Representative cancer-associated U2AF2 mutations alter RNA interactions and splicing

Abstract

High-throughput sequencing of hematologic malignancies and other cancers has revealed recurrent mis-sense mutations of genes encoding pre-mRNA splicing factors. The essential splicing factor U2AF2 recognizes a polypyrimidine-tract splice-site signal and initiates spliceosome assembly. Here, we investigate representative, acquired U2AF2 mutations, namely N196K or G301D amino acid substitutions associated with leukemia or solid tumors, respectively. We determined crystal structures of the wild-type (WT) compared with N196K- or G301D-substituted U2AF2 proteins, each bound to a prototypical AdML polypyrimidine tract, at 1.5, 1.4, or 1.7 Å resolutions. The N196K residue appears to stabilize the open conformation of U2AF2 with an inter-RNA recognition motif hydrogen bond, in agreement with an increased apparent RNA-binding affinity of the N196K-substituted protein. The G301D residue remains in a similar position as the WT residue, where unfavorable proximity to the RNA phosphodiester could explain the decreased RNA-binding affinity of the G301D-substituted protein. We found that expression of the G301D-substituted U2AF2 protein reduces splicing of a minigene transcript carrying prototypical splice sites. We further show that expression of either N196K- or G301D-substituted U2AF2 can subtly alter splicing of representative endogenous transcripts, despite the presence of endogenous, WT U2AF2 such as would be present in cancer cells. Altogether, our results demonstrate that acquired U2AF2 mutations such as N196K and G301D are capable of dysregulating gene expression for neoplastic transformation. High-throughput sequencing of hematologic malignancies and other cancers has revealed recurrent mis-sense mutations of genes encoding pre-mRNA splicing factors. The essential splicing factor U2AF2 recognizes a polypyrimidine-tract splice-site signal and initiates spliceosome assembly. Here, we investigate representative, acquired U2AF2 mutations, namely N196K or G301D amino acid substitutions associated with leukemia or solid tumors, respectively. We determined crystal structures of the wild-type (WT) compared with N196K- or G301D-substituted U2AF2 proteins, each bound to a prototypical AdML polypyrimidine tract, at 1.5, 1.4, or 1.7 Å resolutions. The N196K residue appears to stabilize the open conformation of U2AF2 with an inter-RNA recognition motif hydrogen bond, in agreement with an increased apparent RNA-binding affinity of the N196K-substituted protein. The G301D residue remains in a similar position as the WT residue, where unfavorable proximity to the RNA phosphodiester could explain the decreased RNA-binding affinity of the G301D-substituted protein. We found that expression of the G301D-substituted U2AF2 protein reduces splicing of a minigene transcript carrying prototypical splice sites. We further show that expression of either N196K- or G301D-substituted U2AF2 can subtly alter splicing of representative endogenous transcripts, despite the presence of endogenous, WT U2AF2 such as would be present in cancer cells. Altogether, our results demonstrate that acquired U2AF2 mutations such as N196K and G301D are capable of dysregulating gene expression for neoplastic transformation. Large-scale sequencing projects, together with an emerging plethora of protein structures, have revealed statistically significant clustering of disease-associated mutations at protein–ligand interfaces (1Wang X. Wei X. Thijssen B. Das J. Lipkin S.M. Yu H. Three-dimensional reconstruction of protein networks provides insight into human genetic disease.Nat. Biotechnol. 2012; 30 (22252508): 159-16410.1038/nbt.2106Crossref PubMed Scopus (270) Google Scholar, 2Kamburov A. Lawrence M.S. Polak P. Leshchiner I. Lage K. Golub T.R. Lander E.S. Getz G. Comprehensive assessment of cancer missense mutation clustering in protein structures.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26392535): E5486-E549510.1073/pnas.1516373112Crossref PubMed Scopus (115) Google Scholar, 3Gao M. Zhou H. Skolnick J. Insights into disease-associated mutations in the human proteome through protein structural analysis.Structure. 2015; 23 (26027735): 1362-136910.1016/j.str.2015.03.028Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 4Shao C. Yang B. Wu T. Huang J. Tang P. Zhou Y. Zhou J. Qiu J. Jiang L. Li H. Chen G. Sun H. Zhang Y. Denise A. Zhang D.E. et al.Mechanisms for U2AF to define 3′ splice sites and regulate alternative splicing in the human genome.Nat. Struct. Mol. Biol. 2014; 21 (25326705): 997-100510.1038/nsmb.2906Crossref PubMed Scopus (93) Google Scholar). This revelation explains how mis-sense mutations of the same gene can cause different diseases by affecting distinct functional interfaces of an encoded protein product. Conversely, trans-acting mutations that modify mutually interacting surfaces of a multisubunit complex often produce similar disease symptoms. For example, mis-sense mutations in different domains of the WAS (Wiskott–Aldrich syndrome) protein cause clinically distinct disorders such as Wiskott–Aldrich syndrome or X-linked neutropenia (1Wang X. Wei X. Thijssen B. Das J. Lipkin S.M. Yu H. Three-dimensional reconstruction of protein networks provides insight into human genetic disease.Nat. Biotechnol. 2012; 30 (22252508): 159-16410.1038/nbt.2106Crossref PubMed Scopus (270) Google Scholar). On the other hand, mutations clustered at the mutual interfaces of either complement factor H and component C3 proteins result in hemolytic uremic syndrome (1Wang X. Wei X. Thijssen B. Das J. Lipkin S.M. Yu H. Three-dimensional reconstruction of protein networks provides insight into human genetic disease.Nat. Biotechnol. 2012; 30 (22252508): 159-16410.1038/nbt.2106Crossref PubMed Scopus (270) Google Scholar). Indeed, understanding the 3D aspects of the gene-to-disease process is of great interest to pharmaceutical and medical industries seeking to identify druggable targets for precision medicine. For example, an Phe508 deletion remodels the interactome of the cystic fibrosis transmembrane conductance regulator (ΔF508). Reduced levels of specific ΔF508 cystic fibrosis transmembrane conductance regulator interactors can partially restore channel function (5Pankow S. Bamberger C. Calzolari D. Martínez-Bartolomé S. Lavallée-Adam M. Balch W.E. Yates 3rd, J.R. ΔF508 CFTR interactome remodelling promotes rescue of cystic fibrosis.Nature. 2015; 528 (26618866): 510-51610.1038/nature15729Crossref PubMed Scopus (134) Google Scholar). As a second example, oncogenic mutations of the p53 tumor suppressor protein can stimulate interactions with the transcription factor Nrf2, which in turn increases expression of proteasome genes and confers proteasome inhibitor resistance to cancer cells (6Walerych D. Lisek K. Sommaggio R. Piazza S. Ciani Y. Dalla E. Rajkowska K. Gaweda-Walerych K. Ingallina E. Tonelli C. Morelli M.J. Amato A. Eterno V. Zambelli A. Rosato A. et al.Proteasome machinery is instrumental in a common gain-of-function program of the p53 missense mutants in cancer.Nat. Cell Biol. 2016; 18 (27347849): 897-90910.1038/ncb3380Crossref PubMed Scopus (131) Google Scholar). The premise that disease-relevant mutations tend to cluster at 3D protein interfaces has been incorporated in several recent computational approaches for distinguishing neutral passenger mutations from candidate drivers of human disease (7Shim J.E. Kim J.H. Shin J. Lee J.E. Lee I. Pathway-specific protein domains are predictive for human diseases.PLoS Comput. Biol. 2019; 15 (31075101)e100705210.1371/journal.pcbi.1007052Crossref PubMed Scopus (4) Google Scholar, 8Ashford P. Pang C.S.M. Moya-García A.A. Adeyelu T. Orengo C.A. A CATH domain functional family based approach to identify putative cancer driver genes and driver mutations.Sci. Rep. 2019; 9 (30670742): 26310.1038/s41598-018-36401-4Crossref PubMed Scopus (9) Google Scholar, 9Chen S. Fragoza R. Klei L. Liu Y. Wang J. Roeder K. Devlin B. Yu H. An interactome perturbation framework prioritizes damaging missense mutations for developmental disorders.Nat. Genet. 2018; 50 (29892012): 1032-104010.1038/s41588-018-0130-zCrossref PubMed Scopus (29) Google Scholar). Among hematologic malignancies and certain types of cancers, acquired mis-sense mutations frequently affect pre-mRNA splicing factors involved in the early stages of 3´ splice-site selection (10Dvinge H. Kim E. Abdel-Wahab O. Bradley R.K. RNA splicing factors as oncoproteins and tumour suppressors.Nat. Rev. Cancer. 2016; 16 (27282250): 413-43010.1038/nrc.2016.51Crossref PubMed Scopus (301) Google Scholar). Most often, mutational hot spots affect clusters of residues at the protein–RNA interfaces of SF3B1, SRSF2, and U2AF1 (11). The cancer-associated mutations of SF3B1 further are believed to modify the toroidal structure of the protein and alter its recruitment of RNA helicases (12Tang Q. Rodriguez-Santiago S. Wang J. Pu J. Yuste A. Gupta V. Moldón A. Xu Y.Z. Query C.C. SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing.Genes Dev. 2016; 30 (28087715): 2710-272310.1101/gad.291872.116Crossref PubMed Scopus (46) Google Scholar, 13Carrocci T.J. Zoerner D.M. Paulson J.C. Hoskins A.A. SF3b1 mutations associated with myelodysplastic syndromes alter the fidelity of branchsite selection in yeast.Nucleic Acids Res. 2017; 45 (28062854): 4837-485210.1093/nar/gkw1349PubMed Google Scholar). The recurrent mutations of SF3B1, SRSF2, and U2AF1 in turn dysregulate the functions of the encoded proteins for gene expression and are thought to be drivers of cancer progression. Lower-frequency mutations of other splicing factors targets that alter functional for from such mutations in the of genes have been the of pre-mRNA as for the mutations of S. Lee C. J. recurrent mutations in cancer and mutational Biotechnol. 2016; PubMed Scopus Google Scholar). We cancer-associated mutations for the of the U2AF1 pre-mRNA splicing factor E. A.A. mutations structures of the U2AF2 RNA recognition 2017; PubMed Scopus Google Scholar). U2AF2 mutations affect residues that are in domains of the the RNA recognition and as as an for with U2AF1 and a motif The U2AF2 and are for a polypyrimidine signal the of 3´ splice sites R. J. and functions of polypyrimidine PubMed Google Scholar, R. H. recognition of the polypyrimidine-tract by the splicing factor and the splicing PubMed Scopus Google Scholar). by and demonstrate that the a in an T. S. B. K. A. V. J. M. selection pre-mRNA splicing by PubMed Scopus Google Scholar, J.R. C. T. M. M. interactions the by splicing factor a and 2014; PubMed Scopus Google Scholar, A.A. E. R. S. An domain recognizes the 3′ splice site 2016; PubMed Google Scholar, L. C. T. K. A. M. of the 3′ splice site RNA by the U2AF a Natl. Acad. Sci. U.S.A. 2016; PubMed Scopus Google Scholar). the of the conformation is and can a of A.A. E. R. S. An domain recognizes the 3′ splice site 2016; PubMed Google Scholar, A of to the of the essential splicing factor 2012; PubMed Scopus Google Scholar). A U2AF2 in which the RNA-binding of is by is in the with the U2AF1 C. A splice in U2AF2 is by U2AF1 and its recurrent Acids Res. PubMed Scopus Google Scholar). of the cancer-associated U2AF2 mutations are to cluster the RNA or of the open and E. A.A. mutations structures of the U2AF2 RNA recognition 2017; PubMed Scopus Google Scholar). This that cancer-associated mutations of the U2AF2 could to the and dysregulate pre-mRNA splicing or other the structural and functional of such U2AF2 mutations have to be Here, we RNA-binding and pre-mRNA splicing of minigene and endogenous to investigate the of representative cancer-associated mutations of We an N196K of the that with leukemia and a G301D of the U2AF2 in of and We compared the and 1.7 Å structures of the N196K- and G301D-substituted proteins bound to a prototypical with a Å structure of the wild-type (WT) The U2AF2 the N196K mutation to an hydrogen that appears to stabilize the open The G301D conformation remains to the phosphodiester of the the N196K mutation increases the apparent RNA-binding affinity of the G301D mutation has a The mutations affect splicing of representative transcripts, with the G301D mutation similar to in U2AF2 different structural and functional that the N196K and G301D mutations of U2AF2 have the to gene expression in and cancers in how mutations in the same U2AF2 gene can result in clinically distinct As a to the structural of the N196K and G301D mutations a splice-site we determined the crystal structure of the U2AF2 bound to a and The of the the prototypical transcript which is to the U2AF2 site as determined by in selection R. J. and functions of polypyrimidine PubMed Google Scholar). The from the of our structures A.A. E. R. S. An domain recognizes the 3′ splice site 2016; PubMed Google Scholar). As for structures, a and in the the and The structure determined at Å by similar crystal and the structure of bound to an an for and of structural to the structures bound to and the of the altered and for the are in 21 21 21 where is an for the of a with and is the of of where is the of of the of for the of is for of the from the in the of the program for the are in where is an for the of a with and is the of of where is the of of the of K. and 2012; PubMed Scopus Google for the of is for of the from the in the the program 3rd, structure for PubMed Scopus Google Scholar). in a The structure of the complex is to the Å for of from a Å in the of the are in an M. D. A. 2015; PubMed Scopus Google where are by a of and hydrogen with U2AF2 and The with the are with the of a U2AF2 to alter for the of the A.A. recognition and splicing of a Natl. Acad. Sci. U.S.A. 2014; PubMed Scopus (9) Google Scholar). and similar the of the structure are in the A that hydrogen with the has been by in the a structural at the the interactions and of the that U2AF2 the of the AdML compared with the the structural by a representative of the U2AF2 we determined the crystal structure of an N196K-substituted bound to the AdML at 1.7 Å This N196K is the common U2AF2 mutations, resulting from A in The and crystal is similar to the WT AdML such that structural can be to the amino acid The residue is in a of the the bound and at the from of the structure of the N196K-substituted protein remains similar to the WT complex the WT the is and as alternative of which a hydrogen with the A and the is in the and the residue of alternative in the N196K-substituted interacting with the the position of the has to a hydrogen with conformation of the in the This interaction could stabilize the open U2AF2 conformation for with We the structural by a representative of the U2AF2 by the crystal structure of a G301D-substituted bound to the AdML at Å This G301D results from a A in and Comprehensive of human and 2012; PubMed Scopus Google Scholar, S. C. O. R. M. H. P. et in cancer for Res. 2015; 21 PubMed Scopus Google Scholar). A in The and of the G301D structure similar to the N196K and The residue is the of and the of the bound the residue appears to a structural and with the bound A and the protein remains by the G301D Å The is by hydrogen to the and of from which an of the structure and The of the is of the of the Å This proximity is to the structures of N196K and G301D we that amino acid substitutions would the RNA-binding of the we the of the WT and proteins into a 3´ splice site with and the apparent The N196K increased the apparent affinity of for splice-site RNA by Conversely, the G301D decreased the RNA-binding affinity of by The of the with the and of the residue, as as with the the phosphodiester We that the altered RNA by the N196K and G301D substitutions would in turn affect splice-site We by and of a minigene and which and to an and from AdML T.R. I. M. of the of U2AF for recognition of a 3′ splice Cell Biol. PubMed Scopus Google alternative 3´ splice sites are by or the of the minigene to the AdML for our structures, is for U2AF2 our and the transcript A of splicing at the A of a splice site in to a which a and is to to U2AF2 of a WT U2AF2 increased at the site as A.A. E. R. S. An domain recognizes the 3′ splice site 2016; PubMed Google Scholar). of the N196K-substituted U2AF2 increased of the splice with a in RNA-binding Conversely, expression of the G301D-substituted U2AF2 splicing to results with the in structure and RNA-binding of the proteins, for other interactions of in the altered We the of N196K- and G301D-substituted U2AF2 splicing of representative endogenous in a human We to C. Yang B. Wu T. Huang J. Tang P. Zhou Y. Zhou J. Qiu J. Jiang L. Li H. Chen G. Sun H. Zhang Y. Denise A. Zhang D.E. et al.Mechanisms for U2AF to define 3′ splice sites and regulate alternative splicing in the human genome.Nat. Struct. Mol. Biol. 2014; 21 (25326705): 997-100510.1038/nsmb.2906Crossref PubMed Scopus (93) Google and the of acquired mutations in cancers, we either or G301D in the presence of endogenous We compared the of in U2AF2 levels As C. Yang B. Wu T. Huang J. Tang P. Zhou Y. Zhou J. Qiu J. Jiang L. Li H. Chen G. Sun H. Zhang Y. Denise A. Zhang D.E. et al.Mechanisms for U2AF to define 3′ splice sites and regulate alternative splicing in the human genome.Nat. Struct. Mol. Biol. 2014; 21 (25326705): 997-100510.1038/nsmb.2906Crossref PubMed Scopus (93) Google of U2AF2 increased of the and decreased of and The N196K and G301D substitutions to in splicing of the U2AF2 a similar as U2AF2 that can the splicing The N196K or similar as WT U2AF2 splicing of the and with its RNA-binding with the in demonstrate that the N196K- and U2AF2 can splicing of endogenous gene in the presence of the U2AF2 A of the cancer-associated mutations of U2AF2 affect residues at the RNA of the U2AF2 E. A.A. mutations structures of the U2AF2 RNA recognition 2017; PubMed Scopus Google Scholar). the present we demonstrate structural and functional for different of mutational a N196K mutation and solid G301D The N196K mutation of U2AF2 to the The promotes a and a hydrogen to the which appears to the open U2AF2 conformation for RNA the N196K mutation increases the RNA-binding affinity of The G301D the other hand, a the of the bound in the crystal the G301D protein and RNA similar to the WT the is to the of the G301D mutation the RNA-binding affinity of For a prototypical the N196K a similar as WT by to and the Conversely, the G301D-substituted U2AF2 is to splicing of the in agreement with its RNA-binding U2AF2 in turn expression of its U2AF1 and other splicing factors C. Yang B. Wu T. Huang J. Tang P. Zhou Y. Zhou J. Qiu J. Jiang L. Li H. Chen G. Sun H. Zhang Y. Denise A. Zhang D.E. et al.Mechanisms for U2AF to define 3′ splice sites and regulate alternative splicing in the human genome.Nat. Struct. Mol. Biol. 2014; 21 (25326705): 997-100510.1038/nsmb.2906Crossref PubMed Scopus (93) Google Scholar, T.R. I. M. of the of U2AF for recognition of a 3′ splice Cell Biol. PubMed Scopus Google is that the splicing are the of splicing to U2AF2 levels and substitutions is with a the of The splicing of endogenous in human cells is complex of factors and such as transcription and we the cancer-associated U2AF2 in the presence of U2AF2 to the of cancer cells that have acquired the U2AF2 expression of the N196K or G301D U2AF2 subtly splicing of endogenous such could gene expression to a in the RNA-binding of the proteins to affect splicing in different that could to disease for N196K for G301D An in the of is to cancer mutations from the of neutral that have been in human gene M.S. P. Golub T.R. M. Lander E.S. Getz G. and of cancer genes 21 tumour 2014; PubMed Scopus Google Scholar). For example, can in to cancers often are neutral we significant for cells the U2AF2 proteins in the of our the WT U2AF2 is essential for and its acquired mutations are that the N196K or G301D of U2AF2 splicing of representative gene transcripts, with the apparent of N196K- or mutations U2AF2 L. Lawrence M.S. Y. P. C. K. L. A. Zhang L. Zhang S.M. et and other cancer genes in J. PubMed Scopus Google that functional could of mutations through the that the N196K or G301D mutations of U2AF2 are capable of to the oncogenic of gene Conversely, mutations that affect mutual interfaces of distinct can have functional and cause the same by (1Wang X. Wei X. Thijssen B. Das J. Lipkin S.M. Yu H. Three-dimensional reconstruction of protein networks provides insight into human genetic disease.Nat. Biotechnol. 2012; 30 (22252508): 159-16410.1038/nbt.2106Crossref PubMed Scopus (270) Google Scholar, S. PubMed Scopus Google Scholar, S. M. J. Yang J. J. Wang Y. A. I. G. et interaction in human genetic 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). U2AF2 the of 3´ splice sites C. Yang B. Wu T. Huang J. Tang P. Zhou Y. Zhou J. Qiu J. Jiang L. Li H. Chen G. Sun H. Zhang Y. Denise A. Zhang D.E. et al.Mechanisms for U2AF to define 3′ splice sites and regulate alternative splicing in the human genome.Nat. Struct. Mol. Biol. 2014; 21 (25326705): 997-100510.1038/nsmb.2906Crossref PubMed Scopus (93) Google a signal would have the compared with a mis-sense mutation of the U2AF2 protein acquired mutations affecting protein of U2AF2 SF3B1, or RNA could similar our crystal structure that the N196K the open U2AF2 as for the mutation of U2AF1 in complex with a of splice sites C. A splice in U2AF2 is by U2AF1 and its recurrent Acids Res. PubMed Scopus Google Scholar). As a second example, the cancer-associated mutation of SF3B1 is to and RNA factor mutations in Insights from spliceosome Genet. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar, Q. Rodriguez-Santiago S. Wang J. Pu J. Yuste A. Gupta V. Moldón A. Xu Y.Z. Query C.C. SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing.Genes Dev. 2016; 30 (28087715): 2710-272310.1101/gad.291872.116Crossref PubMed Scopus (46) Google Scholar, 13Carrocci T.J. Zoerner D.M. Paulson J.C. Hoskins A.A. SF3b1 mutations associated with myelodysplastic syndromes alter the fidelity of branchsite selection in yeast.Nucleic Acids Res. 2017; 45 (28062854): 4837-485210.1093/nar/gkw1349PubMed Google which could the of the a J. G. B. Sun Y. A. R. E. E. C. of complex cancer and the PubMed Scopus Google Scholar, E. J. A. E. Y. B. C. The cancer an open for cancer 2012; PubMed Scopus Google that cancers and which are the same cancer types associated with the U2AF2 G301D Indeed, the common mis-sense mutations of and are at its RNA which is to RNA to the as would the G301D of that in certain the N196K and G301D U2AF2 mutations similar as common mutations in other splicing dysregulating pre-mRNA splicing and to neoplastic transformation. the results a for N196K and G301D mis-sense mutations of U2AF2 to to the of malignancies by splice-site signal other 3D interfaces of the U2AF2 protein can explain the of cancer-associated mutations in its splicing factor of splicing factor mutations a of pre-mRNA splicing cancers that be apparent based of the the cluster of mutations at the other of cancer-associated U2AF2 mutations are at the of its conformation or in the are to the of other mutations for U2AF2 and associated our that the cancer-associated N196K and G301D mutations modify the structural and functional of U2AF2 the of U2AF2 and its as a to investigate and and For and RNA-binding the or G301D of the U2AF2 RNA-binding domain the of and the of proteins and as A.A. E. R. S. An domain recognizes the 3′ splice site 2016; PubMed Google Scholar). a of a with 15 the a with a The protein the of and at for from RNA from and to the to or G301D in a with and for The protein from a of of with of a at The of the WT complex The of the N196K or G301D and of to the N196K or G301D to the crystal with a of and and in at K. at the S.M. A. T. A. S. H. P. M. I. M. P. C. J. et for at crystal and Biol. PubMed Scopus Google Scholar). The the and Y. of and Biol. PubMed Scopus Google and C.C. P. A. et of the and Biol. PubMed Scopus Google Scholar). The structures determined the from The P. B. K. and of Biol. PubMed Scopus Google and G. Chen R. et a for structure Google Scholar). The and are in for the RNA-binding as H. conformation and of RNA by the splicing factor Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). A RNA a 3´ splice site with the The proteins and RNA by into a 15 at The RNA in the 30 The of the protein to The a and at 23 by a The at and the at with a of The for the as H. conformation and of RNA by the splicing factor Biol. Full Text Full Text PDF PubMed Scopus Google to the apparent and the of a with The apparent are the of each The or and for each cells at in a The cells in for or to splicing of endogenous transcripts, with and The N196K- and mutations by in the of an U2AF2 in a for A the transcript by the minigene and at a and for The or cells in at with of encoding or G301D human U2AF2 with of or of an as a as by the The cells for RNA and protein For of U2AF2 cells with either U2AF2 and or a and and For protein the cells in a 50 and For and proteins by to a and with specific for U2AF2 or or and signal from a For and and RNA from the cells the from RNA leukemia with The of the minigene for of for 30 for and for The of for of for 30 for and for 15 for of for 30 for and for 15 and for of for 30 for and for 15 The a and with and and a The of with in a by the and to The are in The and structure factors of the and of bound to AdML and have been at the We are to of for the and wild-type of for of for with and for with with transcript leukemia human polypyrimidine RNA recognition motif

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