Various derivatives of the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) RNA have been used to analyze by UV-cross-linking its interaction with mRNA binding proteins from ascites carcinoma Krebs-2 cells. A doublet of proteins with Mr 58 and 60 kD bound to two regions of the IRES. One site is centered at nt 420-421 of EMCV RNA whereas the other is located between nt 315-377. Both sites form hairpin structures, the loops of which contain UCUUU motif, conserved among cardio- and aphthoviruses. The interaction of p58 and p60 with IRES is affected by the integrity of the stem-loop structure proximal to the start AUG codon (nts 680-787), although, under similar conditions, cross-linking of these proteins to this region was not detected. Deletions in the main recognition site of p58 strongly reduce the initiation activity of the IRES in vitro. However, elimination of p58 (p60) binding by these mutations does not completely abolish the ability of the IRES to direct polypeptide synthesis starting from the authentic AUG codon. The IRES can be assembled in vitro from two covalently unlinked transcripts, one containing the target site for p58 and the other encompassing the remaining part of the IRES fused to a reporter gene, resulting in considerable restoration of its activity. Implications of these findings for the mechanism of initiation resulting from internal entry of ribosomes are discussed.

The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) [1]. This six-subunit complex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily conserved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence [4] (ACS) constitutes part of the ORC binding site [2, 5]. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation.

The Nucleolar Localization Elements (NoLEs) of Xenopus laevis U3 small nucleolar RNA (snoRNA) have been defined. Fluorescein-labeled wild-type U3 snoRNA injected into Xenopus oocyte nuclei localized specifically to nucleoli as shown by fluorescence microscopy. Injection of mutated U3 snoRNA revealed that the 5Ј region containing Boxes A and AЈ, known to be important for rRNA processing, is not essential for nucleolar localization. Nucleolar localization of U3 snoRNA was independent of the presence and nature of the 5Ј cap and the terminal stem. In contrast, Boxes C and D, common to the Box C/D snoRNA family, are critical elements for U3 localization. Mutation of the hinge region, Box B, or Box CЈ led to reduced U3 nucleolar localization. Results of competition experiments suggested that Boxes C and D act in a cooperative manner. It is proposed that Box B facilitates U3 snoRNA nucleolar localization by the primary NoLEs (Boxes C and D), with the hinge region of U3 subsequently base pairing to the external transcribed spacer of pre-rRNA, thus positioning U3 snoRNA for its roles in rRNA processing. INTRODUCTIONMany aspects of how macromolecules are targeted to their correct subcellular destination still need to be defined. Although principles governing RNA export to the cytoplasm and import into the nucleus are beginning to be understood, very little is known about signals that direct RNA within the nucleus to the nucleolus. The nucleolus contains a vast array of different RNA species involved in ribosome biogenesis: ribosomal RNA (rRNA) and its precursors and ϳ200 small nucleolar RNAs (snoRNAs). Unlike rRNA whose genes are located within the nucleolus, the other transcripts found in the nucleolus must travel there from their sites of synthesis in the nucleoplasm. What are the "zip codes" for targeting snoRNA to the nucleolus?To address this question, we have studied U3 snoRNA because it is the most abundant snoRNA in the nucleolus, has been sequenced from a large number of animals and plants (Gu and Reddy, 1997), and is well characterized in terms of secondary structure and function in rRNA processing. U3 snoRNA is synthesized in the nucleoplasm in the proximity of coiled bodies (Gao et al., 1997), and formation of its trimethylguanosine cap can occur in the nucleoplasm (Terns and Dahlberg, 1994;Terns et al., 1995), unlike spliceosomal snoRNAs that are exported to the cytoplasm for cap trimethylation (Mattaj, 1986). Once U3 snoRNA is transported to the nucleolus, it is found highly concentrated in the dense fibrillar component (Matera et al., 1994) where initial rRNA processing events are believed to occur, but up to half of the U3 snoRNA is also detected by electron microscopy to be diffuse in the granular component Puvion-Dutilleul et al., 1991Azum-Gélade et al., 1994) where later rRNA processing cleavages take place. On the basis of the localization of U3 snoRNA after various actinomycin D treatments (Puvion-Dutilleul et al., 1992;Rivera-Leó n and Gerbi, 1997), a model has been proposed in which U3 snoRNA trave...