A Specific Set of Exon Junction Complex Subunits Is Required for the Nuclear Retention of Unspliced RNAs in Caenorhabditis elegans

Abstract

The exon junction complex (EJC) is highly conserved in many organisms and is involved in various steps of mRNA metabolism. During the course of investigating the role of EJC in the germ line sex determination of the nematode Caenorhabditis elegans, we found that depletion of one of the three core subunits (Y14, MAG-1, and eukaryotic translation initiation factor 4III [eIF4AIII]) or one auxiliary subunit (UAP56) of EJC resulted in the cytoplasmic leakage of unspliced RNAs from almost all of the C. elegans protein-coding genes examined thus far. This leakage was also observed with the depletion of several splicing factors, including SF3b, IBP160, and PRP19, all of which genetically interacted with Y14. We also found that Y14 physically interacts with both pre-mRNA and spliceosomal U snRNAs, especially U2 snRNA, and that the interaction was abolished when both IBP160 and PRP19 were depleted. Our results strongly suggest that a specific set of EJC subunits is recruited onto introns and interacts with components of the spliceosome, including U2 snRNP, to provide a critical signal for the surveillance and nuclear retention of unspliced RNAs in C. elegans.T he complete removal of introns from pre-mRNA before mRNA export to the cytoplasm is critical to ensure the proper expression of the protein product in higher eukaryotes. Premature export of intron-containing RNAs gives rise to abnormal protein products that are very harmful to various cell functions, although most of such abnormal RNAs are eliminated by mRNA quality control systems, including nonsense-mediated mRNA decay (NMD) (1-4). Thus, cells must have mechanisms to retain partially spliced pre-mRNA in the nucleus. Indeed, most intron-containing RNAs, except in the case of alternative splicing, are normally localized in the nucleus. One mechanism is spliceosome formation around introns, which is confined to the nucleus because most splicing factors are localized in the nucleus. In this mechanism, the spliceosome itself or one or more spliceosomeassociated factors anchor intron-containing RNAs in the nucleus (5-7). Another mechanism for the nuclear retention of introncontaining RNAs is the splicing-dependent recruitment of mRNA export-related factors. The transcription and export (TREX) complex and exon junction complex (EJC) form on the 5= region of the nascent transcript (8, 9) and 20 to 24 nucleotides upstream from splice junctions (10, 11), respectively. The TREX complex contains the proteins UAP56 and Aly/REF (12, 13), both of which are also subunits of EJC (14-16), suggesting the tight coupling of splicing with mRNA export and the involvement of TREX and EJC for preventing the premature export of unspliced RNAs. Both spliceosome-dependent and export-related-factor-coupled mechanisms for the nuclear retention of intron-containing RNAs may operate concurrently, but the molecular details remain to be elucidated.EJC consists of four core subunits (Y14, Mago-nashi, eukaryotic translation initiation factor 4III [eIF4AIII], and Barentz/ MLN51) and several auxiliary...

The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript

“…Two nuclear functions have previously been proposed for the pre-EJC: one in splicing (Ashton-Beaucage et al 2010;Roignant and Treisman 2010;Michelle et al 2012) and one in the control of mRNA export (Le Hir et al 2001;Gatfield and Izaurralde 2002;Shiimori et al 2013). In order to investigate which post-transcriptional function of the EJC was involved in the piRNA pathway, we knocked down a representative set of EJC accessory factors and analyzed both Piwi and transposon levels.…”

The spliceosome as a transposon sensor

“…Intriguingly, the C. elegans UAP56 ortholog acts in cooperation with exon junction complex components, some of which are deposited on transcripts prior to the completion of splicing, 60 in order to divert unspliced transcripts away from the canonical nuclear export pathway. 61 This finding demonstrates the potential for splicing efficiency to dictate a transcript's fate in a UAP56-dependent manner, and may inform studies of the piRNA processing mechanism, which itself requires exon junction complex components. 62 Whether piRNA loci that do not require UAP56 for their processing are also marked by the suppression of splicing signals has not yet been examined.…”

The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

“…Interactions between eIF4AIII and the spliceosome result in eIF4AIII deposition on spliced mRNAs proximal to newly ligated exon junctions, although eIF4AIII is not an essential component of the spliceosome (Ferraiuolo et al, 2004; Shibuya et al, 2004; Shiimori et al, 2013). While eIF4AIII itself has no RNA binding specificity, two associated proteins, Y14 and magoh, coordinate eIF4AIII deposition adjacent to splice junctions (Bono and Gehring, 2011).…”