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RNA Biol. 2015;12(12):1364-71. doi: 10.1080/15476286.2015.1102831.

HuR antagonizes the effect of an intronic pyrimidine-rich sequence in regulating WT1 +/-KTS isoforms.

Li H1,2, Hou S1,2, Hao T1, Azam S1, Liu C3, Shi L1, Lei H1.

Author information

  • 1a Institute of Cancer Stem Cell; Cancer Center; Dalian Medical University ; Dalian , P.R. China.
  • 2c Equal contribution.
  • 3b Breast Disease and Reconstruction Center; Breast Cancer Key Lab of Dalian; the Second Hospital of Dalian Medical University ; Dalian , P.R. China.

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.

KEYWORDS:

+/−KTS isoforms; HuR; RNP purification; WT1; alternative splicing

PMID:
26512748
PMCID:
PMC4829285
DOI:
10.1080/15476286.2015.1102831
[PubMed - indexed for MEDLINE]
Free PMC Article
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