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

ABSTRACT

WT1 +KTS and -KTS isoforms only differ in three amino acids in protein sequence but show significant functional difference. The +/−KTS isoforms were generated by alternative usage of two adjacent 5′ splice sites at RNA level, however, how these two 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.