The multisubunit eukaryotic exosome is an essential RNA processing and degradation machine. In its nuclear form, the exosome associates with the auxiliary factor Rrp6p, which participates in both RNA processing and degradation reactions. The crystal structure of Saccharomyces cerevisiae Rrp6p displays a conserved RNase D core with a flanking HRDC (helicase and RNase D C-terminal) domain in an unusual conformation shown to be important for the processing function of the enzyme. Complexes with AMP and UMP, the products of the RNA degradation process, reveal how the protein specifically recognizes ribonucleotides and their bases. Finally, in vivo mutational studies show the importance of the domain contacts for the processing function of Rrp6p and highlight fundamental differences between the protein and its prokaryotic RNase D counterparts.RNA degradation ͉ RNA processing ͉ x-ray crystallography ͉ RNase D T he RNA exosome participates in a wide range of reactions, including processing and degradation of tRNA and rRNA as well as degradation of both nuclear and cytoplasmic RNA polymerase II-derived transcripts (1-6). The core eukaryotic exosome is present in both the cytoplasm and nucleus and consists of 10 proteins, with at least 7 harboring proven or predicted 3Ј-to-5Ј exonuclease activity (one RNase II and six RNase PH type) (7-10). In the yeast nucleus, the complex is distinguished by three additional proteins, the RNase D-type enzyme Rrp6p (for ribosomal RNA processing), the DEAD-box RNA helicase Mtr4p, and the less well characterized protein Rrp47p (7, 11). Exosomes are found in both eukaryotes and archaea, and, recently, several crystal structures of archaeal subcomplexes were reported (12)(13)(14). The center of the archaeal exosome consists of three Rrp41 and three Rrp42 proteins forming an overall donut-shaped heterohexameric structure. Rrp41 and -42 are each similar to three proteins in the eukaryotic complex, which consists of six different proteins forming the ''donut' ' (12, 14). This ring-like structure is able to bind additional proteins, forming a ''cap'' suggested to constrict and probably regulate the entry of RNA into the central cavity containing the phosphorolytic active sites (12).The nuclear exosome is essential for maturation of eukaryotic ribosomal RNAs (25S, 18S, and 5.8S), which are synthesized as a single transcript (for a review, see ref. 15). Processing is initiated by endonucleolytic cleavage of the external transcribed spacers (ETSs), which are subsequently degraded by Rrp6p (16). This protein is also required for trimming of the two internal transcribed spacers 1 and 2 during maturation to produce the mature rRNAs, and a 30-nt 3Ј-end extended form of 5.8S rRNA appears in ⌬rrp6 cells (17). Similarly, many small nucleolar RNAs (snoRNAs) depend on the exosome during their maturation (18,19), and deletion of Rrp6p in yeast also leads to accumulation of extended forms of both polycistronic snoRNAs (18) and the independently transcribed snoRNAs, such as snR33 and snR40 (20).Rrp6p is homol...

Production of aberrant messenger ribonucleoprotein particles (mRNPs) is subject to quality control (QC). In yeast strains carrying mutations of the THO complex, transcription induction triggers a number of interconnected QC phenotypes: (1) rapid degradation of several mRNAs; (2) retention of a fraction of THO-dependent mRNAs in transcription site-associated foci; and (3) formation of a high molecular weight DNA/protein complex in the 39-ends of THO target genes. Here, we demonstrate that the 39-59 exonucleolytic domain of the nuclear exosome factor Rrp6p is necessary for establishing all QC phenotypes associated with THO mutations. The N terminus of Rrp6p is also important presumably through its binding to the Rrp6p co-factor Rrp47p. Interestingly, the 39-59 exonucleolytic activity of Dis3p, the only other active exonuclease of the nuclear exosome, can also contribute to RNA QC in THO mutants, while other nuclear 39-59 exonucleases cannot. Our data show that exonucleolytic attack by the nuclear exosome is needed both for provoking mRNP QC and for its ensuing elimination of faulty RNA.

Genetic screens in Saccharomyces cerevisiae provide novel information about interacting genes and pathways. We screened for high-copy-number suppressors of a strain with the gene encoding the nuclear exosome component Rrp6p deleted, with either a traditional plate screen for suppressors of rrp6⌬ temperature sensitivity or a novel microarray enhancer/suppressor screening (MES) strategy. MES combines DNA microarray technology with high-copy-number plasmid expression in liquid media. The plate screen and MES identified overlapping, but also different, suppressor genes. Only MES identified the novel mRNP protein Nab6p and the tRNA transporter Los1p, which could not have been identified in a traditional plate screen; both genes are toxic when overexpressed in rrp6⌬ strains at 37°C. Nab6p binds poly(A) ؉ RNA, and the functions of Nab6p and Los1p suggest that mRNA metabolism and/or protein synthesis are growth rate limiting in rrp6⌬ strains. Microarray analyses of gene expression in rrp6⌬ strains and a number of suppressor strains support this hypothesis.Work, primarily in yeast (Saccharomyces cerevisiae), has generated considerable information about the functions of the exosome, a multisubunit complex of proven or predicted 3Ј-5Ј exonucleases. In addition to its role in cytoplasmic-mRNA turnover, the exosome is also involved in a myriad of nuclear events: rRNA, snoRNA, and snRNA processing, as well as the degradation of a variety of stable and unstable nuclear RNAs (1,2,4,12,16,27,28,34,36,44,45). The nuclear exosome also functions in the surveillance and degradation of aberrant mRNAs/mRNPs and pre-mRNAs (9, 14, 15, 24-26, 31, 41). In its nuclear form, the exosome harbors three specific components, one of which (Rrp6p) has been studied in some detail. An rrp6⌬ strain grows slowly and is temperature sensitive and deficient in many of the nuclear-RNA-processing and degradation events mentioned above. Importantly, it is not known which of these are most important for the growth defects of the deletion strain.To address this issue, we identified high-copy-number suppressors of the rrp6⌬ strain. For more than 2 decades, this strategy has been employed to identify interacting genes and pathways of many different mutant phenotypes in S. cerevisiae. Although the approach provides invaluable information, it has stringent requirements. The selection for survival requires a dramatic change in phenotype, i.e., a switch in a life/death discrimination test. The identified genes, therefore, have potent effects, but this suggests that there are probably additional, less potent suppressor genes worth identifying. We therefore also exploited a second approach, which can simultaneously identify enhancer, as well as suppressor, genes, including those that exert much more modest effects on the mutant phenotype. The method, microarray enhancer/suppressor screening (MES), uses DNA microarray technology to rapidly identify genes from plasmid libraries that are either enriched or selected against in a particular test strain during growth in l...

Production of messenger ribonucleoprotein particles (mRNPs) is subjected to quality control (QC). In Saccharomyces cerevisiae, the RNA exosome and its cofactors are part of the nuclear QC machinery that removes, or stalls, aberrant molecules, thereby ensuring that only correctly formed mRNPs are exported to the cytoplasm. The Ccr4-Not complex, which constitutes the major S. cerevisiae cytoplasmic deadenylase, has recently been implied in nuclear exosome-related processes. Consistent with a possible nuclear function of the complex, the deletion or mutation of Ccr4-Not factors also elicits transcription phenotypes. Here we use genetic depletion of the Mft1p protein of the THO transcription/mRNP packaging complex as a model system to link the Ccr4-Not complex to nuclear mRNP QC. We reveal strong genetic interactions between alleles of the Ccr4-Not complex with both the exosomal RRP6 and MFT1 genes. Moreover, Rrp6p-dependent in vivo QC phenotypes of Dmft1 cells can be rescued by codeletion of several Ccr4-Not components. We discuss how the Ccr4-Not complex may connect with the mRNP QC pathway.

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