Splicing of U12-type introns deposits an exon junction complex competent to induce nonsense-mediated mRNA decay

Abstract

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 of a U12-type intron downstream of a premature termination codon within an open reading frame (ORF) induces nonsense-mediated decay of the mRNA in vivo. These findings suggest a common pathway for EJC assembly by the two spliceosomes and highlight the evolutionary age of the EJC and its downstream functions in gene expression.spliceosome ͉ mRNA surveillance ͉ RNA processing T he removal of introns from pre-mRNAs is carried out by a large RNA-protein complex called the spliceosome. Recently, it has been realized that during the course of splicing in metazoan cells, the spliceosome deposits on the nascent mRNA a set of protein factors that shape subsequent events in gene expression (1, 2). The so-called exon junction complex (EJC) resides 20-24 nt upstream of the exon-exon junction that results from completion of the pre-mRNA splicing reaction (1). The EJC has been reported to contain at least seven proteins: REF͞Aly, SRm160, Y14, RNPS1, Magoh, hUpf3, and UAP56 (3). Recently, Pinin (4) and eIF4A3 (5-8) have been added to the list. Among the EJC components, REF͞Aly, Y14, and͞or Magoh have been observed to bind the mRNA export receptor TAP (9, 10), whereas RNPS1 and Y14 interact with hUpf3, a factor required for nonsense-mediated mRNA decay (NMD) (11, 12). These observations suggest a central role for the EJC in the nuclear export and quality control of processed mRNAs (13). In addition, the EJC has been implicated in enhancing translation and in the localization of specific transcripts within the cytoplasm (14, 15). Recently, RNA interference knockdown of many of the above EJC components in Drosophila revealed a defect in mRNA export only for UAP56 (16,17), arguing for the existence of mRNA export adaptors in addition to constituents of the EJC (18).Not only the functions of the EJC but also its pathway of assembly remain to be described in molecular detail. Specifically, it is unclear how the spliceosome orchestrates the deposition of EJC components at a specific position upstream of the exonexon junction. Site-specific crosslinking and proteomic analyses (19) have suggested that some EJC components bind at the C complex stage, which encompasses the first step of the splicing reaction. Recently, eIF4A3, a member of the DExH͞D-box family of RNA helicases, has been implicated as the component most likely to be responsible for the tight but sequenceindependent binding of the EJC to 5Ј exon sequences (5-8). Immunoprecipitation (IP) experiments conducted in a simple in vitro splicing system indicated stepwise binding of successive EJC components to the mRNA (20). However, it is not yet known which splicing factors are involved in...

Flexibility in the site of exon junction complex deposition revealed by functional group and RNA secondary structure alterations in the splicing substrate

“…The amount of variability between experiments and the levels of background were similar to those previously reported (Hirose et al 2004). …”

“…Previously, deposition had been studied for only a handful of splicing substrates and was reported to occur z20 nt upstream of the exon-exon junction (Le Hir et al 2000;Kataoka et al 2001;Hirose et al 2004;Shibuya et al 2004). Here, we have studied chimeric RNA-DNA splicing substrates and RNA substrates containing secondary structure.…”

“…Nuclear extracts were prepared from cells that had been transiently transfected with a plasmid expressing a FLAGtagged EJC protein (Lykke- Andersen et al 2001), namely, eIF4AIII or Magoh. Upon completion of splicing, immunoprecipitation with anti-FLAG antibody confirmed that EJC core proteins specifically associate with the splicing intermediates and spliced mRNA (Lykke- Andersen et al 2001;Hirose et al 2004) relative to the unspliced RNA (Fig. 3, cf.…”

“…Briefly, reactions containing 60% nuclear extract, 0.5 mM ATP, 20 mM creatine phosphate, 2.4 mM MgCl 2 , and z10 fmol of pre-mRNA were incubated at 30°C for 90 min. HEK splicing extracts containing FLAG-tagged proteins were made as previously described (Hirose et al 2004). Cells were harvested 36-48 h after transfection.…”

“…As described (Le Hir et al 2000;Hirose et al 2004), after 90 min of splicing at 30°C, DNA oligonucleotides complementary to each splicing substrate were added to splicing reactions to a final concentration of 20 mM and incubated at 30°C for 15 min. Reactions were terminated by adding 1 mg proteinase K in the presence of 0.5% SDS, 5 mM EDTA, 0.05 mM CaCl 2 and incubating for 15 min at 30°C followed by phenol-chloroform extraction.…”