Genomic Analyses of Musashi1 Downstream Targets Show a Strong Association with Cancer-related Processes

Abstract

Musashi1 (Msi1) is a highly conserved RNA-binding protein with pivotal functions in stem cell maintenance, nervous system development, and tumorigenesis. Despite its importance, only three direct mRNA targets have been characterized so far: m-numb, CDKN1A, and c-mos. Msi1 has been shown to affect their translation by binding to short elements located in the 3′-untranslated region. To better understand Msi1 functions, we initially performed an RIP-Chip analysis in HEK293T cells; this method consists of isolation of specific RNA-protein complexes followed by identification of the RNA component via microarrays. A group of 64 mRNAs was found to be enriched in the Msi1-associated population compared with controls. These genes belong to two main functional categories pertinent to tumorigenesis: 1) cell cycle, cell proliferation, cell differentiation, and apoptosis and 2) protein modification (including ubiquitination and ubiquitin cycle). To corroborate our findings, we examined the impact of Msi1 expression on both mRNA (transcriptomic) and protein (proteomic) expression levels. Genes whose mRNA levels were affected by Msi1 expression have a Gene Ontology distribution similar to RIP-Chip results, reinforcing Msi1 participation in cancer-related processes. The proteomics study revealed that Msi1 can have either positive or negative effects on gene expression of its direct targets. In summary, our results indicate that Msi1 affects a network of genes and could function as a master regulator during development and tumor formation. Musashi1 (Msi1) is a highly conserved RNA-binding protein with pivotal functions in stem cell maintenance, nervous system development, and tumorigenesis. Despite its importance, only three direct mRNA targets have been characterized so far: m-numb, CDKN1A, and c-mos. Msi1 has been shown to affect their translation by binding to short elements located in the 3′-untranslated region. To better understand Msi1 functions, we initially performed an RIP-Chip analysis in HEK293T cells; this method consists of isolation of specific RNA-protein complexes followed by identification of the RNA component via microarrays. A group of 64 mRNAs was found to be enriched in the Msi1-associated population compared with controls. These genes belong to two main functional categories pertinent to tumorigenesis: 1) cell cycle, cell proliferation, cell differentiation, and apoptosis and 2) protein modification (including ubiquitination and ubiquitin cycle). To corroborate our findings, we examined the impact of Msi1 expression on both mRNA (transcriptomic) and protein (proteomic) expression levels. Genes whose mRNA levels were affected by Msi1 expression have a Gene Ontology distribution similar to RIP-Chip results, reinforcing Msi1 participation in cancer-related processes. The proteomics study revealed that Msi1 can have either positive or negative effects on gene expression of its direct targets. In summary, our results indicate that Msi1 affects a network of genes and could function as a master regulator during development and tumor formation. RNA-binding proteins (RBPs) 4The abbreviations used are: RBP, RNA-binding protein; Msi1, Musashi1; UTR, untranslated region; qRT-PCR, quantitative reverse transcription polymerase chain reaction; PABP, Poly(A)-binding protein; RNP, ribonucleoprotein; IP, immunoprecipitation; GFP, green fluorescent protein; BAP, biotin acceptor peptide; CMV, cytomegalovirus; SELEX, systematic evolution of ligands by exponential enrichment; SHH, Sonic Hedgehog. play a central role in the modulation of post-transcriptional events that involve splicing, transport, localization, stability, and translation of mRNAs. RBPs can be classified as either general or specific. Specific RBPs interact with a small subset of mRNAs and can have a restrictive pattern of expression, e.g. being expressed only in certain tissues or during specific developmental stages (reviewed in Refs. 1McKee A.E. Silver P.A. Cell Res. 2007; 17: 581-590Crossref PubMed Scopus (41) Google Scholar, 2Sanchez-Diaz P. Penalva L.O. RNA Biol. 2006; 3: 101-109Crossref PubMed Scopus (57) Google Scholar). Some subsets of functionally related mRNAs may be co-regulated by specific RBPs, thereby setting the basis for the “RNA-operon” model (3Keene J.D. Tenenbaum S.A. Mol. Cell. 2002; 9: 1161-1167Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 4Keene J.D. Nat. Rev. Genet. 2007; 8: 533-543Crossref PubMed Scopus (1011) Google Scholar). One group of specific RBPs comprises “oncofetal” proteins, which play an important role during embryonic development. In adults, their expression is limited to stem and progenitor cells and in some instances are detected in tumor tissues. The coding region determinant-binding protein falls into this category. Transgenic mice overexpressing coding region determinant-binding protein in mammary glands developed tumors in 95% of the cases, suggesting that coding region determinant-binding protein is a proto-oncogene (5Tessier C.R. Doyle G.A. Clark B.A. Pitot H.C. Ross J. Cancer Res. 2004; 64: 209-214Crossref PubMed Scopus (92) Google Scholar). Another RBP with a similar pattern of expression and phenotype is Musashi1 (Msi1), which is the focus of our study. Musashi (Msi) is conserved across species (6Sakakibara S. Nakamura Y. Yoshida T. Shibata S. Koike M. Takano H. Ueda S. Uchiyama Y. Noda T. Okano H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15194-15199Crossref PubMed Scopus (282) Google Scholar) and may have a critical function in stem cell maintenance and self-renewal capability (6Sakakibara S. Nakamura Y. Yoshida T. Shibata S. Koike M. Takano H. Ueda S. Uchiyama Y. Noda T. Okano H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15194-15199Crossref PubMed Scopus (282) Google Scholar, 7Higuchi S. Hayashi T. Tarui H. Nishimura O. Nishimura K. Shibata N. Sakamoto H. Agata K. Mech. Dev. 2008; 125: 631-645Crossref PubMed Scopus (33) Google Scholar, 8Kaneko Y. Sakakibara S. Imai T. Suzuki A. Nakamura Y. Sawamoto K. Ogawa Y. Toyama Y. Miyata T. Okano H. Dev. Neurosci. 2000; 22: 139-153Crossref PubMed Scopus (448) Google Scholar, 9Sanchez-Diaz P.C. Burton T.L. Burns S.C. Hung J.Y. Penalva L.O. BMC Cancer. 2008; 8: 280Crossref PubMed Scopus (59) Google Scholar, 10Siddall N.A. McLaughlin E.A. Marriner N.L. Hime G.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8402-8407Crossref PubMed Scopus (97) Google Scholar). In mammals, Msi1 is mainly expressed in stem and progenitor cells from different tissues (11Sakakibara S. Okano H. J. Neurosci. 1997; 17: 8300-8312Crossref PubMed Google Scholar, 12Potten C.S. Booth C. Tudor G.L. Booth D. Brady G. Hurley P. Ashton G. Clarke R. Sakakibara S. Okano H. Differentiation. 2003; 71: 28-41Crossref PubMed Scopus (422) Google Scholar, 13Clarke R.B. Cell Prolif. 2005; 38: 375-386Crossref PubMed Scopus (63) Google Scholar), and a decrease occurs as cells commit to lineage differentiation (11Sakakibara S. Okano H. J. Neurosci. 1997; 17: 8300-8312Crossref PubMed Google Scholar, 14Sakakibara S. Imai T. Hamaguchi K. Okabe M. Aruga J. Nakajima K. Yasutomi D. Nagata T. Kurihara Y. Uesugi S. Miyata T. Ogawa M. Mikoshiba K. Okano H. Dev. Biol. 1996; 176: 230-242Crossref PubMed Scopus (472) Google Scholar). Overexpression of Msi1 in oligodendrocyte (15Dobson N.R. Zhou Y.X. Flint N.C. Armstrong R.C. Glia. 2008; 56: 318-330Crossref PubMed Scopus (18) Google Scholar) and mammary (16Wang X.Y. Yin Y. Yuan H. Sakamaki T. Okano H. Glazer R.I. Mol. Cell. Biol. 2008; 28: 3589-3599Crossref PubMed Scopus (120) Google Scholar) precursors induces cell proliferation, arrests differentiation, and prevents apoptosis. High levels of Msi1 have been found in different malignancies and, in certain cases, a correlation between Msi1 expression and a cancer cell's proliferative activity has been proposed. Further, small interference RNA-mediated Msi1 knockdown affects cell proliferation, apoptosis, and differentiation and prevents cells from growing as xenographs (9Sanchez-Diaz P.C. Burton T.L. Burns S.C. Hung J.Y. Penalva L.O. BMC Cancer. 2008; 8: 280Crossref PubMed Scopus (59) Google Scholar, 17Sureban S.M. May R. George R.J. Dieckgraefe B.K. McLeod H.L. Ramalingam S. Bishnupuri K.S. Natarajan G. Anant S. Houchen C.W. Gastroenterology. 2008; 134: 1448-1458Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The preferential binding motif of Msi1 (G/A)UnAGU (n = 1–3) has been determined by SELEX (Systematic Evolution of Ligands by Exponential enrichment) (18Imai T. Tokunaga A. Yoshida T. Hashimoto M. Mikoshiba K. Weinmaster G. Nakafuku M. Okano H. Mol. Cell. Biol. 2001; 21: 3888-3900Crossref PubMed Scopus (379) Google Scholar). Three direct Msi1 mRNA targets containing this motif in their 3′-UTRs have been identified so far: m-numb (18Imai T. Tokunaga A. Yoshida T. Hashimoto M. Mikoshiba K. Weinmaster G. Nakafuku M. Okano H. Mol. Cell. Biol. 2001; 21: 3888-3900Crossref PubMed Scopus (379) Google Scholar) and CDKN1A (19Battelli C. Nikopoulos G.N. Mitchell J.G. Verdi J.M. Mol. Cell. Neurosci. 2006; 31: 85-96Crossref PubMed Scopus (148) Google Scholar) in mammals and c-mos in Xenopus laevis (20Charlesworth A. Wilczynska A. Thampi P. Cox L.L. MacNicol A.M. EMBO J. 2006; 25: 2792-2801Crossref PubMed Scopus (137) Google Scholar). Binding of Msi1 to this regulatory element has been shown to modulate mRNA translation either blocking it in the cases of the mammalian targets m-numb and CDKN1A, or enhancing it in the case of X. laevis target c-mos (18Imai T. Tokunaga A. Yoshida T. Hashimoto M. Mikoshiba K. Weinmaster G. Nakafuku M. Okano H. Mol. Cell. Biol. 2001; 21: 3888-3900Crossref PubMed Scopus (379) Google Scholar, 19Battelli C. Nikopoulos G.N. Mitchell J.G. Verdi J.M. Mol. Cell. Neurosci. 2006; 31: 85-96Crossref PubMed Scopus (148) Google Scholar, 20Charlesworth A. Wilczynska A. Thampi P. Cox L.L. MacNicol A.M. EMBO J. 2006; 25: 2792-2801Crossref PubMed Scopus (137) Google Scholar). In mammalian cells, Msi1 represses m-numb 5′-cap-dependent translation by competing with eIF4G for PABP binding (21Kawahara H. Imai T. Imataka H. Tsujimoto M. Matsumoto K. Okano H. J. Cell Biol. 2008; 181: 639-653Crossref PubMed Scopus (154) Google Scholar); the mechanism by which Msi1 activates c-mos translation in X. laevis has yet to be determined. Based on what is known for other specific RBPs, we hypothesize that the three known Msi1 targets represent a small fraction of the RNAs regulated by this protein. To further elucidate the role of Msi1 in stem cell biology and tumor growth, a high throughput study to identify Msi1-associated RNAs is required. In this RIP-Chip is the In this complexes containing the protein of are via or and the RNAs are and identified by analysis S.A. J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, L.O. J.D. 2004; PubMed Google Scholar). has been used to identify RNA targets of RBPs in different (reviewed in Refs. 2Sanchez-Diaz P. Penalva L.O. RNA Biol. 2006; 3: 101-109Crossref PubMed Scopus (57) Google Scholar, J.D. Res. 2005; PubMed Scopus Google Scholar, A. T. Cell Mol. Sci. 2008; PubMed Scopus Google Scholar). RIP-Chip was used to The analysis of 64 Msi1 mRNAs that Msi1 is to be in of cell cycle, cell proliferation, cell differentiation, apoptosis, and analysis and were and into the L.O. J.D. 2004; PubMed Google Scholar) of the biotin acceptor coding gene was L.O. J.D. 2004; PubMed Google Scholar). The 3′-UTRs of the genes and were and into in and reverse in the Another was from the by a Msi1 binding located in and cells were in with and HEK293T cells were Gene to the by the the cells were in with in with biotin to and with of and of the Cell and of of the to the cells, the and the of RNA-protein complexes is in a study L.O. J.D. 2004; PubMed Google Scholar). The RNA were on an as the and of protein was on to and with as L.O. J.D. 2004; PubMed Google Scholar). we performed the isolation of and as with with were in of for and of was for cells were either with the or the as cells were with and RNA cells were with as by for RNA and with the from of RNA was with RNA as the To effects we used the in both RIP-Chip and gene expression of and RNA were and to an gene expression A gene expression analysis was were the and of were the in as and was by a and and other The were was and a method was used for between To genes that are enriched in the Msi1 the model was used Genet. Mol. Biol. 2004; PubMed Scopus Google Scholar). in Msi1 compared with and a or the were as for target were for the genes on the model and the of similar analysis was for the gene expression microarrays. the of the was and was the method Silver J. A. M. D. A. 2007; PubMed Scopus Google Scholar). and between and were expression was and by the model Genet. Mol. Biol. 2004; PubMed Scopus Google Scholar). a of or in Msi1 expression compared with with an for were S. H. Mol. Biol. 2000; PubMed Google Scholar) and from were an system and green as by the of RNA was reverse in a the reverse and a were performed in the was used to the of mRNA the In the of RIP-Chip a gene a in the was used as for RNA of Msi1-associated cells were as with either or for RNA polymerase activity was a of different RNA was and in were performed as The gene expression were to expression and by the of of the of Msi1-associated D. 2003; PubMed Scopus Google Scholar) to by the by of and by of = and are the and of the by a in of from the to the of a = is the of the and is the of the that is the that in the are for in the of Msi1 binding and determined the in the identified Gene Ontology and function of the Msi1 mRNAs identified by analysis was determined by for and G. J. H.C. Biol. 2003; PubMed Google Scholar) and by the an on from was used to and of HEK293T cells with either the or was performed as were Cell were in two were in a with of and and for the was as the Another of was to the were and as to the were as were for analysis a which with with to for and and were into an and in a to via on a of the was with a of from P. K. M. Y. Clarke G. T. T. S.C. T. S. J. S. S. M. R. K. N. A. A. D. D. K. P. A. A. S. D. M. G. D. G. S. J. S. M. Cox T. R. J. A. G. J. A. S. Res. 2008; PubMed Scopus Google Scholar). The results were for analysis by A. R. 2002; PubMed Scopus Google Scholar) and A. R. 2003; PubMed Scopus Google Scholar), and in the P. C. R. X. Nat. 2007; 25: PubMed Scopus Google Scholar, C. Nat. 2008; 3: PubMed Scopus Google Scholar) to and protein expression on of the proteins of proteins as identified their was a to RIP-Chip of Msi1 and of Msi1 to a mRNAs (reviewed in Refs. 1McKee A.E. Silver P.A. Cell Res. 2007; 17: 581-590Crossref PubMed Scopus (41) Google Scholar, 4Keene J.D. Nat. Rev. Genet. 2007; 8: 533-543Crossref PubMed Scopus (1011) Google Scholar, A. T. Cell Mol. Sci. 2008; PubMed Scopus Google Scholar) its in as stem cell maintenance and only three Msi1 mRNA targets have been characterized in to a RIP-Chip analysis to identify Msi1 targets and to a better on the regulated by this protein. to HEK293T cells as a model system for our was developed in this cell system and for a of RBPs L.O. J.D. 2004; PubMed Google Scholar). high levels of Msi1 have been in different tumor and cell we to a cell that levels of and CDKN1A, of was identified this cell and, (21Kawahara H. Imai T. Imataka H. Tsujimoto M. Matsumoto K. Okano H. J. Cell Biol. 2008; 181: 639-653Crossref PubMed Scopus (154) Google Scholar) used HEK293T as the model system to elucidate the mechanism by which Msi1 translation in mammalian are the a RIP-Chip analysis be have been performed either a or is in of and to mRNAs (reviewed in Refs. 2Sanchez-Diaz P. Penalva L.O. RNA Biol. 2006; 3: 101-109Crossref PubMed Scopus (57) Google Scholar, 4Keene J.D. Nat. Rev. Genet. 2007; 8: 533-543Crossref PubMed Scopus (1011) Google Scholar, A. T. Cell Mol. Sci. 2008; PubMed Scopus Google Scholar). To in our results, we two with two different controls. In both cases, Msi1-associated mRNAs were to be enriched in the compared with controls. two were green fluorescent protein and Poly(A)-binding protein is an RNA-binding protein and is expressed in mammalian cells, we that the RNAs represent the In is a protein that a role in mRNA translation via its binding to their indicate that the is similar to the L.O. S.M. H. J.D. Mol. Cancer. 2004; 3: PubMed Scopus Google Scholar). we to as a for mRNAs in is similar to the of RNA polymerase in to identify genes in a with RNA polymerase RIP-Chip a The biotin acceptor to the protein of was in by the and were used to and complexes L.O. J.D. 2004; PubMed Google Scholar). of were In both cases, the the with the One was from cells with the the was from cells with the In cases, a the gene was to of the high protein the of a RIP-Chip we performed a to the levels of protein via were with the and was and characterized L.O. J.D. 2004; PubMed Google Scholar). of both and from cell was highly we RNA an to that the proteins as from of cell of the of RNA we with was for for and for in with the of the three proteins used in our RNAs were and as to of two of or for used to mRNAs that are with a of and or the as compared the results from the two of and identified 64 mRNAs that in both and of the 64 mRNAs binding for Msi1 in the of Msi1-associated cell protein and in and protein protein protein protein protein containing of chain of with protein protein protein protein protein protein binding protein protein protein on RNA-binding A element and protein protein protein of tumor cell protein and in and in a To the results from the we a group of genes enriched in the mRNA a identified Msi1 target in the was as a an of RNA species in the population compared with The Msi1 Binding of Msi1 to its target mRNAs is to in the To we and reverse Msi1-associated mRNAs and in and into HEK293T with and and the controls. the cells were and the mRNAs with were with as The of mRNA with was determined for by a to expressed used the gene determined to be highly with Msi1, as a can be in a in mRNA was in to the as as the In a we a Msi1 binding from the the of in The and the were used in a as of the Msi1 binding an impact on other binding in the of the was the for the in reverse The Msi1 preferential binding motif was determined by SELEX analysis to be = (18Imai T. Tokunaga A. Yoshida T. Hashimoto M. Mikoshiba K. Weinmaster G. Nakafuku M. Okano H. Mol. Cell. Biol. 2001; 21: 3888-3900Crossref PubMed Scopus (379) Google Scholar). a of of K. D. M. Penalva L.O. RNA Biol. 2008; PubMed Scopus Google Scholar) and identified containing Msi1 binding is that Msi1 mRNAs. is that the by is to Msi1 binding To is a of Msi1 binding to be with we D. 2003; PubMed Scopus Google Scholar) to the of mRNAs identified by that Msi1 to be located in a are a of the of Msi1 binding and is the of Msi1 in stem cell differentiation, and tumor growth, the of to its expression levels or function could have The of small that Msi1 binding could be a To a of the and in Msi1 binding and function is A.E. Silver P.A. Cell Res. 2007; 17: 581-590Crossref PubMed Scopus (41) Google Scholar). of Msi1-associated Gene Ontology and to the of the Msi1-associated mRNAs identified by in G. J. H.C. Biol. 2003; PubMed Google Scholar) and were used for this used the with and a gene to an of as the to Gene Ontology processes. found in the cell = gene and and = gene with in the = gene with revealed that a high of the Msi1-associated genes are in cell cell differentiation cell apoptosis and protein modification and being that functions and whose is in used the to targets of the proteins by Msi1-associated mRNAs. The shown in was by for the proteins by Msi1-associated mRNAs and targets with a participation in in cell cycle, apoptosis, proliferation, and that we used the and to the was used to and the proteins by the Msi1-associated targets in and processes. A of the found is In a we as a to identify genes that are to of the proteins by the Msi1-associated mRNAs. In other we to identify a was determined to be the with of Based on the high of we a between and the tumor gene three proteins, two of which functions and which is to the The protein can interact in or complexes with and and its results in and transcription T. J.D. G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. N. A. J. K. C. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Y. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). is by by and T. Genes Dev. PubMed Scopus Google Scholar) and S. Clark P.A. G. PubMed Scopus Google Scholar). Another identified gene with to is protein was identified as a target mainly in the to and R. H. J. T. T. T. T. N. 2000; PubMed Scopus Google Scholar, T. K. H. H. A. Y. S. T. N. Genes Dev. 2003; 17: PubMed Scopus Google Scholar). between Msi1 and is with Msi1 represses and D. P. G. S. 2008; PubMed Scopus Google Scholar), and the cell regulator is a target of our study that Msi1 and are of the affects two important and Sonic (9Sanchez-Diaz P.C. Burton T.L. Burns S.C. Hung J.Y. Penalva L.O. BMC Cancer. 2008; 8: 280Crossref PubMed Scopus (59) Google Scholar) the between Msi1 and used to further identify with the direct between and a the important identified was a that as a T. Y. J. Cell. 2003; PubMed Scopus Google Scholar). found other in the to the and protein A is shown as a direct target of SHH, its of the ubiquitin is in thereby modification and cancer development (reviewed in N. M. Mol. Cancer Res. 2006; PubMed Scopus Google Scholar). In our RIP-Chip analysis we found that of the Msi1-associated mRNAs were in protein modification and and of and were related to ubiquitin results a between Msi1, and the modulation of processes. of Msi1 corroborate our findings, we a gene expression analysis was to the impact of Msi1 on the study is important for a better of the network of genes regulated by Msi1 and of Msi1 effects in tumor formation. compared the RNA of and cells gene expression microarrays. two of of in for genes and were determined to be expressed between and to an between RIP-Chip and results in of gene of the in the be by the of genes identified in the RIP-Chip The results in and this by a between Msi1 and cell and apoptosis. the RIP-Chip results, we determined a with a between a subset of genes that to Msi1 and and in with our (9Sanchez-Diaz P.C. Burton T.L. Burns S.C. Hung J.Y. Penalva L.O. BMC Cancer. 2008; 8: 280Crossref PubMed Scopus (59) Google Scholar). Msi1 and mRNA a between Msi1 binding and function on protein we HEK293T cells with either or and characterized their protein expression a method P. C. R. X. Nat. 2007; 25: PubMed Scopus Google Scholar). proteins were were proteins from the of Msi1 mRNAs by proteins, expression in cell The for identified proteins is from the The expression in a protein in and which is an protein. in protein expression in both suggesting that Msi1 can function either as or of of translation was in the case of and (18Imai T. Tokunaga A. Yoshida T. Hashimoto M. Mikoshiba K. Weinmaster G. Nakafuku M. Okano H. Mol. Cell. Biol. 2001; 21: 3888-3900Crossref PubMed Scopus (379) Google Scholar, 19Battelli C. Nikopoulos G.N. Mitchell J.G. Verdi J.M. Mol. Cell. Neurosci. 2006; 31: 85-96Crossref PubMed Scopus (148) Google Scholar), was in the case of c-mos (20Charlesworth A. Wilczynska A. Thampi P. Cox L.L. MacNicol A.M. EMBO J. 2006; 25: 2792-2801Crossref PubMed Scopus (137) Google Scholar). we for the that Msi1 can and protein expression in the cell that of the mRNA and of Msi1 binding the of or that is the RIP-Chip and the gene expression we that genes identified in the RIP-Chip have mRNA expression These in mRNA levels may be a direct of Msi1 with RBPs that gene expression, Msi1 could both the levels of translation and mRNA expression levels were in the cells in the suggesting that Msi1 prevents mRNA and genes to this HEK293T cells with either or we transcription by the cells with and mRNA levels a of In both cases, we that Msi1 to a of the mRNAs is a function for it to be determined Msi1 One is that Msi1 with RNAs and affects their with mRNAs by blocking or the of their binding In our are with the “RNA-operon” a high of the Msi1-associated targets to be related to a small of functional a similar pattern is for targets. In a was found between Msi1-associated mRNAs and cell cycle, proliferation, cell differentiation, apoptosis, and protein modification as as with of and thereby the that Msi1 functions as a master regulator in stem cell biology and tumor