Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing
RNA binding proteins control post-transcriptional processing such as alternative RNA splicing , polyadenylation and protein translation , which is a prevalent part of gene regulation in normal cell differentiation and in cancer development ( Cabezas-Wallscheid et al . , 2014; Chen et al . , 2014; de Klerk and Hoen , 2015; Shapiro et al . , 2011 ) . Arginine methylation of RNA binding proteins by protein arginine methyltransferases ( PRMTs ) regulates RNA splicing ( Bedford and Clarke , 2009; Bezzi et al . , 2013; Cheng et al . , 2007; Sinha et al . , 2010 ) , subcellular localizations ( Matsumoto et al . , 2012; Nichols et al . , 2000; Tradewell et al . , 2012 ) as well as the binding affinity to RNA molecules ( Rho et al . , 2007 ) . Nevertheless , the role of arginine methylation in regulating protein stability remains unknown . Here we demonstrate that an RNA binding protein , RBM15 , is methylated by PRMT1 , which triggers its ubiquitylation by an E3 ligase CNOT4 . RBM15 belongs to the split ends ( spen ) family of proteins . Spen proteins are evolutionally conserved RNA binding proteins , which are involved in transcriptional regulation of Notch , Wnt and mitogen-activated protein kinase signal transduction pathways ( Chang et al . , 2008; Chen and Rebay , 2000; Oswald et al . , 2002; Rebay et al . , 2000; Su et al . , 2015 ) . Recently SPEN and RBM15 have been shown to be essential for Xist-mediated X chromosome inactivation ( Chu et al . , 2015; McHugh et al . , 2015; Minajigi et al . , 2015; Moindrot et al . , 2015; Monfort et al . , 2015 ) . Genetic studies in Drosophila have shown that spen is required for cell-fate decision during development ( Kolodziej et al . , 1995 ) . FPA , the RBM15 homolog in Arabidopsis , controls flowering via regulating alternative polyadenylation of antisense RNAs at the FLC locus ( Hornyik et al . , 2010 ) . RBM15 is essential for the development of multiple tissues in mouse knockout models , in particular , for the maintenance of the homeostasis of long-term hematopoietic stem cells and for megakaryocyte ( MK ) and B cell differentiation ( Niu et al . , 2009; Raffel et al . , 2009; Xiao et al . , 2015 ) . Furthermore , RBM15 is involved in the chromosome translocation t ( 1;22 ) , which produces the RBM15-MKL1 fusion protein associated with acute megakaryoblastic leukemia ( AMKL ) ( Ma et al . , 2001; Mercher et al . , 2001 ) . Spen proteins consist of two domains: an RNA binding domain and a Spen Paralog and Ortholog C-terminal ( SPOC ) domain . Previously , spen proteins such as RBM15 and SHARP have been shown to use the SPOC domains to recruit histone deacetylases for transcriptional regulation of Notch pathway and steroid receptor-dependent transcriptional regulation , and recruit mixed lineage leukemia ( MLL ) complexes to promoters for histone H3K4 methylation ( Ariyoshi and Schwabe , 2003; Lee and Skalnik , 2012; Ma et al . , 2007; Oswald et al . , 2002; Shi et al . , 2001; Xiao et al . , 2015 ) . Additionally , RBM15 is also involved in RNA export ( Uranishi et al . , 2009; Zolotukhin et al . , 2008; Zolotukhin et al . , 2009 ) . RBM15 resides mainly within nuclear RNA splicing speckles by confocal microscopy ( Horiuchi et al . , 2013 ) , suggesting that RBM15 is involved in RNA splicing . However , how spen proteins control cell differentiation is not described at molecular level . In this report , we linked cellular differentiation to RBM15-regulated RNA metabolism using MK differentiation as a model . We demonstrated that RBM15 binds to specific introns of pre-messenger RNA ( mRNA ) of genes such as RUNX1 , GATA1 and TPOR ( aka c-MPL or MPL ) , which play critical roles in MK differentiation , and to 3’UTRs of genes involved in RNA splicing and metabolic regulation . Reducing RBM15 protein concentration by PRMT1-mediated methylation favors the production of the alternatively spliced isoforms: RUNX1a , GATA1s and c-MPL-exon9- , a truncated c-MPL isoform . We also found that RBM15 promotes MK maturation in human primary cells . Therefore , PRMT1-RBM15 pathway fine-tunes cell differentiation via controlling the RBM15 protein concentration .
In AMKL , MK differentiation is blocked . Gene expression data from AMKL patient samples ( Bourquin et al . , 2006 ) shows a higher PRMT1 expression level than other types of acute myeloid leukemia . Furthermore , high expression of PRMT1 is correlated with poor survival rate in acute myeloid leukemia ( Figure 1—figure supplement 1 ) . These clinical data strongly support that PRMT1 might be a key player in leukemogenesis . We applied bio-orthogonal profiling of protein methylation ( BPPM ) technology ( Figure 1—figure supplement 2 ) ( Wang et al . , 2011 ) to identify proteins methylated by PRMT1 in MKs . We found that RBM15 is methylated at R578 by mass spectrometry analysis ( Figure 1—figure supplement 3A ) . Alignment of the RBM15 sequences covering the methylation site shows that the methylation site is conserved across diverse species ( Figure 1A ) and downstream of the RBM15 RNA binding domains ( Figure 1—figure supplement 3B ) . 10 . 7554/eLife . 07938 . 003Figure 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: http://dx . doi . org/10 . 7554/eLife . 07938 . 00310 . 7554/eLife . 07938 . 004Figure 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: http://dx . doi . org/10 . 7554/eLife . 07938 . 00410 . 7554/eLife . 07938 . 005Figure 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 methyltransferasesDOI: http://dx . doi . org/10 . 7554/eLife . 07938 . 00510 . 7554/eLife . 07938 . 006Figure 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: http://dx . doi . org/10 . 7554/eLife . 07938 . 00610 . 7554/eLife . 07938 . 007Figure 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 typeDOI: http://dx . doi . org/10 . 7554/eLife . 07938 . 007 We validated that two generic antibodies against mono-methyl arginine proteins and against di-methyl arginine proteins ( Cell Signaling Inc . ) , can recognize the methylated peptides from RBM15 ( NP_073605 ) R578 region , specifically in dot blots ( Figure 1—figure supplement 3C&D ) . We observed that affinity purified Flag-tagged RBM15 protein is more efficiently methylated than the R578K mutant in western blots ( WBs ) in transfected 293T cells ( Figure 1B , compare lanes 1 and 2 ) as well as in leukemia cells ( Figure 1—figure supplement 3E ) . Given that the R578 is the last arginine in the RDRDRD repeat , we mutated all the three arginines , but still detected low methylation background , indicating other arginine residues not in the repeat are methylated ( Figure 1—figure supplement 3F ) . To determine whether PRMT1 is responsible , we overexpressed the two major isoforms of PRMT1 ( V1 i . e . Q99873-3 , V2 i . e . Q99783-1 ) in 293T cells together with wild type ( WT ) RBM15 ( Figure 1C ) . Overexpressing either isoform enhanced the mono-methylation of RBM15 ( Figure 1C , compare lanes 1 , 2 and 3 ) and the V2 dimethylated RBM15 more effectively than the V1 . Given that the V2 contains additional amino acids in the N terminal region relative to V1 ( Figure 1D ) , it is striking that the PRMT1 V2 is primarily responsible for the dimethylation of RBM15 in vivo ( Figure 1C ) . In vitro methylation assays showed that the RBM15 peptide was dimethylated as efficiently as the histone H4 peptide without monomethylated peptides detected ( Figure 1—figure supplement 3G ) . We then used purified Flag-RBM15 from 293T cells to conduct in vitro methylation assays . Only the WT RBM15 protein but not the R578K mutant was effectively dimethylated by PRMT1 in vitro ( Figure 1E ) . These results together with in vivo methylation data collectively argue that mono-methylated RBM15 is a transient intermediate in vivo . To confirm that PRMT1 is responsible for in vivo methylation , we made MEG-01 stable cell lines expressing doxycycline-inducible shRNA against PRMT1 . We detected the methylated form of RBM15 with both mono-methyl and dimethyl antibodies . Reducing the expression of PRMT1 decreased the methylation level of the endogenous RBM15 ( Figure 1F ) . When the leukemia cells were treated with DB75 ( a PRMT1 inhibitor ) ( Yan et al . , 2014 ) , RBM15 methylation was reduced ( Figure 1G ) . The methylation of RBM15 was further confirmed by an antibody raised against dimethylated RBM15 at R578 ( Figure 1—figure supplement 4 ) . Therefore , we concluded that PRMT1 enzymatic activity is responsible for the methylation of RBM15 at R578 . RBM15 might be methylated by other PRMTs and at other sites since some methylation signals , especially the monomethylation signals , are present despite PRMT1 knock-down or inhibition and in the R578K mutant . Nevertheless , the PRMT1-dependent R578 methylation forms the majority of dimethylated species . When RBM15 was co-expressed together with PRMT1 , RBM15 protein level was reduced . Strikingly , the V2 isoform ( detected by V2-specific antibody ) caused a greater reduction of RBM15 protein level ( Figure 2A ) , which coincided with the PRMT1 V2’s capability for RBM15 dimethylation ( Figure 1C ) . To further confirm that methylation triggers protein degradation , we used non-specific methyltransferase inhibitors ( adenosine dialdehyde [adox] + methylthioadenosine [MTA] ) ( Figure 2B , left panel ) as well as DB75 ( Figure 2B right panel ) to treat MEG-01 cells . Consistently , PRMT1 knockdown increased RBM15 protein level ( Figure 2C , left panel ) but not the RBM15 mRNA level ( Figure 2C , right panel ) in MEG-01 cells as well as in other leukemia cells ( data not shown ) . Conversely , overexpression of PRMT1 V2 ( Figure 2D , left panel ) reduced RBM15 protein level . Given that the RBM15 mRNA levels in PRMT1 knockdown cells and in PRMT1 overexpressing cells were the same as in the control MEG-01 cells ( Figure 2D middle and right panels ) , we concluded that PRMT1 controls RBM15 at protein level . Finally , we observed that the specific knockdown of PRMT1 V2 resulted in higher RBM15 protein level ( Figure 2E ) . Taken together , we concluded that the PRMT1 V2 enzymatic activity plays a major role in regulating the stability of the RBM15 protein . 10 . 7554/eLife . 07938 . 008Figure 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: http://dx . doi . org/10 . 7554/eLife . 07938 . 008 When we co-expressed RBM15 R578K together with PRMT1 V2 protein in 293T cells , we found that the mutant protein remained stable regardless of overexpression of PRMT1 V2 ( Figure 2F , compare lanes 4 and 2 ) . Thus , methylation of RBM15 at R578 is the direct cause for protein degradation . To further determine whether RBM15 degradation is affected by methylation on R578 , we compared the half-life of the WT versus the mutant RBM15 proteins . The Flag-tagged RBM15 mutant has longer half-life compared with the WT RBM15 ( Figure 2G ) . Tag does not affect protein stability as the tagged RBM15 and endogenous RBM15 have similar half-life . We showed that the proteasome inhibitor MG132 stabilized the RBM15 protein , which indicates that the degradation is mediated by ubiquitylation ( Figure 3A ) . To further confirm that the RBM15 protein is ubiquitylated , we used nickel beads to purify the poly-ubiquitylated form of RBM15 under denaturing conditions from 293T cells expressing the poly-histidine-tagged ubiquitin and Flag-tagged RBM15 ( Figure 3B ) . 10 . 7554/eLife . 07938 . 009Figure 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: http://dx . doi . org/10 . 7554/eLife . 07938 . 00910 . 7554/eLife . 07938 . 010Figure 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: http://dx . doi . org/10 . 7554/eLife . 07938 . 01010 . 7554/eLife . 07938 . 011Figure 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: http://dx . doi . org/10 . 7554/eLife . 07938 . 011 To investigate whether methylation of RBM15 by PRMT1 is required for RBM15 ubiquitylation , we mutated the methylation site to lysine . Compared with the WT RBM15 protein , significantly less R578K RBM15 protein was modified by ubiquitin ( Figure 3C compare lanes 4 and 5 , ) when the respective plasmids were co-expressed in 293T cells . Thus , methylation on R578 for protein ubiquitylation is one major pathway for RBM15 degradation . To validate that endogenous RBM15 is also ubiquitylated in a methylation-dependent manner , we treated the MEG-01 cells with PRMT1 inhibitor DB75 . After DB75 treatment , less ubiquitylated RBM15 proteins were observed ( Figure 3D ) . PRMT1 has been shown to physically interact with the CCR4-NOT complex via the CAF1 ( aka CNOT7 ) subunit for RNA export ( Kerr et al . , 2011; Robin-Lespinasse et al . , 2007 ) . Furthermore , CNOT4 , a subunit of the CCR4-NOT complex , has been shown to be an E3 ligase ( Albert et al . , 2002 ) . To examine whether CNOT4 might be responsible for ubiquitylation of RBM15 , we co-expressed CNOT4 with RBM15 in 293T cells . Strikingly , when RBM15 was co-expressed with PRMT1 and CNOT4 , RBM15 was more severely ubiquitylated than RBM15 expressed with PRMT1 or CNOT4 alone ( Figure 3E ) . We also detected the interaction between PRMT1 and RBM15 WT and mutant proteins in 293T cells ( Figure 3E lanes 3 , 4 and 7 , 8 ) . Moreover , only the WT and not the mutant RBM15 interacted with HA-tagged CNOT4 ( Figure 3E lane 3 , 4 ) . In Figure 3E and Figure 3C , we showed that the RBM15 R578K , which cannot be methylated by PRMT1 , had significantly less ubiquitylated RBM15 protein . This data strongly suggest that methylation is required for subsequent ubiquitylation by the CNOT4 complex . To further confirm that CNOT4 is the enzyme responsible for endogenous RBM15 ubiquitylation , we showed that CNOT4 knockdown ( WB in Figure 3F and real-time polychromase chain reaction [PCR] in Figure 3—figure supplement 2A ) increased the amount of endogenous RBM15 protein in MEG-01 cells ( Figure 3F ) . When MEG-01 cells were treated with DB75 , CNOT4 knockdown did not further stabilize the RBM15 protein ( Figure 3F ) . These results confirm that methylation is required for the subsequent degradation of the RBM15 protein . We used the clustered regularly interspaced short palindromic repeat ( CRISPR ) technology to knockout one allele of CNOT4 gene in 293T cells . We also found that removal of one allele of CNOT4 stabilized the endogenous RBM15 protein levels ( Figure 3—figure supplement 2B&C ) . We then purified the RBM15 and CNOT4 proteins ( Figure 3—figure supplement 1 ) to do in vitro ubiquitylaton assay . The methylated RBM15 protein ( Flag-tagged ) by PRMT1 was more efficiently ubiquitylated by CNOT4 than the purified RBM15 R578 in vitro ( Figure 3G compare lane 5 to lane 3 ) . Taken together , we demonstrated that the RBM15 protein stability is negatively regulated by PRMT1-mediated methylation at R578 , and CNOT4 is an E3 ligase responsible for degrading the methylated RBM15 . To understand further whether the methylation site on RBM15 is sufficient to bind to CNOT4 complex , we synthesized two RBM15 peptides covering the arginine methylation sites to pull-down CNOT4 from cell extract . The di-methylated peptide bound specifically to the CNOT4 protein ( Figure 3H ) , which is agreeable with the coimmunprecipitation data in Figure 3E showing that CNOT4 only interacted with WT RBM15 proteins . This result implies that either CNOT4 or subunits in the CNOT4 complex directly interacts with RBM15 via the methylation mark . PRMT1 RNA levels were measured in isolated mouse long-term hematopoietic stem cells ( LT-HSC ) , short-term hematopoietic stem cells ( ST-HSC ) , progenitor cells and terminally differentiated lineages . PRMT1 expression level was the lowest in LT-HSC and higher especially in myeloid progenitor cells ( Figure 4—figure supplement 1A–D ) . Megakaryocyte-–erythrocyte progenitor ( MEP ) cells expressed the highest level of PRMT1 . We also checked the expression of PRMT1 and RBM15 in human cells with HemaExplorer ( Bagger et al . , 2013 ) . The expression profiles of PRMT1 and RBM15 in different lineages are shared between humans and mice ( Figure 4—figure supplement 1E–G ) . The mRNA level of RBM15 does not change as dramatically as that of PRMT1 among different lineages , which implies that post-transcriptional modification such as arginine methylation of RBM15 might contribute to the regulation of RBM15 protein levels . To understand the biological functions of PRMT1 in MK differentiation , we used phorbol myristate acetate ( PMA ) to stimulate MEG-01 cells into mature MK cells ( Ogura et al . , 1988 ) . Upon PMA stimulation , we observed upregulation of the RBM15 protein within 12 hr and simultaneous down-regulation of the PRMT1 V2 protein ( Figure 4A left and middle panels ) . During the incubation period , neither the RBM15 mRNA level ( Figure 4A , right panel ) nor the total PRMT1 protein level ( mainly V1 isoform ) changed more than 1 . 5-folds , nevertheless we found that RBM15 protein level was significantly increased . The methyl-RBM15 level reduced coinciding with the decrease of the isoform V2 protein ( detected by V2-specific antibody ) . Thus we conclude the isoform V2 –mediated methylation is one mechanism for upregulating RBM15 protein level , although we cannot rule out other possibilities such as enhanced protein translation . To assess RBM15 effects on MK differentiation , we ectopically expressed the WT and the R578K mutant proteins in MEG-01 cells by retroviruses . After stimulation with PMA for 7 days , more CD41+ cells ( Figure 4B ) as well as higher levels of polyploidy ( Figure 4C ) were detected . In comparison to WT RBM15 , the RBM15 R578K generated more CD41+ cells , which may be explained by increased RBM15 R578K stability ( Figure 3 ) . 10 . 7554/eLife . 07938 . 012Figure 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: http://dx . doi . org/10 . 7554/eLife . 07938 . 01210 . 7554/eLife . 07938 . 013Figure 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: http://dx . doi . org/10 . 7554/eLife . 07938 . 01310 . 7554/eLife . 07938 . 014Figure 4—figure supplement 2 . RBM15 protein levels in MEG-01 cell lines expressing two short hairpin RNA constructs against RBM15 by western blots . DOI: http://dx . doi . org/10 . 7554/eLife . 07938 . 014 However , the cell line differentiation system can only mimic certain aspects of normal megakaryopoiesis . For example , we cannot detect CD42 in MEG-01 cells upon PMA stimulation . Therefore , we performed MK differentiation assays with adult human CD34+ cells . Compared with the control , DB75-treated cells produced a higher percentage of CD61+CD42+ mature MK cells ( Figure 4D ) . Conversely , ectopic expression of PRMT1 V2 in CD34+ cells promoted the generation of single positive CD61+ MK progenitor cells but blocked MK cell maturation ( Figure 4E ) . Like PRMT1 overexpression , knockdown of RBM15 by shRNAs blocked MK maturation ( Figure 4F , Figure 4—figure supplement 2 ) , and overexpression of both RBM15 and its mutant promoted the maturation of CD34+ cells ( Figure 4G ) . Overexpression of RBM15 or RBM15 R578K rescued MK differentiation in CD34+ cells expressing PRMT1 V2 ( Figure 4H ) . Taken together , PRMT1 controls megakaryopoiesis via controlling RBM15 protein levels . To understand how RBM15 protein level controls megakaryocytic differentiation , we affinity purified RBM15-associated proteins from 293T cells , which express endogenous RBM15 protein . Mass spectrometry analysis showed that RBM15 was associated with ASH2 and WTAP , two known RBM15-bound proteins ( Horiuchi et al . , 2013; Lee and Skalnik , 2012 ) as well as proteins involved in RNA splicing ( Figure 7—source data 1 ) , which is consistent with prior findings that RBM15 resides in nuclear splicing speckles ( Horiuchi et al . , 2013 ) . Thus , we reasoned that RBM15 might regulate alternative splicing . We performed RNA immunoprecipitation assay ( RIP ) under stringent wash conditions with an RBM15 antibody recognizing the N-terminal region , followed by real-time polychromase chain reaction ( RT-PCR ) to detect genes known to be important for MK differentiation . We detected the bindings of RUNX1 , GATA1 and c-MPL mRNAs with RBM15 , while we did not detect that of control GAPDH mRNA ( Figure 5A ) . Then we took an unbiased approach to identify global RBM15 target genes using RIP-seq . We have identified 1297 genes to which RBM15 binds directly ( Figure 5—source data 1 ) . Fifty-five percent of RBM15 binding sites are in intronic regions and 41% are in 3’UTR regions , with the remainder in 5’UTRs and CDS elements ( Figure 5B ) . Gene ontology ( GO ) pathway analysis showed RBM15 regulates genes involved in differentiation and signal transduction via binding to intronic regions , whereas genes involved in RNA splicing and metabolic pathways were regulated via binding to 3’UTRs ( Figure 5C&D ) . We found that overexpression of PRMT1 V1 and V2 isoforms as well as reducing the expression level of RBM15 enhanced mitochondria biogenesis in MEG-01 cells ( Figure 5—figure supplement 3 ) , which implies that PRMT1-RBM15 axis regulates metabolism . Among the RBM15 targeting genes with binding sites in introns are transcription factors known to be important for MK differentiation: RUNX1 , GATA1 , and TAL1 ( Figure 5—figure supplement 1 , 2 ) . Although the transcription factor LEF1 has not yet been linked to MK differentiation , LEF1 has been shown to interact with RUNX1 genetically and biochemically ( Daga et al . , 1996; Mayall et al . , 1997; McNerney et al . , 2013 ) . RBM15 binding peaks on c-MPL pre-mRNA in the RIP-seq data ( Figure 5—figure supplement 2 ) . 10 . 7554/eLife . 07938 . 015Figure 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: http://dx . doi . org/10 . 7554/eLife . 07938 . 01510 . 7554/eLife . 07938 . 016Figure 5—source data 1 . Identification of RNAs associated with RBM15 by RNA immunoprecipitation assay with anti-RBM15 antibody . Genes related to MK differentiation are highlighted . DOI: http://dx . doi . org/10 . 7554/eLife . 07938 . 01610 . 7554/eLife . 07938 . 017Figure 5—source data 2 . Analysis of gene expression profile changes with RNA-seq data from RBM15 knockdown MEG-01 cells . Genes related to MK differentiation are highlighted . MK , megakaryocyte; RNA-seq , RNA sequencing . DOI: http://dx . doi . org/10 . 7554/eLife . 07938 . 01710 . 7554/eLife . 07938 . 018Figure 5—source data 3 . Analysis of differential exon usage regulated by RBM15 with RNA-seq data from RBM15 knockdown MEG-01 cells . Genes related to MK differentiation are highlighted . MK , megakaryocyte; RNA-seq , RNA sequencing . DOI: http://dx . doi . org/10 . 7554/eLife . 07938 . 01810 . 7554/eLife . 07938 . 019Figure 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: http://dx . doi . org/10 . 7554/eLife . 07938 . 01910 . 7554/eLife . 07938 . 020Figure 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 07938 . 02010 . 7554/eLife . 07938 . 021Figure 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 methyltransferaseDOI: http://dx . doi . org/10 . 7554/eLife . 07938 . 02110 . 7554/eLife . 07938 . 022Figure 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: http://dx . doi . org/10 . 7554/eLife . 07938 . 022 To further investigate how RBM15 regulates alternative RNA splicing , we performed RNA-seq assays with RNA isolated from MEG-01 cells with or without RBM15 knockdown . The gene expression profile analysis showed that RBM15 knock-down alters metabolism and endoplasmic reticulum stress response pathways as well as proteins involved in chromatin assembly ( Figure 5—source data 2 ) . We then used two programs ( DEXSeq and MISO ) to detect the alterations of exon usage . DEXSeq detected 9704 differential exon usage events with P value lower than 0 . 05 and fold change higher than 1 . 2 fold . The MISO program detected 2027 differential exon usage events with Bayes factor higher than 2 . RBM15 regulates differential exon usage in all eight categories in either directions ( Figure 5E , F ) . Significant exon usage changes of 156 genes in the RIP-seq group were detected by both programs ( Figure 5—source data 3 ) including transcription factors RUNX1 , GATA1 , STAT5A , TAF9 , TAL1 , LEF1 and ZNF160 as well as chromatin remodeling factors such as macroH2A , DEK , BRD9 , TLE3 , NCOR1 and HDAC4 . RUNX1 , GATA1 , STAT5A and TAL1 ( SCL ) are well-studied transcription factors for their roles in MK differentiation ( Crispino , 2005; Olthof et al . , 2008; Tijssen et al . , 2011 ) . Signal transduction genes important for hematopoiesis such as RPTOR and CDC42 ( Kaushansky and Kaushansky , 2014 ) were found on the list as well . Therefore , RBM15 protein level may affect hematopoiesis via multiple pathways . At a molecular level , RBM15 not only affects alternative RNA splicing such as RUNX1 , GATA1 , TAL1 , STAT5A , LEF1 but also affects UTR utilization ( e . g . TAL1 ) , mixed exon usage ( e . g . CDC42 ) and intron retention ( e . g . macroH2A ) ( Figure 5—figure supplement 4 ) . The MISO program was used to analyze how GATA1 is alternatively spliced . After knocking down RBM15 , we found skipping of exon 2 on GATA1 ( Figure 5G ) and validated our results by resolving GATA1 isoforms in an agarose gel ( Figure 5H ) . GATA1 has two different isoforms: full-length GATA1 ( GATA1fl ) , and short-form GATA1 ( GATA1s ) , which is generated by skipping the exon 2 ( Rainis et al . , 2003 ) . In Down syndrome leukemia , GATA1fl mRNA translates the GATA1s protein when mutations on GATA1fl mRNA create an alternative translation start site . Using isoform specific probes targeting to the exon junctions of GATA1s and GATA1fl to perform real-time PCR assays , we found that reducing RBM15 protein level favored the accumulation of GATA1s as the ratio of GATA1fl/GATA1s was reduced ( Figure 6A top panel ) . Conversely , overexpression of RBM15 reversed the ratio of GATA1fl/GATA1s in favor of GATA1fl . The RBM15 R578K protein , which is more stable , altered the ratio more than the WT RBM15 . Furthermore , overexpression of V2 in MEG-01 cells reduced the ratio of GATA1fl to GATA1s , similar to the effect of RBM15 knockdown . When we used inducible cell lines to knockdown PRMT1 , we observed a higher ratio of GATA1fl/GATA1s-like in cells overexpressing RBM15 ( Figure 6A ) . To further probe the importance of PRMT1 enzymatic activity in alternative splicing , we used PRMT1 inhibitor DB75 to treat MEG-01 cells , CMK cells and CMY cells , which all derived from AMKL leukemia patients . All three AMKL cell lines had higher GATA1fl/GATA1s ratio when treated with DB75 ( Figure 6—figure supplement 1 ) . Both isoforms of GATA1 can support MK differentiation , but only GATA1s supports unrestricted proliferation due to losing the interaction with E2F1 ( Li et al . , 2000 ) . Therefore , fine-tuning the ratio between two different GATA1 isoforms regulates the balance between proliferation and differentiation of MK progenitors . 10 . 7554/eLife . 07938 . 023Figure 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 methyltransferaseDOI: http://dx . doi . org/10 . 7554/eLife . 07938 . 02310 . 7554/eLife . 07938 . 024Figure 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 07938 . 02410 . 7554/eLife . 07938 . 025Figure 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: http://dx . doi . org/10 . 7554/eLife . 07938 . 02510 . 7554/eLife . 07938 . 026Figure 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 reactionDOI: http://dx . doi . org/10 . 7554/eLife . 07938 . 026 RUNX1 is required for MK differentiation as shown in a conditional RUNX1 knockout mouse model ( Ichikawa et al . , 2004 ) . Individual RUNX1 isoforms regulate hematopoiesis differently ( Komeno et al . , 2014; Tsuzuki and Seto , 2012 ) . RBM15 bound to RUNX1 pre-mRNA in intronic regions and in 3’UTR ( Figure 5—figure supplement 2 ) . The MISO program found that RUNX1 is alternatively spliced by skipping exon 6 , which generates RUNX1-Exon 6- protein . Overexpression of RUNX1-Exon 6- in transgenic mice leads to reduced pool of hematopoietic stem cells ( Komeno et al . , 2014 ) ( Figure 5—figure supplement 4 ) . Given that RBM15 binds to the 3’UTR in exon 8 as well as to intronic regions flanking exon 7a , we investigated whether knockdown of RBM15 causes alternative 3’UTR usage to generate RUNX1a , which uses exon7a’ 3’UTR . In RBM15 knock-down MEG-01 cells , the ratio between RUNX1a and RUNX1b , c switched in favor of RUNX1a . RUNX1a , which lacks the transactivation domain , acts as a dominant negative regulator to antagonize the function of RUNX1b , c ( Ran et al . , 2013a ) . Increasing the amount of RUNX1a may interfere with the differentiation into mature MKs . Consistent with RBM15 knockdown data , we showed that overexpression of RBM15 as well as knockdown of PRMT1 tilted the isoform balance toward RUNX1b , c . In agreement with the PRMT1 knockdown data , overexpression of PRMT1 V2 switched the ratio in favor of RUNX1a ( Figure 6A middle panel ) . The thrombopoietin receptor ( TPOR , aka . c-MPL ) is upregulated during MK differentiation . c-MPL has at least four described isoforms in humans ( Figure 6—figure supplement 2 ) . Using reported primers ( Li et al . , 2000 ) to amplify cDNA from MEG-01 cells , we found an additional band on the PAGE gel , which was identified as an undescribed isoform lacking exon 9 ( c-MPL-exon9-– ) through sequencing . The c-MPL-exon9– mRNA was predicted to yield a truncated protein ( Figure 6—figure supplement 2 ) . Thus we designed specific primer spanning the junction between exon 8 and 10 to detect c-MPL-exon9– mRNA . We also used a pair of primers , the forward primer spanning exon 8 and 9 and the reverse primer annealing to exon 9 , to detect the full-length MPL ( i . e . c-MPL-exon 9+ ) . The c-MPL-exon9-–/c-MPL-exon9+ ratio was higher in favor of c-MPL-exon9- ( Figure 6A , bottom panel ) when RBM15 protein level is reduced . Furthermore , when RBM15 or RBM15 R578K was overexpressed , the ratio was down in favor of c-MPL-exon9+ . Given that c-MPL exon9+ is required for MK differentiation , the data further support for the positive role of RBM15 at MK maturation . All other isoforms with part of exon 9 or with both exon 9 and 10 missing were spliced in the same way by RBM15-PRMT1 pathway ( Figure 6—figure supplement 3 ) . Thus , RBM15 enhances the inclusion of exons to produce full-length functional c-MPL like in RUNX1’s case . Consistent with our data , mouse Rbm15 regulates c-Mpl alternative splicing in the same fashion ( Xiao et al . , 2014 ) . We monitored the isoform ratios using human adult CD34+ cells in pro-MK differentiation medium over a time course . We found that the GATA1fl/GATA1s ratio was increased , and the RUNX1a/RUNX1b , c ratio and c-MPL-exon9–/c-MPL-exon9+ ratio were decreased during the course of differentiation ( Figure 6B ) . We observed that the isoform ratio changes in the DB75-treated CD34+ cells ( Figure 6C ) exactly like in the DB75-treated MEG-01 cells ( Figure 6—figure supplement 1 ) . Based on these data , we concluded that RBM15 regulates alternative RNA splicing of genes important for MK differentiation . By controlling the RBM15 protein dosage , PRMT1 thereby regulates MK differentiation . To understand the detailed mechanism of how RBM15 regulates alternative splicing , we identified RBM15-associated splicing factors such as SF3B1 and U2AF , known proteins in committed RNA splicing A complex that bind to branch point region ( Figure 7—source data 1 ) . Furthermore , we confirmed the interaction between SF3B1 and RBM15 by co-immunoprecipitation with anti-Flag antibody for Flag-tagged RBM15 proteins ( Figure 7A ) and with anti-RBM15 antibody ( Figure 7B ) . To further confirm that PRMT1 enzymatic activity is important for alternative RNA splicing , we inhibited PRMT1 activity with DB75 or shPRMT1 . The interaction between SF3B1 and RBM15 was subject to methylation regulation . One simple explanation is that RBM15 might recruit more SF3B1 because of more RBM15 and SF3B1 proteins available . It is also possible that methylation might be directly involved in RBM15 and SF3B1 interaction . Given that co-immunoprecipitation assays cannot distinguish the direct and indirect protein-–protein interaction in a protein complex , interaction of RBM15 with SF3B1 only implies that RBM15 recruits the 3’RNA splicing complex . Since SF3B1 is often mutated in leukemia cells , we sequenced the SF3B1 gene in MEG-01 cells and confirmed that SF3B1 was not mutated in this cell line . Thus the interaction is between two WT proteins . 10 . 7554/eLife . 07938 . 027Figure 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 methyltransferaseDOI: http://dx . doi . org/10 . 7554/eLife . 07938 . 02710 . 7554/eLife . 07938 . 028Figure 7—source data 1 . Mass spectrometry analysis of RBM15-associated proteins . DOI: http://dx . doi . org/10 . 7554/eLife . 07938 . 028 From the RIP-seq data , we found that RBM15 was specifically associated with intronic region close to 5’end of intron 1 of GATA1 ( Figure 7C ) . We validated the RIP-seq data by real-time PCR with primer sets covering the GATA1 intron 1 . Analysis of immunoprecipitated RNA associated with SF3B1 indicated that SF3B1 also preferentially bound to the same region that RBM15 bound to . Furthermore , with RBM15 knockdown , less SF3B1 was associated with GATA1 pre-mRNA ( Figure 7D ) . On the other hand , GAPDH has similar level of SF3B1 binding to introns regardless of RBM15 expression level . Thus , RBM15 is responsible for recruiting SF3B1 to pre-mRNA molecules . In this case , RBM15 facilitates the inclusion of GATA1 exon 2 . For c-MPL pre-mRNA , RBM15 preferentially bound to the intron 8 and 10 of the pre-mRNA molecules with two distinct peaks ( Figure 7E left ) , while SF3B1 ( Figure 7E right ) did bind to regions between the two RBM15 bound peaks at higher level than other regions . When we used RBM15 knockdown MEG-01 cells to do SF3B1 RIP on c-MPL pre-mRNA , we found that SF3B1 binding was significantly reduced in RBM15 bound regions ( Figure 7E right ) . In this case , RBM15 functions as a splicing enhancer for inclusion of exons 9 and 10 . In aggregate , these data show that RBM15 is responsible for recruiting SF3B1 containing branch point recognition complex to introns , and methylation controls the RBM15 dosage thereby controlling alternative RNA splicing ( Figure 7F ) .
We report here that PRMT1 V2 dimethylates RBM15 within the sequence ( LYRDRDR ( me2 ) DLY ) . Although the most common sequence for arginine methylation is arginine flanked by glycines , PRMT1 has been previously reported to methylate arginines in PGC1-α and in RUNX1 flanked by other amino acids ( Teyssier et al . , 2005; Zhao et al . , 2008 ) . Recent proteomics work further proves that the methylation motif can be very diverse ( Guo et al . , 2014 ) . Since we can still detect a low level of mono-methylation signal after R578K mutation , we believe that the mono-methylation signals come from additional arginines methylated by PRMT1 or other arginine methyltransferases . Protein mono-methylation sites , in general , are redundant and prevalent and are catalyzed by multiple protein arginine methyltransferases ( Dhar et al . , 2013 ) . Nevertheless , these potential sites are not involved in methylation-mediated ubiquitylation . Another surprising result is that the V2 isoform , which contains additional amino acids on the N-terminus , primarily dimethylates RBM15 in vivo ( Figure 1C ) . Data from PMA-treated MEG-01 cells argue that the V2 isoform is more relevant to MK maturation ( Figure 4A ) . Although it has been demonstrated that V1 and V2 have different substrate specificities in vivo ( Goulet et al . , 2007 ) , RBM15 is the first V2-specific substrate identified . Of note , the V2 isoform only comprises less than 5% of total PRMT1 level , but V2 is mainly responsible for oncogenic activities in colon and breast cancers ( Baldwin et al . , 2012; Papadokostopoulou et al . , 2009 ) . We cannot completely exclude a role for V1 in RBM15 methylation due to the inability to knockdown V1 specifically . Because developing inhibitors specific for PRMT1 V2 isoform will potentially alleviate off-target effects , which remain a major impediment for developing anti-PRMT1 inhibitors , our data offers new clues on how to achieve V2-specific inhibition . RBM15 is involved in chromosome translocation t ( 1;22 ) , which produces the fusion product RBM15-MKL1 ( Ma et al . , 2001; Mercher et al . , 2001 ) . Whether the fusion protein , which retains R578 , is methylated by PRMT1 is still an unanswered question . It was previously reported that methylation of a transcriptional coactivator p/CIP by PRMT4 or CARM1 leads to its degradation , however the mechanism was incomplete ( Naeem et al . , 2007 ) . Here we report that PRMT1 methylation triggers protein degradation via ubiquitylation by the E3 ligase CNOT4 . CNOT4 is loosely associated with the other subunits of CCR4-NOT complex ( Lau et al . , 2009 ) . Apart from deadenylation activity of other CCR4-NOT complex , CNOT4 has been shown to ubiquitylate a histone demethylase Jhd2 , which is responsible for erasing the histone H3K4 trimethylation marks in yeast ( Mersman et al . , 2009 ) . Given that RBM15 interacts with histone methyltransferases complexes to maintain histone H3K4 methylation level ( Lee and Skalnik , 2012; Xiao et al . , 2015 ) , our data provide new insights into a potential crosstalk between methyltransferases and demethylases . Recently , the E3 ligase WWP2 has been shown to recognize the methylated lysine in SOX2 through its HECT domain ( Fang et al . , 2014 ) . Given that CNOT4 is within a large CCR4-NOT complex with nine subunits , we have not determined which domain in CNOT4 or other subunits of CCR4-NOT complex recognizes the methylated RBM15 . The peptide pull-down experiments strongly support that methylated region on RBM15 is sufficient for binding to CNOT4 . Since we found that the RING domain on CNOT4 alone was not sufficient for binding to methylated RBM15 ( data not shown ) , it is possible that more than one region on CNOT4 or other subunits in CCR4-NOT complex might interact with RBM15 . In order to investigate the biological significance of PRMT1-mediated methylation of RBM15 , we analyzed the role of PRMT1-RBM15 axis in hematopoiesis . Normal MK differentiation can be divided into two phases: from hematopoietic stem cells to MK progenitor cells and from MK progenitor cells into mature polyploidy MK cells . Genes such as ARHGEF1 ( ARHGEF1 , 2 are in Figure 5—source data 1 ) play opposite roles at different phases of MK differentiation ( Gao et al . , 2012; Smith et al . , 2012 ) . The number of MEP progenitor cells increases , while the polypoidy of mature MKs decreases in Rbm15 knockout mice ( Niu et al . , 2009 ) . Consistent with this , RBM15 knockdown in human primary cells like Rbm15 knockdown in mice produces low percentage of mature MKs ( Figure 4F ) . PRMT1 promotes the production of MEP cells , but PRMT1 has to be turned off to generate mature , polyploid CD41+/CD61+CD42+ MK cells . Because both PRMT1 and RBM15 are ubiquitously expressed in all tissues , PRMT1-–RBM15 might regulate differentiation in the same fashion in other tissues such as cardiac tissue , spleen and placenta where RBM15 has been shown to be essential for development ( Raffel et al . , 2009 ) . According to the Oncomine database ( www . oncomine . com ) , PRMT1 is highly expressed in acute myeloid and lymphoid leukemia ( AML and ALL ) as well as in solid tumors ( Rhodes et al . , 2007 ) . PRMT1 accounts for 4% gene mutations , amplifications and deletions in solid tumors ( see www . cbioportal . org ) ( Gao et al . , 2013 ) . The relevance of PRMT1 inhibition in cancer therapies is summarized in reviews ( Greenblatt and Nimer , 2014; Yang and Bedford , 2013 ) . The roles of PRMT1 in supporting cancer cell proliferation are shown in AML1-ETO and MLL-EEN-initiated leukemia ( Cheung et al . , 2007; Shia et al . , 2012 ) . A PRMT1-mediated MK block may therefore be contributory to AMKL leukemogenesis . Apart from their functions in MK differentiation , RUNX1 , GATA1 and c-MPL as well as other epigenetic factors such as BRD4 found in this study are dysregulated in a broad spectrum of hematological malignancies as well as solid tumors ( Baratta et al . , 2015; Crawford et al . , 2008; Crispino , 2005; Harada et al . , 2004; Pikman et al . , 2006 ) . Thus , dysregulation of PRMT1-–RBM15 pathway might be a common mechanism in leukemia and solid tumors . The role of RNA splicing is increasingly appreciated in hematological malignancies ( Cazzola et al . , 2013 ) . The RNA splicing patterns vary widely from hematopoietic stem cells to progenitor cells in humans ( Chen et al . , 2014 ) . Our studies offer mechanisms on how RNA splicing is regulated during hematopoiesis . RBM15 directly recruits the SF3B1 branch point recognition complex to introns . SF3B1 has been found to have mutations in myelodysplastic syndrome and in leukemia ( Yoshida et al . , 2011 ) . Given that SF3B1 knockout mouse cannot mimic defects seen in myelodysplastic syndromes ( Wang et al . , 2014 ) , mutations in SF3B1 are likely gain-of-function mutations . Whether altered interactions between RBM15 and SF3B1 mutants contribute to hematological malignancies warrants future study . One direct consequence of RNA splicing is the altered concentration ratios between functional proteins and their alternatively spliced dominant negative counterparts . The physiological significance for multiple isoforms of c-MPL is to attenuate signaling from full-length c-MPL during MK maturation . In transgenic mouse exclusively expressing full-length c-MPL , excessive production of platelets is observed ( Tiedt et al . , 2009 ) . Abnormal expression of GATA1s and RUNX1a are associated with leukemia ( Crispino , 2005; Liu et al . , 2009 ) . By controlling isoform ratios , HSCs fine-tune the lineage preference and the magnitude of lineage production during differentiation . This type of regulation is very important for HSCs to respond quickly to ever-changing environmental clues . RBM15 by binding to introns flanking the alternatively spliced exons enhances the inclusion of the exons to produce full length c-MPL mRNA ( Figure 7E ) . In the case of GATA1 , RBM15 binds to intron 1 to recruit SF3B1 to the same location ( Figure 7D ) . As a result , RBM15 enhances the inclusion of exon 2 to generate the GATA1fl mRNA ( Figure 6A ) . RBM15 facilitates the production of more full-length RUNX1b , c by skipping the exon that encodes RUNX1a . Knockdown RBM15 also facilitate the generation of RUNX1-exon6-– . Based on our proteomic data , RBM15 binds to RNA binding proteins that are involved in RNA surveillance , transport and degradation ( Figure 7—source data 1 ) . Consistently , we also detected association of RBM15 with matured mRNAs ( Figure 5A ) . In addition , RBM15 associates with transcriptional elongation factors such as Tho2 and CTR9 besides its role in transcription initiation by positively regulating histone H3K4 methylation ( Xiao et al . , 2015 ) . We speculate that RBM15 participates in regulating the whole lifecycle of a messenger RNA . This is reminiscent of the functions of CCR4-NOT complex ( Miller and Reese , 2012 ) . RBM15 has been shown to be a part of RBM20-mediated RNA splicing network in cardiomyocytes ( Maatz et al . , 2014; Raffel et al . , 2009 ) . Thus , RBM15 is involved in the development of other tissues in addition to hematopoiesis . Further experiments to define the specificity and affinity of RBM15 binding sequences will help us to understand the mechanisms of alternative splicing mediated by RBM15 . Apart from binding to introns , RBM15 binds to 3’UTR of metabolic enzymes , splicing factors as well as a few long noncoding RNA molecules and microRNAs ( Figure 5—source data 2 ) . Given that splicing factors can be involved in alternative RNA polyadenylation and RNA export , it is not surprising that RBM15 binds to 3’UTRs for alternative RNA polyadenylation , export as well as for RNA splicing ( Castelo-Branco et al . , 2004; Kyburz et al . , 2006 ) . Analysis by the MISO program indicates that RBM15 regulates alternative RNA splicing of genes such as HK1 by binding to 3’UTR . In the future , deeper sequencing of RNA samples and performing high-resolution PAR-CLIP assays ( Hafner et al . , 2010 ) with RBM15 antibodies will further define the RBM15-mediated RNA metabolism . In summary , our findings demonstrated a new role for PRMT1 in regulating RNA metabolism . Given that both RBM15 and PRMT1 are evolutionarily conserved proteins from plants to mammals , our studies on the PRMT1-–RBM15 pathway offer mechanistic insights into how RBM15 may regulate cell fate decision in multi-cellular organisms at post-transcriptional levels .
To reveal proteome-wide substrates of protein methyltransferases such as PRMT1 in the context of complex cellular components , we took advantage of the emerging BPPM technology ( Luo , 2012; Wang and Luo , 2013 ) . In BPPM , designated methyltransferases are engineered to process sulfonium-alkyl SAM analogues as alternative cofactors and thus transfer the distinct sulfonium alkyl handles to substrates ( Figure 1—figure supplement 2A ) . Because the alkyl handles contain a terminal-alkyne for the azide-alkyne Huisgen cycloaddition ( the click reaction ) , we can then couple the chemical moiety with a biotin-containing azide probe for amenable target enrichment and characterization . With the previously-identified Y39F-M48G PRMT1 mutant and the matched 4-propargyloxy-but-2-enyl SAM ( Pob-SAM ) as BPPM reagents ( Wang et al . , 2011 ) , RBM15 in the context of Meg-01 cells was readily labeled by the enzyme-–cofactor pair , modified by a biotin-containing azide probe , and enriched by streptavidin beads ( Figure 1—figure supplement 2B ) . As the negative control , the label efficiency dropped significantly in the absence of the Y39F-M48G PRMT1 variant . The tagged proteins were subsequently purified with streptavidin beads for mass spectrometry analysis . RBM15 was identified among the methylated proteins . We verified the mass spectrometry data with direct WB with RBM15 antibody against biotinylated proteins ( Figure 1—figure supplement 2C ) . To further map the RBM15 methylation sites , Flag-tagged RBM15 protein was purified from transient transfected 293T cells for tandem mass spectrometry analysis . A mono-methylation site was found at R578 , which lies between the RNA-binding domain and the SPOC domain ( Figure 1—figure supplement 3A ) . The human RBM15 cDNA in pCDNA3 vector was kindly provided by Dr . Barbara Felber ( NIH ) . The nucleotide sequences of all constructs were verified by sequencing . The retrovirus expression vector Migr1 was used to overexpress RBM15 and its mutant . pTripZ ( Openbiosystems ) was used to overexpress the PRMT1-V1 and PRMT1-V2 proteins by replacing RFP with PRMT1-V1 or PRMT1-V2 cDNA . The lentivirus expressing shRNA against PRMT1 , RBM15 , and CNOT4 were purchased from Openbiosystems ( ThermoScientific Inc . ) . The sequences are available in Supplementary file 1 . pRS and pRS-shPRMT1-V2 plasmids were reported previously ( Baldwin et al . , 2012 ) . For virus production , 293T cells were cotransfected with retroviral or lentiviral plasmids with helper plasmids as described ( Vu et al . , 2013 ) . Cells stably expressing PRMT1-V1 and PRMT1-V2 or knockdown RBM15 or PRMT1 were selected with 5 µg/ml puromycin , and GFP-positive cells stably expressing Flag-RBM15 and Flag-RBM15-R were sorted on a FACSAria cell sorter . BD FACS LSRFortessa was used to do FACS analysis . The gene bank number for this new c-MPL isoform is KF964490 . HEK293T cells were cultured in DMEM supplemented with 10% FBS and transient transfected by lipofectamine 2000 ( Invitrogen , Grand Island , USA ) . The pcDNA3 empty vectors were added to all transfections to balance total DNA . The human leukemic cell line MEG-01 was cultured in RPMI1640 medium containing 10% FBS with 100U/ml penicillin and 100ug/ml streptomycin . The generic methyl-transferase inhibitors: Adox ( adenosine , periodate oxidized , Sigma , #7154 ) and MTA ( 5’-Deoxy-5’- ( methylthio ) adenosine , Sigma , #D5011 ) were purchased from Sigma , St . Louis , MO . PMA ( 20 nM ) was added to stimulate Meg-01 cells to differentiate into MKs . The differentiated cells were labeled with anti-CD41 antibody and CD42 antibody ( BD ) and analyzed by FACS Fortessa machine . Transfected 293T cells from 10-cm dish were lysed in 1 ml of lysis buffer ( 20 mM Hepes , pH 7 . 9 , 150 mM NaCl , 1mM MgCl2 , 1% NP40 , 10 mM NaF , 0 . 2 mM NaVO4 , 10 mM β-glycerol phosphate ) with freshly added dithiothreitol ( DTT; 1 mM ) and a proteinase inhibitor cocktail ( Roche , Indianapolis , IN ) . The cells were incubated for 30 min on ice and sonicated with a Bioruptor Sonicator ( Diagenode , Denville , NJ ) . The extracts were cleared by centrifugation at 12 , 000×g for 15 min at 4°C . Immunoprecipitations were performed at 4°C in lysis buffer in the presence of RNaseA ( 10µg/ml ) using indicated antibody and 50 µl of 50% ( V/V ) slurry of Protein A agarose ( Roche , Indianapolis , IN ) or anti-Flag M2 agarose ( Sigma , St . Louis , MO ) for 4 hr . The precipitates were extensively washed and resuspended in 2×SDS-PAGE sample buffer directly . The immunopurified protein and cell lysates were resolved by 10% SDS-PAGE and transferred to PVDF membranes ( Millipore , Billerica , MA ) for WB assays . The proteins were visualized by the Immobilon Western Chemiluminescent horseradish peroxidase substrate detection kit ( Millipore ) . PRMT1-V2 specific antibody was reported ( Baldwin et al . , 2012 ) . The other antibodies used were commercially available , including anti-Flag M2 Ab ( #F1804 , Sigma ) , RBM15 monoclonal antibody ( #66059-1-1g , Proteintech , Chicago ) , RBM15 polyclonal antibody ( #: 10587-1-AP , Proteintech , Chicago ) , PRMT1 ( #07404 , Upstate Biotechnology ) , SF3B1 ( #PA5-19679 , Thermo Scientific ) , Ub ( #U5397 , Sigma ) and GAPDH ( #MA5-15738 , Thermo Scientific , Waltham ) . HEK293T cells transfected with Flag-RBM15 and His-ubiquitin expression plasmids for 40 hr were treated with 10 µM MG132 for 6 hr before harvesting . Cells were lysed in buffer A ( 6 M guanidine-HCl , 0 . 1 M Na2HPO4/NaH2PO4 , pH 7 . 5 , 10 mM imidazole ) at 4°C for 15 min . The lysate was sonicated for 20 cycles in Bioruptor Sonicator . After cleaning by centrifugation at 12 , 000g for 30 min at 4°C , the supernatant was incubated with Ni2+-NTA beads ( QIAGEN , Valencia , CA ) for 3 hr at room temperature . The beads were sequentially washed with buffers A , B ( 1 . 5 M guanidine-HCl , 25 mM Na2HPO4/NaH2PO4 , 20 mM Tris-Cl [pH 6 . 8] , 17 . 5 mM imidazole ) , and C ( 25 mM Tris-Cl [pH 6 . 8] , 20 mM imidazole ) . The ubiquitin-conjugated proteins were boiled in 2×SDS-PAGE loading buffer containing 200 mM imidazole and subjected to immunoblot analysis . Total RNA was prepared using RNeasy plus Kit ( QIAGEN ) . cDNA was generated by the Verso cDNA synthesis Kit ( Thermo Scientific ) with random hexamer primers . Real-time PCR assays were performed with Absolute Blue qPCR SYBR green Mix ( Thermo Scientific ) or Taqman Universal Master Mix II ( Applied Biosystems , for GATA1-FL and GATA1-S ) on a ViiA 7 system ( Applied Biosystems ) . Primers are listed in Supplementary file 2 . GAPDH was used as an internal control for normalization . Relative expression level was calculated by ∆ ( ∆ct ) method and all results were expressed as mean values ± standard errors from at least three independent experiments . GATA1 isoforms can be detected by regular PCR reactions with the primers: GATA1 Ex1 F: ATCACACTGAGCTTGCCACA , GATA1 Ex3 R: AGCTTGGGAGAGGAATAGGC in Figure 5H . For identification of methylation sites of targeted proteins , samples were separated by 10% of SDS-PAGE . The gel band were excised from the gel , reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide . Then in-gel digestion was carried out with the sequencing grade modified trypsin ( Promega , Fitchburg , WI ) in 50 mM ammonium bicarbonate at 37°C overnight . The peptides were extracted twice with 1% trifluoroacetic acid in 50% acetonitrile aqueous solution for 30 min . The extractions were then centrifuged in a speedvac to reduce the volume . For Liquid chromatography-tandem mass spectrometry ( LC-MS/MS ) analysis , the digested product was separated by a 60 min gradient elution at a flow rate 0 . 30 µL/min using an UltiMate 3000 RSLCnano System ( Thermo Scientific , USA ) which was directly interfaced with a Thermo Q Exactive benchtop mass spectrometer . The analytical column was a home-made fused silica capillary column ( 75 µm ID , 150 mm length; Upchurch , Oak Harbor , WA ) packed with C-18 resin ( 300 Å , 5 µm , Varian , Lexington , MA ) . Mobile phase A consisted of 0 . 1% formic acid and mobile phase B consisted of 100% acetonitrile and 0 . 1% formic acid . The mass spectrometer was operated in the data-dependent acquisition mode using the Xcalibur 2 . 2 . 0 software and there is a single full-scan mass spectrum in the Orbitrap ( 400–1800 m/z , 70 , 000 resolution ) followed by 8 MS/MS scans under the higher energy collision dissociation ( HCD ) . The MS/MS spectra from each LC-MS/MS run were searched against the selected database using an in-house Mascot or Proteome Discovery searching algorithm . The peptide pulldown method is published before ( Vu et al . , 2013 ) . Briefly , two peptides ( Biotin-LLYRDRDRME2ADLYPDSDWV and Biotin-LLYRDRDRDLYPDSDWV ) with or without methylation on R578 were synthesized . Biotin was added for binding to streptavidin beads . 200 μl of the whole cell extract ( 10mg/ml in H lysis buffer ) prepared as mentioned in co-immunoprecipitation assays was used to incubate with peptides ( at final concentration of 1 μM ) overnight at 4°C . The beads were washed with wash buffer ( 300mM NaCl , 1mM DTT , 20mM Tris pH 7 . 4 and 0 . 1% NP40 ) five times . Protein bound to the beads were boiled in SDS sample buffer and resolved in 10% SDS PAGE . RNA-seq data of 200 AML samples were downloaded from the Cancer Genome Atlas ( TCGA ) . The PRMT1 expression levels were used for survival analysis using Kaplan Meier ( K-–M ) curves and log-rank tests . Samples with available clinical outcomes ( survival month , n=103 ) data were included in the analysis . Logarithm 2-based transformations of each gene were performed prior to any analysis . Significant association was determined at 5% type I error level . Two guide RNAs were designed to target exon 2 of human CNOT4 at two different sites using the CRISPR design tool ( Zhang’s lab ) ( Ran et al . , 2013b ) . The distance between target sites is roughly 100bp . Two gRNAs were cloned into pX330 vector , and transient transfected into 293T cells . Single cells were selected by serial dilution . Single cell colonies were screened for internal deletion by PCR using 2 primers: F: TTCCCCTAAAATGTGTTATGATGA; R: CCAGTGCAGTGTTCTTTCCA . Guide RNA sequences: ( 1 ) CACCG GGATGTCATGTCCTCAGCGT; ( 2 ) CACCGGTGGATGCCAAAGTGTGCGT . RBM15 N-terminal sequence ( Acetyl-RTAGRDPVPRRSPRWRRAVPLC ) was used to develop rabbit polyclonal antibody against RBM15 by GeneMed Synthesis Inc . ( St . Antonio , Texas ) for RNA immunoprecipitation assay . Ten million MEG-01 cells grown at the exponential phase were cross-linked by 1% of formaldehyde in 37°C incubator for 10 min . Cells were washed twice with ice-cold phosphate-buffered saline lysed in H-lysis buffer ( 20mM HEPES pH 7 . 9 , 300 mM NaCl , 1mM MgCl2 , 1% NP40 , NaF 10 mM , 0 . 2 mM NaVO4 , 10 mM β-glycerol phosphate , 5% glycerol , 1 mM DTT , 40 U/ml RNaseOUT , and proteinase inhibitors ) , and then sonicated by Bioruptor ( Diagenode ) to fragments with average sizes of 200bp ( DNA ) for 30 min at 4oC . After the lysate was spun down , the supernatant was incubated with RBM15 antibody overnight at 4oC . Beads were washed five times with stringent buffer ( 50mM Tris pH7 . 5 , 1M NaCl , 1mM ethylenediaminetetraacetic acid [EDTA] , 0 . 1%SDS , 1% Na Deoxycholate , 1M Urea , and 1% NP40 ) , and once with TE buffer ( 10mM Tris pH 7 . 9 , 1mM EDTA ) . Immunoprecipitated RNA were eluted by incubating with 150μl RIP elution buffer ( 50mM Tris-Cl pH 7 . 9 , 5mM EDTA , 1% SDS , 200mM NaCl and 267 μg/ml of proteinase K ) for 1h at 45°C . Genomic DNA was removed by DnaseI Turbo ( Ambion ) . RNA was extracted by Trizol ( Invitrogen ) and cDNA was synthesized with random priming ( Thermo Verso cDNA synthesis kit ) . The cDNA was analyzed by qRT-PCR . We did two biological replicates for RIP-seq experiments with anti-RBM15 antibody and normal IgG . The RNA was converted into cDNA using SureSelect single strand RNA selection kit ( Illumina ) and sequenced with Illumina 2000 sequencer . Granulocyte-colony stimulating factor mobilized peripheral human blood cells were used to purify CD34+ cells . These CD34+ cells were spinfected with lentiviruses and grown in basic cytokine mix ( 100ng/ml of stem cell factor ( SCF ) , 100ng/ml FLt3 ligand , 50ng/ml of IL-6 and 20ng/ml of thrombopoietin[TPO] ) with Iscove's Modified Dulbecco's Medium medium plus 20% BIT ( Stem Cell Technology , Canada ) . For MK differentiation , we cultured the CD34+ cells with 50ng/ml TPO and 2ng/ml of SCF . The maturation of MK cells was assessed for the percentage of CD41+CD42+ or CD61+CD42+ populations as well as the percentage of polyploidy with PI staining by FACS analysis . We first trimmed adaptor sequences and low quality bases from paired end RIP sequencing reads using Trimmomatic ( Bolger et al . , 2014 ) . Processed reads were then mapped to hg19 genome with the STAR ( Dobin et al . , 2013 ) aligner . RIP reads from all samples were summarized into read coverage profiles on all chromosomes , and peaks were identified with a custom algorithm ( Loeb et al . , 2012 ) by convolving the read coverage signal with the second derivative of a Gaussian filter ( bandwidth = 300 ) . For each peak , we used DESeq ( Anders and Huber , 2010 ) to test if there was significant difference in read counts within the peak region between RBM15 samples and control samples after correcting for library size differences . 1515 peaks with more reads in RBM15 samples and FDR < 5% were defined as significant RBM15 binding sites . Each peak was annotated with information including gene name and region ( i . e . intron , 3’UTR , 5’UTR , CDS ) according to the overlapping RefSeq transcript . We obtained about 100 million paired-end reads for each RNA sample . All of the reads were mapped to the human reference genome ( GRCh37/hg19 ) using the STAR aligner . A gene annotation file was used to guide the alignments ( Ensembl GTF version GRCh37 . 70 ) . The mean insert sizes and the standard deviations were calculated using Picard-tools ( v1 . 126 ) ( http://broadinstitute . github . io/picard/ ) . Differential exon usage was calculated by DEXSeq ( Anders et al . , 2012 ) and MISO ( Katz et al . , 2010 ) programs to measure the abundance of event types: skipped exon ( SE ) , mixed exon usage ( MXE ) , alternative first exon ( AFE ) , alternative last exon ( ALE ) , alternative 5’ or 3’ splicing sites ( A5SS or A3SS ) and tandom 3’UTR use ( T3UTR ) . The Read Per Million ( RPM ) normalized BigWig files were generated using BEDTools ( v2 . 17 . 0 ) and bedGraphToBigWig tool ( v4 ) . And all the downstream statistical analyses and generating plots were performed in R ( v3 . 1 . 1 ) ( http://www . r-project . org/ ) . All the RNA-seq and RIP-seq data were deposited in NIH ( GSE73893 ) . In vitro ubiquitination assay was performed essentially as described ( Wang et al . , 2004 ) . Briefly , RBM15 or RBM15 R578K with or without PRMT1-mediated methylation were incubated with HA-CNOT4 in 32 . 5 μl reaction mixture containing 50 mM Tris ( pH7 . 5 ) , 5 mM MgCl2 , 2 mM NaF , 2 mM ATP , 10 mM Okadaic acid , 0 . 6 M DTT , 0 . 1 mg E1 , 0 . 2 mg UBCH5C and1 mg recombinant ubiquitin ( Sigma , St . Louis , MO ) . Reactions were carried out for 1 hr at 37°C before stopping with SDS sample loading buffer and resolving by 8% SDS-PAGE . Ubiquitination was detected by Western blot with anti-Flag antibody .
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 .
The many different cell types in an adult animal all develop from a single fertilized egg . The development of cells into more specialized cell types is called ‘differentiation’ . Proteins and other molecules from both inside and outside of the cells regulate the differentiation process . RNA is a molecule that is similar to DNA , and performs several important roles inside cells . Perhaps most importantly , RNA molecules act as messengers and carry genetic instructions during gene expression . RBM15 is an RNA-binding protein that is found throughout nature , and is involved in a number of developmental processes . Previous research has linked the incorrect control of RBM15 with an increased risk of certain cancers , including megakaryocytic leukemia . However , it is not clear what role RNA-binding proteins such as RBM15 play during differentiation . Now , Zhang , Tran , Su et al . have investigated the role of RBM15 during the development of large cells found in human bone marrow ( called megakaryocytes ) . First , the experiments demonstrated that an enzyme called PRMT1 modifies RBM15 . This enzyme adds a chemical mark called a methyl group at a specific site ( an arginine amino acid ) on the RNA-binding protein . Next , Zhang , Tran , Su et al . showed that the addition of this methyl group earmarks RBM15 for destruction . This means that an increase in PRMT1 levels reduces the amount of RBM15 in cells , while decreases in PRMT1 have the opposite effect . Further experiments showed that RBM15 normally processes the RNA messengers that carry the genetic instructions needed for the differentiation of bone marrow cells . An excess of PRMT1 enzyme leads to a lack of this RNA-binding protein . This in turn interferes with the differentiation process , and can contribute to the development of cancers such as megakaryocytic leukemia . Future work will therefore explore whether targeting PRMT1 with drugs could represent an effective treatment for these kinds of cancers .