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Drosophila has two pairs of Pax genes, ey/toy and eyg/toe, that play different functions during eye development. ey specifies eye fate, while eyg promotes cell proliferation. We have determined the molecular basis for the functional diversity of Eyg and Ey. Eyg and Ey act by distinct transcriptional mechanisms. They use different DNA-binding domains for target recognition. Most interestingly, Eyg acts exclusively as a repressor, whereas Ey is an activator. Several vertebrate Pax proteins are known to switch between activator and repressor activities, but none as repressors only. Eyg may be the first Pax protein as a dedicated repressor. Vertebrates produce a Pax6 isoform, Pax6-5a, differing from Pax6 in DNA-binding properties and functions and structurally similar to Eyg/Toe. We found that Pax6-5a acts as an activator like Ey, but has DNA-binding specificity like Eyg. [ABSTRACT FROM AUTHOR]

The DEAH/RNA helicase A (RHA) helicase family comprises proteins involved in splicing, ribosome biogenesis and transcription regulation. We report the structure of yeast Prp43p, a DEAH/RHA helicase remarkable in that it functions in both splicing and ribosome biogenesis. Prp43p displays a novel structural architecture with an unforeseen homology with the Ski2-like Hel308 DNA helicase. Together with the presence of a beta-hairpin in the second RecA-like domain, Prp43p contains all the structural elements of a processive helicase. Moreover, our structure reveals that the C-terminal domain contains an oligonucleotide/oligosaccharide-binding (OB)-fold placed at the entrance of the putative nucleic acid cavity. Deletion or mutations of this domain decrease the affinity of Prp43p for RNA and severely reduce Prp43p ATPase activity in the presence of RNA. We also show that this domain constitutes the binding site for the G-patch-containing domain of Pfa1p. We propose that the C-terminal domain, specific to DEAH/RHA helicases, is a central player in the regulation of helicase activity by binding both RNA and G-patch domain proteins.

Prp43p is a RNA helicase required for pre-mRNA splicing and for the synthesis of large and small ribosomal subunits. The molecular functions and modes of regulation of Prp43p during ribosome biogenesis remain unknown. We demonstrate that the G-patch protein Pfa1p, a component of pre-40S pre-ribosomal particles, directly interacts with Prp43p. We also show that lack of Gno1p, another G-patch protein associated with Prp43p, specifically reduces Pfa1p accumulation, whereas it increases the levels of the pre-40S pre-ribosomal particle component Ltv1p. Moreover, cells lacking Pfa1p and depleted for Ltv1p show strong 20S pre-rRNA accumulation in the cytoplasm and reduced levels of 18S rRNA. Finally, we demonstrate that Pfa1p stimulates the ATPase and helicase activities of Prp43p. Truncated Pfa1p variants unable to fully stimulate the activity of Prp43p fail to complement the 20S pre-rRNA processing defect of Deltapfa1 cells depleted for Ltv1p. Our results strongly suggest that stimulation of ATPase/helicase activities of Prp43p by Pfa1p is required for efficient 20S pre-rRNA-to-18S rRNA conversion.

The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non-coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of 'orphan' H/ACA RNAs participate in pre-rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 'orphan' H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre-rRNA processing, two evolutionarily conserved sequence elements in the 3'-hairpin of snR30 base-pair with short pre-rRNA sequences located in the eukaryote-specific internal region of 18S rRNA. The newly discovered snR30-18S base-pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3'-hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein-binding site.

Drosophila has two pairs of Pax genes, ey/toy and eyg/toe, that play different functions during eye development. ey specifies eye fate, while eyg promotes cell proliferation. We have determined the molecular basis for the functional diversity of Eyg and Ey. Eyg and Ey act by distinct transcriptional mechanisms. They use different DNA-binding domains for target recognition. Most interestingly, Eyg acts exclusively as a repressor, whereas Ey is an activator. Several vertebrate Pax proteins are known to switch between activator and repressor activities, but none as repressors only. Eyg may be the first Pax protein as a dedicated repressor. Vertebrates produce a Pax6 isoform, Pax6-5a, differing from Pax6 in DNA-binding properties and functions and structurally similar to Eyg/Toe. We found that Pax6-5a acts as an activator like Ey, but has DNA-binding specificity like Eyg.

The small nucleolar ribonucleoprotein particles containing H/ACA-type snoRNAs (H/ACA snoRNPs) are crucial trans-acting factors intervening in eukaryotic ribosome biogenesis. Most of these particles generate the site-specific pseudouridylation of rRNAs while a subset are required for 18S rRNA synthesis. To understand in detail how these particles carry out these functions, all of their protein components have to be characterized. For that purpose, we have affinity-purified complexes containing epitope-tagged Gar1p protein, previously shown to be part of H/ACA snoRNPs. Under the conditions used, three polypeptides of 65, 22 and 10 kDa apparent molecular weight specifically copurify with epitope-tagged Gar1p. The 22 and 10 kDa polypeptides were identified as Nhp2p and a novel protein we termed Nop10p, respectively. Both proteins are conserved, essential and present in the dense fibrillar component of the nucleolus. Nhp2p and Nop10p are specifically associated with all H/ACA snoRNAs and are essential to the function of H/ACA snoRNPs. Cells lacking Nhp2p or Nop10p are impaired in global rRNA pseudouridylation and in the A1 and A2 cleavage steps of the pre-rRNA required for the synthesis of mature 18S rRNA. These phenotypes are probably a direct consequence of the instability of H/ACA snoRNAs and Gar1p observed in cells deprived of Nhp2p or Nop10p. Our results suggest that Nhp2p and Nop10p, together with Cbf5p, constitute the core of H/ACA snoRNPs.

Site-specific 2'-O-ribose methylation of eukaryotic rRNAs is guided by small nucleolar RNAs (snoRNAs). The methylation guide snoRNAs carry long perfect complementaries to rRNAs. These antisense elements are located either in the 5' half or in the 3' end region of the snoRNA, and are followed by the conserved D' or D box motifs, respectively. An uninterrupted helix formed between the rRNA and the antisense element of the snoRNA, in conjunction with the adjacent D' or D box, constitute the recognition signal for the putative methyltransferase. Here, we have identified an additional essential box element common to methylation guide snoRNAs, termed the C' box. We show that the C' box functions in concert with the D' box and plays a crucial role in the methyltransfer reaction directed by the upstream antisense element and the D' box. We also show that an internal fragment of U24 methylation guide snoRNA, encompassing the upstream antisense element and the D' and C' box motifs, can support the site-specific methylation of rRNA. This strongly suggests that the C box of methylation guide snoRNAs plays an essential role in the methyltransfer reaction guided by the 3'-terminal antisense element and the D box of the snoRNA.

Eukaryotic rRNAs possess numerous post-transcriptionally modified nucleotides. The most abundant modifications, 2'-O-ribose methylation and pseudouridylation, occur in the nucleolus during rRNA processing. The nucleolus contains a large number of small nucleolar RNAs (snoRNAs) most of which can be classified into two distinct families defined by conserved sequence boxes and common associated proteins. The C and D box-containing snoRNAs are associated with fibrillarin, and most of them function as guide RNAs in site-specific ribose methylation of rRNAs. The nucleolar function of the other class of snoRNAs, which share box H and ACA elements and are associated with a glycine- and arginine-rich nucleolar protein, Gar1p, remains elusive. Here we demonstrate that the yeast Saccharomyces cerevisiae Gar1 snoRNP protein plays an essential and specific role in the overall pseudouridylation of yeast rRNAs. These results establish a novel function for Gar1 protein and indicate that the box H/ACA snoRNAs, or at least a subset of these snoRNAs, function in the site-specific pseudouridylation of rRNAs.

Three of the four eukaryotic ribosomal RNA molecules (18S, 5.8S and 25-28S rRNA) are transcribed as a single precursor, which is subsequently processed into the mature species by a complex series of cleavage and modification reactions. Early cleavage at site A1 generates the mature 5'-end of 18S rRNA. Mutational analyses have identified a number of upstream regions in the 5' external transcribed spacer (5' ETS), including a U3 binding site, which are required in cis for processing at A1. Nothing is known, however, about the requirement for cis-acting elements which define the position of the 5'-end of the 18S rRNA or of any other eukaryotic rRNA. We have introduced mutations around A1 and analyzed them in vivo in a genetic background where the mutant pre-rRNA is the only species synthesized. The results indicate that the mature 5'-end of 18S rRNA in yeast is identified by two partially independent recognition systems, both defining the same cleavage site. One mechanism identifies the site of cleavage at A1 in a sequence-specific manner involving recognition of phylogenetically conserved nucleotides immediately upstream of A1 in the 5' ETS. The second mechanism specifies the 5'-end of 18S rRNA by spacing the A1 cleavage at a fixed distance of 3 nt from the 5' stem-loop/pseudoknot structure located within the mature sequence. The 5' product of the A1 processing reaction can also be identified, showing that, in contrast to yeast 5.8S rRNA, the 5'-end of 18S rRNA is generated by endonucleolytic cleavage.

We have developed techniques for the detailed analysis of cis-acting sequences in the pre-rRNA of Saccharomyces cerevisiae and used these to study the processing of internal transcribed spacer 1 (ITS1) leading to the synthesis of 5.8S rRNA. As is the case for many eukaryotes, the 5' end of yeast 5.8S rRNA is heterogeneous; we designate the major, short form 5.8S(S), and the minor form (which is seven or eight nucleotides longer) 5.8S(L). These RNAs do not have a precursor/product relationship, but result from the use of alternative processing pathways. In the major pathway, a previously unidentified processing site in ITS1, designated A3, is cleaved. A 10 nucleotide deletion at site A3 strongly inhibits processing of A3 and the synthesis of 5.8S(S); processing is predominantly transferred to the alternative 5.8S(L) pathway. Site A3 lies 76 nucleotides 5' to the end of 5.8S(S), and acts as an entry site for 5'-->3' exonuclease digestion which generates the 5' end of 5.8S(S). This pathway is inhibited in strains mutant for XRN1p and RAT1p. Both of these proteins have been reported to have 5'-->3' exonuclease activity in vitro. Formation of 5.8S(L) is increased by mutations at A3 in cis or in RAT1p and XRN1p in trans, and is kinetically faster than 5.8S(S) synthesis.