No abstract

The factors involved in rRNA processing in eukaryotes assemble cotranscriptionally onto the nascent prerRNAs and include endonucleases, exonucleases, RNA helicases, GTPases, modifying enzymes and snoRNPs (small nucleolar ribonucleoproteins). The precursor of three of the four eukaryotic mature rRNAs contains the rRNA sequences flanked by two internal (ITS1 and ITS2) and two external (5¢-ETS and 3¢-ETS) spacer sequences that are removed during processing [1,2]. The pre-rRNA is first assembled into a 90S particle that contains U3 snoRNP and 40S subunit-processing factors [3,4]. The early pre-rRNA endonucleolytic cleavages at sites A 0 , A 1 and A 2 occur within the 90S particles [3,5]. A 2 cleavage releases the first pre60S particle, which differs in composition from the known 90S particle. Pre60S particles contain 27S rRNA, ribosomal L proteins and many nonribosomal proteins [6].As they mature, pre60S particles migrate from the nucleolus to the nucleoplasm and their content of nonribosomal factors changes [7,8]. Nip7p was among the proteins identified in the early pre60S particle [6][7][8], and has been shown to participate in the processing of 27S pre-rRNA to the formation of 25S [9]. Interestingly, Nip7p also binds the exosome subunit Rrp43p [10]. The exosome complex is responsible for the degradation of the excised 5¢-ETS and for the 3¢)5¢ exonucleolytic processing of 7S pre-rRNA to form the mature 5.8S rRNA. The exosome is also involved in the processing of snoRNAs and in mRNA degradation [11][12][13].During processing, pre-rRNA undergoes covalent modifications that include isomerization of some uridines into pseudouridines and addition of methyl groups to specific nucleotides, mainly at the 2¢-O posi- In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form large and intriguingly organized complexes. A novel nucleolar protein, Nop53p, was isolated by using Nop17p as bait in the yeast two-hybrid system. Nop53p also interacts with a second nucleolar protein, Nip7p. A carbon source-conditional strain with the NOP53 coding sequence under the control of the GAL1 promoter did not grow in glucose-containing medium, showing the phenotype of an essential gene. Under nonpermissive conditions, the conditional mutant strain showed rRNA biosynthesis defects, leading to an accumulation of the 27S and 7S pre-rRNAs and depletion of the mature 25S and 5.8S mature rRNAs. Nop53p did not interact with any of the exosome subunits in the yeast twohybrid system, but its depletion affects the exosome function. In pull-down assays, protein A-tagged Nop53p coprecipitated the 27S and 7S pre-rRNAs, and His-Nop53p also bound directly 5.8S rRNA in vitro, which is consistent with a role for Nop53p in pre-rRNA processing.Abbreviations ETS, external transcribed spacer; b-Gal, b-galactosidase; GFP, green fluorescent protein; GST, glutathione S-transferase; ITS, internal transcribed spacer; RFP, red fluorescent protein; snoRNP, small nucleolar ribonucleoprotein.

Initially identified in yeast, the exosome has emerged as a central component of the RNA maturation and degradation machinery both in Archaea and eukaryotes. Here we describe a series of high-resolution structures of the RNase PH ring from the Pyrococcus abyssi exosome, one of them containing three 10-mer RNA strands within the exosome catalytic chamber, and report additional nucleotide interactions involving positions N5 and N7. Residues from all three Rrp41-Rrp42 heterodimers interact with a single RNA molecule, providing evidence for the functional relevance of exosome ring-like assembly in RNA processivity. Furthermore, an ADP-bound structure showed a rearrangement of nucleotide interactions at site N1, suggesting a rationale for the elimination of nucleoside diphosphate after catalysis. In combination with RNA degradation assays performed with mutants of key amino acid residues, the structural data presented here provide support for a model of exosome-mediated RNA degradation that integrates the events involving catalytic cleavage, product elimination, and RNA translocation. Finally, comparisons between the archaeal and human exosome structures provide a possible explanation for the eukaryotic exosome inability to catalyze phosphate-dependent RNA degradation.Initially described as a multisubunit RNase complex required for maturation of 5.8 S rRNA in yeast (1), the 3Ј 3 5Ј exoribonuclease complex exosome was subsequently shown to play a central role on numerous pathways related to RNA processing and degradation, both in the nucleus and in the cytoplasm (reviewed in Ref. 2). These include 3Ј end processing of rRNAs, small nuclear RNAs, and small nucleolar RNAs (3-5), degradation of aberrant pre-mRNAs, pre-tRNAs, and pre-rRNAs (6 -9), normal turnover of cytosolic mRNAs (10), and degradation of RNA fragments produced during RNA interference processes (11, 12).Increasing structural and genetic studies with archaeal and eukaryotic exosomes have unveiled the evolutionary conservation of its molecular architecture (1, 13-18). The archaeal exosome consists of two RNase PH subunits (Rrp41 and Rrp42) and two proteins containing the S1/KH (Rrp4) or S1/zinc ribbon (Csl4) RNA-binding domains (17)(18)(19). Determination of the three-dimensional structure of the Sulfolobus solfataricus and Archaeoglobus fulgidus exosomes revealed a PNPase-like fold (20), composed by alternating RNase PH subunits assembled into a hexameric ring capped by a trimer of RNA-binding proteins, which can be formed by either Rrp4 or Csl4 or possibly by a mixture of both (19,21,22). Such an architecture encloses the exoribonucleolytic active sites at the bottom of the RNase PH ring catalytic chamber and restricts the entry to only unstructured RNA through the S1 pore, formed by the RNAbinding subunits of the exosome cap placed at the top of the ring (19,(21)(22)(23). Both Rrp41 and Rrp42 subunits possess the same RNase PH-fold and are involved in substrate binding, but the amino acid residues contributing to the interactions with the phosphate nucle...

A new modular gene-expression system for use in studies of translational control in Saccharomyces cerevisiae was constructed. A GAL::PGK fusion promoter (GPF) directed the inducible synthesis of mRNAs initiated at a single major site. A series of leader sequences were tested in combination with each of two reporter genes (encoding chloramphenicol acetyl transferase (cat) and luciferase (luc)). Stem-loop structures of three different sizes and predicted stabilities were inserted into each of two different unique restriction sites in the leader. After correction for relative mRNA abundance, a stem-loop of predicted stability equivalent to approximately -18 kcal mol-1 inhibited translation by up to 89%. The degree of inhibition exerted by the other stem-loops correlated positively with their predicted stabilities. Combinations of two stem-loops at different sites yielded an inhibitory effect greater than that of either individual stem-loop alone. Similar inhibitory effects were observed with both reporter genes. However, inhibition of translation, particularly of the cat gene, was more effective when the stem-loop was positioned close to the start codon rather than at the 5' end of the leader. The observed results reflect an important form of post-transcriptional control that is expected to act on a large number of genes in yeast.

No abstract