-RBM15 antibody.
A) Dot blot for the anti-methyl-RBM15 antibody. Synthesized RBM15 peptides with different methylation status on arginine 578 were spotted for blotting with the anti-methyl RBM15 antibody. ( B to D) RBM15 methylation status was measured by anti-methyl-RBM15 antibody by western blotting. ( B) The whole cell extracts from 293T cells expressing PRMT1 were used for western blotting with the anti-methyl RBM15 antibody. Empty vector was used as a control. ( C) The whole cell lysates from 293T cells expressing WT RBM15 or R578K mutant were used for western blotting. ( D) A stable MEG-01 cell line expressing Dox-inducible shPRMT1 was used to knock down PRMT1. The expression of methyl-RBM15 was measured by western blotting with anti-methyl RBM15 antibody, RBM15 antibody, PRMT1 antibody and GAPDH antibody. Dox, doxycycline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PRMTs, protein arginine methyltransferases; WT, wild type DOI:
Figure 2.. PRMT1 V2 isoform destabilizes RBM15 via methylation.
A) WB for RBM15 in 293T cells overexpressing PRMT1 V1 and V2. PRMT1 V2 was detected by anti-V2 specific antibody (PRMT1 V2). PRMT1 V1 and V2 were detected by an antibody for all isoforms (labeled as PRMT1). ( B) The level of the RBM15 protein as detected by WB in MEG-01 cells treated with methyltransferase inhibitors (Adox and MTA mix) or DB75. ( C) RBM15 protein level was measured by WB in MEG-01 cells with two doxycycline-inducible shRNA against PRMT1 (on the left). In the middle and right sides are real-time PCR results to show the mRNA levels of total amount of PRMT1, PRMT1 V2, and RBM15 in shPRMT1#1 stable MEG-01 cell line (middle) and in shPRMT1#2 stable MEG-01 cells (right). All data are presented as mean ± standard deviation from three independent experiments. ( D) RBM15 protein level was measured by WB in MEG-01 cells induced by Dox to express PRMT1 V2 isoform. On the right are the real-time PCR charts for PRMT1 V2 and RBM15 mRNA levels. Data are presented as mean ± standard deviation from three independent experiments. ( E) RBM15 protein level was accessed by WB in a MEG-01 stable cell line expressing shRNA against V2. The names of antibodies are listed on right. The pRS vector retrovirus infected MEG-01 cells were used as control. ( F) WB with anti-Flag antibody to detect the protein levels of RBM15 wild type and R578K mutant proteins in 293T cells overexpressing PRMT1 V2 and RBM15 proteins. ( G) The half-life of the RBM15 proteins in MEG-01 cells, and stable cell lines overexpressing Flag-tagged RBM15 and RBM15 R578K were assessed by WB. Cyclohemixide were added to stop protein synthesis 30 min before harvesting cells as the 0 time point. The half-life curves were plotted by GraphPad Prism 6. Adox, adenosine dialdehyde; DMSO, dimethyl sulfoxide; Dox, doxycycline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; MTA, methylthioadenosine; PCR; polymerase chain reaction; PRMTs, protein arginine methyltransferases; shRNA; short hairpin RNA; WB, western blot. DOI:
Figure 3.. The RBM15 is ubiquitylated in a methylation-dependent manner.
A) WB for the RBM15 protein from MEG-01 cells treated with the proteasome inhibitor MG132. ( B) The ubiquitylated RBM15-Flag was detected by anti-Flag antibody. The poly-ubiquitylated RBM15 was purified with nickel beads under denaturing conditions (6M of guanidine-HCl) from 293T cells expressing RBM15-Flag and poly-histidine-tagged ubiquitin. ( C) The ubiquitylated RBM15 was measured by anti-ubiquitin antibody after affinity purified with Flag antibody from MG132 treated 293T cells expressing RBM15-Flag or R578K-Flag and ubiquitin. ( D) IP-WB for poly-ubiquitylated RBM15 in DB75 treated MEG-01 cells. The endogenous RBM15 protein was immunoprecipitated by anti-RBM15 antibody and then blotted with anti-ubiquitin antibody and anti-RBM15 antibody. ( E) The ubiquitylated RBM15 was detected by anti-Ub antibody after RBM15-Flag as well as its mutant was affinity purified from 293T cells transfected with combinations of plasmids shown above the gel. CNOT4 was detected via its HA tag. ( F) WB to detect RBM15 protein levels in two MEG-01 cell lines expressing two different shCNOT4. PRMT1 inhibitor (DB75) was used to treat the cells expressing shCNOT4 RNAs. The efficiency of shCNOT4 knockdown was checked by real-time PCR. ( G) In vitro ubiquitylation assays with CNOT4 and RBM15. Purified PRMT1 was added to methylate RBM15 in vitro in lanes 5 and 6 first before incubating with CNOT4 for in vitro ubiquitylation assays. All components were affinity purified from 293T cells. The ubiquitylated RBM15-Flag was detected by WB with anti-Flag antibody. ( H) CNOT4 from MEG-01 whole cell extract was pulled down with methylated and nonmethylated peptides of RBM15. CNOT4 was detected by WB with anti-CNOT4 antibody. DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; IP, immunoprecipitation protocol; PCR, polymerase chain reaction; PRMTs, protein arginine methyltransferases; WB, western blot. DOI:
Figure 3—figure supplement 1.. Purified proteins used in vitro methylation and ubiquitylation assays.
A) The Flag-tagged RBM15 protein was affinity purified by anti-Flag antibody from 293T cells overexpressing FLAG RBM15 protein as shown by Coomassie staining of 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. ( B) Affinity purified HA-CNOT4 and HA-PRMT1 proteins were shown by silver staining. Both proteins were produced in 293T cells transfected with pCDNA3-HA-PRMT1 and pCDNA3-HA-CNOT4, respectively. Anti-HA antibody (12Ca5) column was used to purify these proteins. Proteins were eluted by 0.5mg/ml of HA peptide (Sigma) in PBS. HA, hemagglutinin; PBS, phosphate-buffered saline; PRMTs, protein arginine methyltransferases. DOI:
Figure 3—figure supplement 2.. The efficiency of CNOT4 knockdowns.
A) Real-time PCR assays for CNOT4 in MEG-01 cells expressing shCNOT4. ( B) WBs show the knockdown efficiencies of two shRBM15 constructs in MEG-01 cells. ( C) Using CRISPR to knockdown CNOT4 in 293T cells (A5 is a 293T cell line with only one wild type allele of CNOT4) as shown by real-time PCR on the left and by WB on the right. RBM15 protein level goes up in CNOT4 +/-− cells by WB assays. CRISPR, clustered regularly interspaced short palindromic repeat; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; PCR, polyerase chain reaction; WB, western blot. DOI:
Figure 4.. PRMT1 controls the protein level of RBM15 in MK maturation.
A) WB to measure the protein levels in MEG-01 cells stimulated to maturation by PMA. The left panel (WB results) showed the protein levels by antibodies against GAPDH, RBM15, methyl-RBM15, PRMT1 with a PRMT1 antibody against all isoforms and PRMT1 V2 with specific V2 antibody during the course of maturation. The middle panel shows the quantitation of the protein bands in the WBs on the left normalized to GAPDH. The right panel showed the decrease of PRMT1 V2 by real-time PCR during maturation with GAPDH mRNA as an internal control. Real-time PCR data were presented as means ± standard deviation from three independent experiments. ( B) Histograms of CD41 + cells on PMA-treated MEG-01 cells overexpressing RBM15 and RBM15R578K mutant proteins on day 3. The percentage of CD41 + cells was calculated according to matched antibody isotype control. Three independent experiments were done with statistics shown on the left. P*** <0.001, P** <0.01. ( C) FACS analysis of the polyploid status of PMA-treated cells overexpressing RBM15 and R578K mutant proteins by PI staining. Vector: lentivirus vector. P*** <0.001. ( D) The matured MK cells were measured by CD61 +CD42 +. Human adult CD34 + cells in pro-MK differentiation medium were treated with DB75 for 3 days. Three independent experiments were done with P*< 0.05. ( E) Human adult CD34 + cells were infected with lentivirus expressing PRMT1 V2 or lentivirus vector and grown in pro-MK differentiation medium for 5 days. Three independent experiments were done with P***<0.001. ( F) Human adult CD34 + cells were infected with two lentiviruses expressing shRNAs against RBM15 and grown in pro-MK differentiation medium. Three biological replicates were used for P value. P** <0.01. ( G) Human adult CD34 + cells were infected with lentiviruses expressing RBM15 or R578K proteins and grown in pro-MK differentiation medium for 5 days. Three independent experiments were done with P**<0.01. ( H) Human adult CD34 + cells were infected with lentiviruses expressing RBM15 or R578K together with a lentivirus expressing PRMT1 V2 and grown in pro-MK differentiation medium. Three biological replicates were used for P value. P**<0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PI, propidium iodide; PMA, phorbol myristate acetate; PRMTs, protein arginine methyltransferases; WB, western blot; WT, wild type. DOI:
Figure 4—figure supplement 1.. Relative expression levels of PRMT1 isoforms and RBM15 in different hematopoietic lineages derived from mouse and human.
A-C) The relative expression levels of total PRMT1 (including V1 and V2) ( A), PRMT1 V2 ( B) and RBM15 ( C) in different mouse lineages were measured by real-time polychromase chain reaction. The expression level is normalized to GAPDH, then normalized to the level in LT-HSC. ( D) The surface markers we used to sort mouse lineages. ( E) The expression level of PRMT1 in different human hematopoietic lineages. ( F) The expression level of RBM15 in different human hematopoietic lineages based on database from HemaExplorer. ( G) The abbreviations for the different human hematopoietic lineages used in panels ( E) and ( F). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LT-HSC, long-term hematopoietic stem cells; PRMTs, protein arginine methyltransferases. DOI:
Figure 4—figure supplement 2.. RBM15 protein levels in MEG-01 cell lines expressing two short hairpin RNA constructs against RBM15 by western blots.
Figure 5.. Analysis of RBM15 target genes.
A) Real-time PCR assays for detecting RNA associated with RBM15 in MEG-01 cells by RIP with the RBM15 antibody. The levels of RBM15-associated mRNAs were calculated as mean ± standard deviation from three independent experiments. ( B) The distribution of RBM15 binding sites. All the RBM15 target genes were listed in Figure 5—source data 2. ( C) GO pathway analysis (FDR<0.01) showed pathways associated with genes that have RBM15 binding sites in introns. ( D) GO pathway analysis (FDR <0.01) revealed pathways associated with genes containing RBM15 binding sites in 3’UTR regions. ( E) Differential exon usage events detected by the MISO program. ( F) The changes of percentage splice-in events in different splicing categories when RBM15 is knocked down. ( G) MISO plot for skipping of GATA1 exon 2 when RBM15 was knocked down. ( H) Isoforms of GATA1fl and GATA1s were detected by PCR using RNA extracted from MEG-01 cells with or without RBM15 knockdown. ALE, alternative last exon; AFE, alternative first exon; A5SS, alternative 5’ splicing sites; A3SS, alternative 3’ splicing sites; GO, gene ontology; MXE, mutually exclusive exon usage; PCR, polymerase chain reaction; RI, retention intron; RIP, RNA immunoprecipitation assay; SE, skipped exon; T3UTR, tandem UTR. DOI:
Figure 5—figure supplement 1.. RBM15 binding to pre-mRNA of genes known important for hematopoiesis.
A) RBM15 binding peaks on pre-mRNA of CDC42. ( B) RBM15 binding sites on pre-mRNA of macroH2A (H2AFZ). ( C) RBM15 binding on pre-mRNA of TAL1. ( D) RBM15 binding peaks on pre-mRNA of LEF1. mRNA, messenger RNA. DOI:
Figure 5—figure supplement 2.. The RBM15 binding profiles on the c-MPL (
A) and RUNX1 ( B) pre-mRNAs.
The red bar is calculated as significant peaks in bioinformatics analysis. mRNA, messenger RNA.
Figure 5—figure supplement 3.. The mitochondria biogenesis is regulated by the PRMT1-RBM15 pathway.
The mitotracker (MitoTracker deep red FM, Invitrogen) was used to stain the active mitochondria in MEG-01 stable cell lines infected with lentivirus vector, lentivirus expressing shRBM15, and lentivirus overexpressing PRMT1 V1 and V2. The cell nuclei were stained with Hoechst. PRMT, protein arginine methyltransferase
Figure 5—figure supplement 4.. Representative genes detected by MISO and DEXSeq in the genes detected by RIP.
In the MISO diagram, the red graph is for MEG-01 cells, and the orange diagrams are for two MEG-01 cell lines expressing two shRBM15 RNAs. (
A) TAL1 (aka SCL) skips exon 2 and 3 as detected by DEXSeq and use alternative short 3’UTR after RBM15 was knocked down. TAL1 has the exon 2 (1:47691115–47691561) skipped in RBM15 knockdown cells, which losses protein coding ability for transcript (bottom panel). ( B) MacroH2A (H2AFZ) retains intron (4:100870543–100870820) when RBM15 protein level is reduced. ( C) RUNX1 has multiple exon usage alteration events as demonstrated by DEXSeq on the top. The E017 marks the change for generating RUNX1a (E7a is included) when RBM15 is knocked down. The bottom is MISO analysis showing that Exon 6 is skipped to generate a RUNX1 protein without the transcriptional repression region. This isoform was reported (Komeno et al. 2014). ( D) CDC42 skips the exon 2a (1:22400587–22400712) or exon 2b (1:22400647–22400712). Although the open reading frame is still intact with the skipping events, the change of 5’UTR might cause changing in efficiency of protein translation. DOI:
Figure 6.. Methylation of RBM15 controls alternative splicing of genes (
RUNX1, GATA1 and c-MPL) important for MK differentiation.
A) Alternative splicing of RUNX1, GATA1, c-MPL in MEG-01 cells and MEG-01-derived stable cell lines overexpressing RBM15 and PRMT1 V2 or knocking down RBM15 and PRMT1 V2. The ratios of different isoforms were calculated from real-time PCR assays with isoform specific primers. At least three independent experiments were performed. P****: <0.01; P***: <0.05; P**: <0.2 and P*: <0.3 compared to their respective vector control groups. ( B) Time course for alternative splicing of RUNX1, GATA1 and c-MPL in human adult CD34 + cells grown in pro-MK differentiation medium. Three independent experiments were used to calculate the standard deviation. ( C) The alternative splicing of GATA1, RUNX1 and c-MPL was measured as ratio change in human adult CD34 + cells treated with DB75 overnight in basic cytokine mix. Three independent experiments were used to calculate the P values. PCR, polymerase chain reaction; PRMT, protein arginine methyltransferase DOI:
Figure 6—figure supplement 1.. Alternative splicing of GATA1 is regulated by PRMT1 in acute megakaryoblastic leukemia cell lines.
DMSO, dimethyl sulfoxide; PRMT, protein arginine methyltransferase. P value was calculated from three independent repeats. ****P < 0.01, *** P < 0.05.
Figure 6—figure supplement 2.. Schematic diagram of MPL isoforms.
A) A new isoform of MPL (c-MPL-exon9-) (Genebank No. KF964490) was detected in MEG-01 cells. We synthesized cDNA from Meg-01 cells using Verso cDNA synthesis kit (Thermo Scientific) and performed regular PCR with primers as reported (Li et al., 2000). Direct ligation of exon 8 with exon 10 generates a truncated MPL protein as shown below. ( B) Schematic diagram of c-MPL isoforms generated by alternative splicing. cDNA, complementary DNA; PCR, polymerase chain reaction. DOI:
Figure 6—figure supplement 3.. Alternative splicing of c-MPL is measured as the ratios of different isoforms by real-time PCR assays.
The data are the averages from three independent experiments with standard deviation. (
A) the ratio between c-MPL72minus-exon9/c-Mpl-exon9+. ( B) the ratio between c-MPL-tr/c-MPL-exon 9+. The c-MPL-tr stands for c-MPL isoform without exon 9 and exon 10. We also observed that the ratio of the c-MPL isoform with 72bp missing in the exon 9 (i.e. c-MPL-72minus-exon9+) to c-MPL-exon 9+ mRNA is similarly changed like the ratio of c-MPL-exon 9-/c-MPL-exon 9+ mRNA upon overexpression or knockdown of RBM15 or of PRMT1. We discovered that the ratio of c-MPL-tr (which misses both exon 9 and exon 10) to c-MPL-exon9+ was changed likewise when the RBM15 level is changed as shown in Figure 6. Dox, doxycycline; PCR, polymerase chain reaction DOI:
Figure 7.. RBM15 directly recruits the intron-binding splicing factor, SF3B1, for alternative RNA splicing.
A) The interaction between SF3B1 and RBM15 in the context of PRMT1-mediated methylation. RBM15-Flag and RBM15 R578K-Flag expressed from two MEG-01 cell lines with or without DB75 treatment were immunoprecipitated with anti-Flag antibody for detecting interaction with SF3B1 by WB. ( B) The endogenous SF3B1 was co-immunopreciptiated with anti-RBM15 antibody in MEG-01 cells expressing inducible shPRMT1. Normal mouse serum was used as a negative control. ( C) RBM15 binding profile on GATA1 pre-mRNA based on RIP-seq data. The green peaks are the binding sites for RBM15 and the blue profile is the binding profile for normal IgG. Two biological replicates were used for bioinformatic analysis. The significant peaks were shaded with pink squares. ( D) The regions where RBM15 (RIP with RBM15 antibody, left panel) and SF3B1 (RIP with SF3B1 antibody right panel) bound on GATA1 pre-mRNA in MEG-01 cells (solid bar) and RBM15 knockdown MEG-01 cells (open bar) were mapped by real-time PCR assays. The locations of primers on the pre-mRNA of GATA1 were shown on the bottom. Three biological replicates were used to calculate the standard deviations. GAPDH intron 1 was used as negative controls for both antibodies. ( E) The regions on c-MPL pre-mRNA, where RBM15 and SF3B1 bound in MEG-01 cell lines expressing shRBM15 (square line) or expressing pLKO vector (solid dot line), were assessed by RIP with RBM15 (left panel) and SF3B1 antibodies (right panel). The locations of primers on the pre-mRNA of c-MPL are shown on the bottom. Three biological replicates were used for standard deviations. ( F) A model for RBM15-mediated regulation of alternative RNA splicing. RBM15 and SF3B1 cooperate to produce GATA1fl and low level of RBM15 leads to lower SF3B1 binding and skipping of the exon 2. PRMT1-mediated methylation of RBM15 controls the ubiquitylation of RBM15 by CNOT4, thus controlling the balance between proliferation and differentiation in megakaryopoiesis. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; RIP, RNA immunoprecipitation assay; PCR, polymerase chain reaction; PRMT, protein arginine methyltransferase DOI:
Author response image 1.. Knockdown RUNX1 in CD34
+ cord blood cells block megakaryocyte differentiation.
Two shRNA against RUNX1 were expressed by retroviruses. The cells were grown in pro-megakaryocyte differentiation medium and harvested on day five.
Author response image 2.
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