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.
Highlights
More than 90% of human coding genes undergo alternative splicing in specific tissues or developmental stages,[1] whereas alternative splicing is a major determinant for the diversity of proteins in higher eukaryotes.[2]
There are 2 major alternative splicing events in Wilms’ tumor gene 1 (WT1) pre-mRNA, the first one refers to inclusion or skipping of exon 5, and the second one involves alternative usage of 2 adjacent 5’ splice sites at the end of exon 9 which results in insertion or removal of 3 amino acids Lys-Thr-Ser (KTS) in between of zinc fingers 3 and 4, these 2 isoforms have been designated as CKTS and ¡KTS isoforms.[4]
We show that a pyrimidine-rich sequence in intron 9 close to the 50 splice sites serves as a splicing regulatory elements (SREs) favoring the production of the CKTS isoform
Summary
More than 90% of human coding genes undergo alternative splicing in specific tissues or developmental stages,[1] whereas alternative splicing is a major determinant for the diversity of proteins in higher eukaryotes.[2] Due to alternative splicing and usage of different translation starting codons, many variants have been detected for Wilms’ tumor gene 1 (WT1), one of the first cloned tumor suppressor genes.[3] WT1 locates at chromosome 11p13 and contains 10 exons. WT1 expression is upregulated, while at later stages of differentiation its expression is downregulated.[13] WT1 was first discovered as a tumor suppressor in Wilms’
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