A general consequence of pre-mRNA splicing is the stable deposition of several proteins 20-24 nucleotides (nt) upstream of exon-exon junctions on spliced mRNAs. This exon junction complex (EJC) contains factors involved in mRNA export, cytoplasmic localization, and nonsense-mediated mRNA decay. Here we probed the mechanism and timing of EJC assembly. Over the course of splicing, the 5 exon is subject to numerous dynamic protein-RNA interactions involving at least nine distinct polypeptides. Within the fully assembled spliceosome, these interactions afford protection to the last 25-27 nt of the 5 exon intermediate. Throughout their lifetimes in vivo, mRNAs exist as mRNA-protein particles (mRNPs). The associated proteins control every aspect of mRNA metabolism, from subcellular transport to translational efficiency to rate of decay. Exactly which proteins associate with a particular mRNA depends on its sequence, its subcellular localization, and its synthetic history. Furthermore, the complement of mRNP proteins evolves as the mRNA moves to different locations and is acted on by such processes as nuclear export and translation. Many proteins join the mRNP in the nucleus and then accompany it to the cytoplasm, where they influence subsequent mRNA metabolism ( These splicing-specific mRNP proteins constitute the exon junction complex (EJC), which associates with spliced mRNAs in a sequence-independent manner a fixed distance upstream of exon-exon junctions (Le Hir et al. 2000b). Using coimmunoprecipitation strategies, we and others recently reported that the EJC assembled in HeLa nuclear extract contains numerous epitopes, including those recognized by antibodies against REF/Aly, Y14, SRm160, DEK, RNPS1, UAP56, and Magoh (Blencowe et al. 1998;Mayeda et al. 1999;Kataoka et al. 2000Kataoka et al. , 2001Le Hir et al. 2000a,b, 2001bMcGarvey et al. 2000;Zhou et al. 2000;Gatfield et al. 2001;Luo et al. 2001). When assembled in vivo, the EJC is additionally precipitated by antibodies against Upf2, Upf3, and the TAP:p15 heterodimer (Kim et al. 2001b;Le Hir et al. 2001a;Lykke-Andersen et al. 2001). Finally, REF/Aly, Y14, SRm160, RNPS1, Upf2, Upf3, and TAP were all present in mRNPs immunopurified from the nuclear fraction of COS cells with antibodies against a subunit of the nuclear cap binding complex (Lejeune et al. 2002).The known EJC components function at multiple levels of mRNA metabolism. SRm160 and RNPS1 were originally characterized as activators of pre-mRNA splicing (Blencowe et al. 1998;Mayeda et al. 1999

Alfalfa mosaic virus (AMV) RNA replication requires the viral coat protein (CP). AMV CP is an integral component of the viral replicase; moreover, it binds to the viral RNA 3'-termini and induces the formation of multiple new base pairs that organize the RNA conformation. The results described here suggest that AMV coat protein binding defines template selection by organizing the 3'-terminal RNA conformation and by positioning the RNA-dependent RNA polymerase (RdRp) at the initiation site for minus strand synthesis. RNA-protein interactions were analyzed by using a modified Northwestern blotting protocol that included both viral coat protein and labeled RNA in the probe solution ("far-Northwestern blotting"). We observed that labeled RNA alone bound the replicase proteins poorly; however, complex formation was enhanced significantly in the presence of AMV CP. The RNA-replicase bridging function of the AMV CP may represent a mechanism for accurate de novo initiation in the absence of canonical 3' transfer RNA signals.

We demonstrate here that replacing potassium chloride (KCl) with potassium acetate (KAc) or potassium glutamate (KGlu) routinely enhances the yield of RNA intermediates and products obtained from in vitro splicing reactions performed in HeLa cell nuclear extract. This effect was reproducibly observed with multiple splicing substrates. The enhanced yields are at least partially due to stabilization of splicing precursors and products in the KAc and KGlu reactions. This stabilization relative to KCl reactions was greatest with KGlu and was observed over an extended potassium concentration range. The RNA stability differences could not be attributed to heavy metal contamination of the KCl, since ultrapure preparations of this salt yielded similar results. After testing various methods for altering the salts, we found that substitution of KAc or KGlu for KCl and MgAc 2 for MgCl 2 in splicing reactions is the simplest and most effective. Since the conditions defined here more closely mimic in vivo ionic concentrations, they may permit the study of more weakly spliced substrates, as well as facilitate more detailed analyses of spliceosome structure and function.