Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an Affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71+ erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation.
References
[1]
Burgess DJ (2012) Alternative splicing: proteomic rewiring through transcriptomic diversity. Nat Rev Genet 13: 518-519. doi:10.1038/nrg3299. PubMed: 22805699.
[2]
Chen M, Manley JL (2009) Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat Rev Mol Cell Biol 10: 741-754. PubMed: 19773805.
[3]
Nilsen TW, Graveley BR (2010) Expansion of the eukaryotic proteome by alternative splicing. Nature 463: 457-463. doi:10.1038/nature08909. PubMed: 20110989.
[4]
Hallegger M, Llorian M, Smith CW (2010) Alternative splicing: global insights. FEBS J 277: 856-866. doi:10.1111/j.1742-4658.2009.07521.x. PubMed: 20082635.
[5]
Warzecha CC, Sato TK, Nabet B, Hogenesch JB, Carstens RP (2009) ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing. Mol Cell 33: 591-601. doi:10.1016/j.molcel.2009.01.025. PubMed: 19285943.
[6]
Irimia M, Blencowe BJ (2012) Alternative splicing: decoding an expansive regulatory layer. Curr Opin Cell Biol 24: 323-332. doi:10.1016/j.ceb.2012.03.005. PubMed: 22465326.
Ohno G, Ono K, Togo M, Watanabe Y, Ono S et al. (2012) Muscle-Specific Splicing Factors ASD-2 and SUP-12 Cooperatively Switch Alternative Pre-mRNA Processing Patterns of the ADF/Cofilin Gene in Caenorhabditis elegans. PLOS Genet 8: e1002991. PubMed: 23071450.
[9]
Kuroyanagi H, Ohno G, Mitani S, Hagiwara M (2007) The Fox-1 family and SUP-12 coordinately regulate tissue-specific alternative splicing in vivo. Mol Cell Biol 27: 8612-8621. doi:10.1128/MCB.01508-07. PubMed: 17923701.
[10]
Anyanful A, Ono K, Johnsen RC, Ly H, Jensen V et al. (2004) The RNA-binding protein SUP-12 controls muscle-specific splicing of the ADF/cofilin pre-mRNA in C. elegans. J Cell Biol 167: 639-647. doi:10.1083/jcb.200407085. PubMed: 15545320.
[11]
Yan W, Zhang J, Zhang Y, Jung YS, Chen X (2012) p73 expression is regulated by RNPC1, a target of the p53 family, via mRNA stability. Mol Cell Biol 32: 2336-2348. doi:10.1128/MCB.00215-12. PubMed: 22508983.
[12]
Xu E, Zhang J, Chen X (2012) MDM2 expression is repressed by the RNA-binding protein RNPC1 via mRNA stability. Oncogene, 32: 2169–78. PubMed: 22710720.
[13]
H？tte GJ, Linam-Lennon N, Reynolds JV, Maher SG (2012) Radiation sensitivity of esophageal adenocarcinoma: the contribution of the RNA-binding protein RNPC1 and p21-mediated cell cycle arrest to radioresistance. Radiat Res 177: 272-279. doi:10.1667/RR2776.1. PubMed: 22214381.
[14]
Cho SJ, Jung YS, Zhang J, Chen X (2012) The RNA-binding protein RNPC1 stabilizes the mRNA encoding the RNA-binding protein HuR and cooperates with HuR to suppress cell proliferation. J Biol Chem 287: 14535-14544. doi:10.1074/jbc.M111.326827. PubMed: 22371495.
[15]
Zhang J, Cho SJ, Shu L, Yan W, Guerrero T et al. (2011) Translational repression of p53 by RNPC1, a p53 target overexpressed in lymphomas. Genes Dev 25: 1528-1543. doi:10.1101/gad.2069311. PubMed: 21764855.
[16]
Zhang J, Jun Cho S, Chen X (2010) RNPC1, an RNA-binding protein and a target of the p53 family, regulates p63 expression through mRNA stability. Proc Natl Acad Sci U S A 107: 9614-9619. doi:10.1073/pnas.0912594107. PubMed: 20457941.
[17]
Cho SJ, Zhang J, Chen X (2010) RNPC1 modulates the RNA-binding activity of, and cooperates with, HuR to regulate p21 mRNA stability. Nucleic Acids Res 38: 2256-2267. doi:10.1093/nar/gkp1229. PubMed: 20064878.
[18]
Shu L, Yan W, Chen X (2006) RNPC1, an RNA-binding protein and a target of the p53 family, is required for maintaining the stability of the basal and stress-induced p21 transcript. Genes Dev 20: 2961-2972. doi:10.1101/gad.1463306. PubMed: 17050675.
[19]
Yamamoto ML, Clark TA, Gee SL, Kang JA, Schweitzer AC et al. (2009) Alternative pre-mRNA splicing switches modulate gene expression in late erythropoiesis. Blood 113: 3363-3370. doi:10.1182/blood-2008-05-160325. PubMed: 19196664.
[20]
Yang G, Huang SC, Wu JY, Benz EJ Jr (2008) Regulated Fox-2 isoform expression mediates protein 4.1R splicing during erythroid differentiation. Blood 111: 392-401. doi:10.1182/blood-2007-01-068940. PubMed: 17715393.
[21]
Ponthier JL, Schluepen C, Chen W, Lersch RA, Gee SL et al. (2006) Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16. J Biol Chem 281: 12468-12474. doi:10.1074/jbc.M511556200. PubMed: 16537540.
[22]
Gimm JA, An X, Nunomura W, Mohandas N (2002) Functional characterization of spectrin-actin-binding domains in 4.1 family of proteins. Biochemistry 41: 7275-7282. doi:10.1021/bi0256330. PubMed: 12044158.
[23]
Horne WC, Huang SC, Becker PS, Tang TK, Benz EJ Jr (1993) Tissue-specific alternative splicing of protein 4.1 inserts an exon necessary for formation of the ternary complex with erythrocyte spectrin and F-actin. Blood 82: 2558-2563. PubMed: 8400303.
[24]
Discher D, Parra M, Conboy JG, Mohandas N (1993) Mechanochemistry of the alternatively spliced spectrin-actin binding domain in membrane skeletal protein 4.1. J Biol Chem 268: 7186-7195. PubMed: 8463254.
[25]
Conboy J, Kan YW, Shohet SB, Mohandas N (1986) Molecular cloning of protein 4.1, a major structural element of the human erythrocyte membrane skeleton. Proc Natl Acad Sci U S A 83: 9512-9516. doi:10.1073/pnas.83.24.9512. PubMed: 3467321.
[26]
Angers S, Thorpe CJ, Biechele TL, Goldenberg SJ, Zheng N et al. (2006) The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-beta-catenin pathway by targeting Dishevelled for degradation. Nat Cell Biol 8: 348-357. doi:10.1038/ncb1381. PubMed: 16547521.
[27]
De Gregorio E, Preiss T, Hentze MW (1999) Translation driven by an eIF4G core domain in vivo. EMBO J 18: 4865-4874. doi:10.1093/emboj/18.17.4865. PubMed: 10469664.
[28]
Newman EA, Muh SJ, Hovhannisyan RH, Warzecha CC, Jones RB et al. (2006) Identification of RNA-binding proteins that regulate FGFR2 splicing through the use of sensitive and specific dual color fluorescence minigene assays. RNA 12: 1129-1141. doi:10.1261/rna.34906. PubMed: 16603716.
[29]
van Zalen S, Jeschke GR, Hexner EO, Russell JE (2012) AUF-1 and YB-1 are critical determinants of beta-globin mRNA expression in erythroid cells. Blood 119: 1045-1053. doi:10.1182/blood-2011-10-387316. PubMed: 22134169.
[30]
Tehranchi R, Invernizzi R, Grandien A, Zhivotovsky B, Fadeel B et al. (2005) Aberrant mitochondrial iron distribution and maturation arrest characterize early erythroid precursors in low-risk myelodysplastic syndromes. Blood 106: 247-253. doi:10.1182/blood-2004-12-4649. PubMed: 15755901.
[31]
Marturana F, Timmins NE, Nielsen LK (2011) Short-term exposure of umbilical cord blood CD34+ cells to granulocyte-macrophage colony-stimulating factor early in culture improves ex vivo expansion of neutrophils. Cytotherapy 13: 366-377. doi:10.3109/14653249.2010.518610. PubMed: 20860426.
[32]
Warzecha CC, Jiang P, Amirikian K, Dittmar KA, Lu H et al. (2010) An ESRP-regulated splicing programme is abrogated during the epithelial-mesenchymal transition. EMBO J 29: 3286-3300. doi:10.1038/emboj.2010.195. PubMed: 20711167.
[33]
Warzecha CC, Shen S, Xing Y, Carstens RP (2009) The epithelial splicing factors ESRP1 and ESRP2 positively and negatively regulate diverse types of alternative splicing events. RNA Biol 6: 546-562. doi:10.4161/rna.6.5.9606. PubMed: 19829082.
[34]
Keefe AD, Wilson DS, Seelig B, Szostak JW (2001) One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag. Protein Expr Purif 23: 440-446. doi:10.1006/prep.2001.1515. PubMed: 11722181.
[35]
Geng J, Carstens RP (2006) Two methods for improved purification of full-length mammalian proteins that have poor expression and/or solubility using standard Escherichia coli procedures. Protein Expr Purif 48: 142-150. doi:10.1016/j.pep.2006.01.021. PubMed: 16529945.
[36]
Dittmar KA, Jiang P, Park JW, Amirikian K, Wan J et al. (2012) Genome-wide determination of a broad ESRP-regulated posttranscriptional network by high-throughput sequencing. Mol Cell Biol 32: 1468-1482. doi:10.1128/MCB.06536-11. PubMed: 22354987.
[37]
Shen S, Warzecha CC, Carstens RP, Xing Y (2010) MADS+: discovery of differential splicing events from Affymetrix exon junction array data. Bioinformatics 26: 268-269. doi:10.1093/bioinformatics/btp643. PubMed: 19933160.
[38]
Su AI, Wiltshire T, Batalov S, Lapp H, Ching KA et al. (2004) A gene atlas of the mouse and human protein-encoding transcriptomes. Proc Natl Acad Sci U S A 101: 6062-6067. doi:10.1073/pnas.0400782101. PubMed: 15075390.
[39]
Ross DT, Scherf U, Eisen MB, Perou CM, Rees C et al. (2000) Systematic variation in gene expression patterns in human cancer cell lines. Nat Genet 24: 227-235. doi:10.1038/73432. PubMed: 10700174.
[40]
Sun Y, Li T, Ma K, Tian Z, Zhu Y et al. (2009) The impacts of ERCC1 gene exon VIII alternative splicing on cisplatin-resistance in ovarian cancer cells. Cancer Invest 27: 891-897. doi:10.3109/07357900902744536. PubMed: 19832035.
[41]
R？nnstrand L (2004) Signal transduction via the stem cell factor receptor/c-Kit. Cell Mol Life Sci 61: 2535-2548. doi:10.1007/s00018-004-4189-6. PubMed: 15526160.
[42]
Liu P, Barb J, Woodhouse K, Taylor JGGt, Munson PJ, et al (2011) Transcriptome profiling and sequencing of differentiated human hematopoietic stem cells reveal lineage-specific expression and alternative splicing of genes. Physiol Genomics 43: 1117-1134. doi:10.1152/physiolgenomics.00099.2011. PubMed: 21828245.
[43]
Schischmanoff PO, Winardi R, Discher DE, Parra MK, Bicknese SE et al. (1995) Defining of the minimal domain of protein 4.1 involved in spectrin-actin binding. J Biol Chem 270: 21243-21250. doi:10.1074/jbc.270.36.21243. PubMed: 7673158.
[44]
Hou VC, Lersch R, Gee SL, Ponthier JL, Lo AJ et al. (2002) Decrease in hnRNP A/B expression during erythropoiesis mediates a pre-mRNA splicing switch. EMBO J 21: 6195-6204. doi:10.1093/emboj/cdf625. PubMed: 12426391.
[45]
Jolma A, Kivioja T, Toivonen J, Cheng L, Wei G et al. (2010) Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities. Genome Res 20: 861-873. doi:10.1101/gr.100552.109. PubMed: 20378718.
[46]
Léveillé N, Elkon R, Davalos V, Manoharan V, Hollingworth D et al. (2011) Selective inhibition of microRNA accessibility by RBM38 is required for p53 activity. Nat Commun 2: 513. doi:10.1038/ncomms1519. PubMed: 22027593.
[47]
Ray D, Kazan H, Cook KB, Weirauch MT, Najafabadi HS et al. (2013) A compendium of RNA-binding motifs for decoding gene regulation. Nature 499: 172-177. doi:10.1038/nature12311. PubMed: 23846655.
[48]
Ule J, Stefani G, Mele A, Ruggiu M, Wang X et al. (2006) An RNA map predicting Nova-dependent splicing regulation. Nature 444: 580-586. doi:10.1038/nature05304. PubMed: 17065982.
[49]
Yeo GW, Coufal NG, Liang TY, Peng GE, Fu XD et al. (2009) An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat Struct Mol Biol 16: 130-137. doi:10.1038/nsmb.1545. PubMed: 19136955.
[50]
Baron-Benhamou J, Gehring NH, Kulozik AE, Hentze MW (2004) Using the lambdaN peptide to tether proteins to RNAs. Methods Mol Biol 257: 135-154. PubMed: 14770003.
