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The exon junction complex (EJC), a set of proteins deposited on mRNAs as a consequence of pre-mRNA splicing, is a key effector of downstream mRNA metabolism. We have identified eIF4AIII, a member of the eukaryotic translation initiation factor 4A family of RNA helicases (also known as DExH/D box proteins), as a novel EJC core component. Crosslinking and antibody inhibition studies suggest that eIF4AIII constitutes at least part of the platform anchoring other EJC components to spliced mRNAs. A nucleocytoplasmic shuttling protein, eIF4AIII associates in vitro and in vivo with two other EJC core factors, Y14 and Magoh. In mammalian cells, eIF4AIII is essential for nonsense-mediated mRNA decay (NMD). Finally, a model is proposed by which eIF4AIII represents a new functional class of DExH/D box proteins that act as RNA clamps or 'place holders' for the sequence-independent attachment of additional factors to RNAs. [ABSTRACT FROM AUTHOR]

The spliceosome is a multimegadalton RNA-protein machine that removes noncoding sequences from nascent pre-mRNAs. Recruitment of the spliceosome to splice sites and subsequent splicing require a series of dynamic interactions among the spliceosome's component U snRNPs and many additional protein factors. These dynamics present several challenges for structural analyses, including purification of stable complexes to compositional homogeneity and assessment of conformational heterogeneity. We have isolated spliceosomes arrested before the second chemical step of splicing (C complex) in which U2, U5 and U6 snRNAs are stably associated. Using electron microscopy, we obtained images of C complex spliceosomes under cryogenic conditions and determined a three-dimensional structure of a core complex to a resolution of 30 Å. The structure reveals a particle of dimensions 27 × 22 × 24 nm with a relatively open arrangement of three primary domains. [ABSTRACT FROM AUTHOR]

What''s in a spliceosome? More than we ever imagined, according to recent reports employing proteomics techniques to analyze this multi-megadalton machine. As of 1999, around 100 splicing factors were identified (Burge et al., 1999); however, that number has now nearly doubled due primarily to improved purification of spliceosomes coupled with advances in mass spectrometry analyses of complex mixtures. Gratifyingly, most of the previously identified splicing factors were found in the recent mass spec studies. Nonetheless, the number of new proteins emerging with no prior connection to splicing was surprising. Without functional validation, it would be premature to label these proteins as bona fide splicing factors. Yet many were identified multiple times in complexes purified under diverse conditions or from different organisms. Another recurring theme regards the dynamic nature of spliceosomal complexes, which may be even more intricate than previously thought. [Copyright &y& Elsevier]

We recently reported that spliceosomes alter messenger ribonucleoprotein particle (mRNP) composition by depositing several proteins 20-24 nucleotides upstream of mRNA exon-exon junctions. When assembled in vitro, this so-called exon-exon junction complex' (EJC) contains at least five proteins: SRm160, DEK, RNPS1, Y14 and REF. To better investigate its functional attributes, we now describe a method for generating spliced mRNAs both in vitro and in vivo that either do or do not carry the EJC. Analysis of these mRNAs in Xenopus luevis oocytes revealed that this complex is the species responsible for enhancing nucleocytoplasmic export of spliced mRNAs. It does so by providing a strong binding site for the mRNA export factors REF and TAP/p15. Moreover, by serving as an anchoring point for the factors Upf2 and Up13, the EJC provides a direct link between splicing and nonsense-mediated mRNA decay. Finally, we show that the composition of the EJC is dynamic in vivo and is subject to significant evolution upon mRNA export to the cytoplasm. [ABSTRACT FROM AUTHOR]

Summary: Unlike DNA, RNA abundances can vary over several orders of magnitude. Thus, identification of RNA–protein binding sites from high-throughput sequencing data presents unique challenges. Although peak identification in ChIP-Seq data has been extensively explored, there are few bioinformatics tools tailored for peak calling on analogous datasets for RNA-binding proteins. Here we describe ASPeak (abundance sensitive peak detection algorithm), an implementation of an algorithm that we previously applied to detect peaks in exon junction complex RNA immunoprecipitation in tandem experiments. Our peak detection algorithm yields stringent and robust target sets enabling sensitive motif finding and downstream functional analyses.Availability: ASPeak is implemented in Perl as a complete pipeline that takes bedGraph files as input. ASPeak implementation is freely available at https://sourceforge.net/projects/as-peak under the GNU General Public License. ASPeak can be run on a personal computer, yet is designed to be easily parallelizable. ASPeak can also run on high performance computing clusters providing efficient speedup. The documentation and user manual can be obtained from http://master.dl.sourceforge.net/project/as-peak/manual.pdf.Contact: alper.kucukural@umassmed.edu or ccenik@stanford.edu [ABSTRACT FROM AUTHOR]

Over the past decade many studies have revealed a complex web of interconnections between the numerous steps required for eukaryotic gene expression. One set of interconnections link nuclear pre-mRNA splicing and the subsequent metabolism of the spliced mRNAs. It is now apparent that the means of connection is a set of proteins, collectively called the exon junction complex, which are deposited as a consequence of splicing upstream of mRNA exon–exon junctions. [Copyright &y& Elsevier]

Summary: Eukaryotes possess numerous quality control systems that monitor both the synthesis of RNA and the integrity of the finished products. We previously demonstrated that Saccharomyces cerevisiae possesses a quality control mechanism, nonfunctional rRNA decay (NRD), capable of detecting and eliminating translationally defective rRNAs. Here we show that NRD can be divided into two mechanistically distinct pathways: one that eliminates rRNAs with deleterious mutations in the decoding site (18S NRD) and one that eliminates rRNAs containing deleterious mutations in the peptidyl transferase center (25S NRD). 18S NRD is dependent on translation elongation and utilizes the same proteins as those participating in no-go mRNA decay (NGD). In cells that accumulate 18S NRD and NGD decay intermediates, both RNA types can be seen in P-bodies. We propose that 18S NRD and NGD are different observable outcomes of the same initiating event: a ribosome stalled inappropriately at a sense codon during translation elongation. [Copyright &y& Elsevier]

Abstract: The exon junction complex (EJC) is a macromolecular complex deposited at splice junctions on mRNAs as a consequence of splicing. At the core of the EJC are four proteins: eIF4AIII, a member of the DExH/D-box family of NTP-dependent RNA binding proteins, Y14, Magoh, and MLN51. These proteins form a stable heterotetramer that remains bound to the mRNA throughout many different cellular environments. We have determined the three-dimensional (3D) structure of this EJC core using negative-stain random-conical tilt electron microscopy. This structure represents the first structure of a DExH/D-box protein in complex with its binding partners. The EJC core is a four-lobed complex with a central channel and dimensions consistent with its known RNA footprint of about ten nucleotides. Using known X-ray crystallographic structures and a model of three of the four components, we propose a model for complex assembly on RNA and explain how Y14:Magoh may influence eIF4AIII''s RNA binding. [Copyright &y& Elsevier]