We previously found that the splicing of exon 5 to exon 6 in the rat beta-TM gene required that exon 6 first be joined to the downstream common exon 8 (Helfman et al., Genes and Dev. 2, 1627-1638, 1988). Pre-mRNAs containing exon 5, intron 5 and exon 6 are not normally spliced in vitro. We have carried out a mutational analysis to determine which sequences in the pre-mRNA contribute to the inab...

Introduction: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic kidney disorders with the incidence of 1 in 1,000 births. ADPKD is genetically heterogeneous with two genes identified: PKD1 (16p13.3, 46 exons) and PKD2 (4q21, 15 exons). Eighty five percent of the patients with ADPKD have at least one mutation in the PKD1 gene. Genetic studies have demonstrate...

We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)-labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)-depend...

The exon junction complex (EJC) is loaded onto mRNAs as a consequence of splicing and regulates multiple posttranscriptional events. MLN51, Magoh, Y14, and eIF4A3 form a highly stable EJC core, but where this tetrameric complex is assembled in the cell remains unclear. Here we show that EJC factors are enriched in domains that we term perispeckles and are visible as doughnuts around nuclear spe...

RNA export factor (REF) is a component of the exon junction complex (EJC) that is deposited on mRNA in a splicing-dependent manner, and targets spliced mRNA for export. In this study, analysis of the RNA-binding protein complexes revealed that REF associates with beta-globin mRNA at the region other than the EJC deposition site. Comparison between RNA polymerase II and T7 transcription and furt...

The proteins Mago and Y14 are evolutionarily conserved binding partners. Y14 is a component of the exon-exon junction complex (EJC), deposited by the spliceosome upstream of messenger RNA (mRNA) exon-exon junctions. The EJC is implicated in post-splicing events such as mRNA nuclear export and nonsense-mediated mRNA decay. Drosophila Mago is essential for the localization of oskar mRNA to the po...

Metazoan cells have two pathways for intron removal involving the U2- and U12-type spliceosomes, which contain mostly nonoverlapping sets of small nuclear ribonucleoproteins. We show that in vitro splicing of a U12-type intron assembles an exon junction complex (EJC) that is comparably positioned and contains many of the same components as that deposited by the U2-type spliceosome. The presence...

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that recognizes and degrades transcripts containing NMD cis elements in their 3'untranslated region (UTR). In yeasts, unusually long 3'UTRs act as NMD cis elements, whereas in vertebrates, NMD is induced by introns located >50 nt downstream from the stop codon. In vertebrates, splicing leads to deposition of exon junction...