title: HuR antagonizes the effect of an intronic pyrimidine-rich sequence in regulating WT1 +/-KTS isoforms.
abstract: WT1 + KTS and -KTS isoforms only differ in 3 amino acids in protein sequence but show significant functional difference. The +/-KTS isoforms were generated by alternative usage of 2 adjacent 5' splice sites at RNA level, however, how these 2 isoforms are regulated is still elusive. Here we report the identification of an intronic pyrimidine-rich sequence that is critical for the ratio of +/-KTS isoforms, deletion or partial replacement of the sequence led to full/significant shift to -KTS isoform. To identify trans-factors that can regulate +/-KTS isoforms via the binding to the element, we performed RNP assembly using in vitro transcribed RNA with or without the pyrimidine-rich sequence. Mass spectrometry analysis of purified RNPs showed that the element associated with many splicing factors. Co-transfection of these factors with WT1 reporter revealed that HuR promoted the production of -KTS isoform at the reporter level. RNA immuno-precipitation experiment indicated that HuR interacted with the pyrimidine-rich element in WT1 intron 9. We further presented evidence that transient or stable over-expression of HuR led to enhanced expression of endogenous -KTS isoform. Moreover, knockdown of HuR resulted in decreased expression of endogenous -KTS isoform in 293T, SW620, SNU-387 and AGS cell lines. Together, these data indicate that HuR binds to the pyrimidine-rich sequence and antagonize its effect in regulating WT1 +/-KTS isoforms.
authors.affiliation: a Institute of Cancer Stem Cell; Cancer Center; Dalian Medical University ; Dalian , P.R. China.|c Equal contribution.
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.affiliation: a Institute of Cancer Stem Cell; Cancer Center; Dalian Medical University ; Dalian , P.R. China.|c Equal contribution.
authors.forename: Shuai
authors.initials: S
authors.lastname: Hou
authors.affiliation: a Institute of Cancer Stem Cell; Cancer Center; Dalian Medical University ; Dalian , P.R. China.
authors.forename: Tian
authors.initials: T
authors.lastname: Hao
authors.affiliation: a Institute of Cancer Stem Cell; Cancer Center; Dalian Medical University ; Dalian , P.R. China.
authors.forename: Sikandar
authors.initials: S
authors.lastname: Azam
authors.affiliation: b Breast Disease and Reconstruction Center; Breast Cancer Key Lab of Dalian; the Second Hospital of Dalian Medical University ; Dalian , P.R. China.
authors.forename: Caigang
authors.initials: C
authors.lastname: Liu
authors.affiliation: a Institute of Cancer Stem Cell; Cancer Center; Dalian Medical University ; Dalian , P.R. China.
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.affiliation: a Institute of Cancer Stem Cell; Cancer Center; Dalian Medical University ; Dalian , P.R. China.
authors.forename: Haixin
authors.initials: H
authors.lastname: Lei
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D000067780:ELAV-Like Protein 1 / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics; D012261:Ribonucleoproteins / Q000378:metabolism; D025721:WT1 Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D000067780:ELAV-Like Protein 1; D020033:Protein Isoforms; D011743:Pyrimidines; D012322:RNA Precursors; D012261:Ribonucleoproteins; D025721:WT1 Proteins; C030986:pyrimidine
keywords: +/−KTS isoforms; HuR; RNP purification; WT1; alternative splicing