The SR Protein 9G8 and the Wilms' Tumor Suppressor Protein WT1 Promote Translation of mRNAs with Retained Introns

Recent data indicate that the various steps in post-transcriptional gene expression are linked. In the studies presented in this thesis, we show further evidence of the interconnection between the multiple steps of mRNA metabolism. In one study, we show a link between splicing with nucleocytoplasmic export and translation. In another study, we show that the products of an alternatively spliced pre-mRNA have different roles in mRNA metabolism. Specifically, SR proteins, Tap, and WT1(+KTS) function similarly to link nuclear and cytoplasmic events in the expression of mRNAs with retained introns. The first study examines the cytoplasmic role of the splicing regulatory SR protein, 9G8, which has recently been proposed to function in mRNA export in conjunction with the export protein, Tap/NXF1. We investigated the effect of 9G8 on cytoplasmic RNA fate. 9G8 was shown to enhance expression of unspliced RNA containing either the MPMV-CTE or the recently discovered Tap-CTE. 9G8 also enhanced polyribosome association of unspliced RNA containing a CTE. Hyperphosphorylated 9G8 was present in monosomes and small polyribosomes, whereas soluble fractions contained only hypophosphorylated protein. Our results are consistent with a model in which hypophosphorylated SR proteins remain stably associated with mRNP complexes during export and are released during translation initiation concomitant with increased phosphorylation. The second study examines the Wilms' tumor 1 (WT1) gene, which plays an important role in mammalian urogenital development. Dysregulation of this gene is observed in many human cancers. Alternative splicing of WT1 RNA leads to the expression of two major protein isoforms, WT1(+KTS) and WT1(-KTS). Whereas WT1(-KTS) acts as a transcriptional regulator, no clear function has been ascribed to WT1(+KTS), despite the fact that this protein is crucial for normal development. Here we show that WT1(+KTS) functions to enhance expression from RNA possessing a retained intron and containing either a cellular or viral constitutive transport element (CTE). WT1(+KTS) expression increases the levels of unspliced RNA containing a CTE and specifically promotes the association of this RNA with polyribosomes. These studies provide further support for links between different steps in RNA metabolism and for the existence of post-transcriptional operons.

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