Although the U3 small nucleolar RNA (snoRNA), a member of the box C/D class of snoRNAs, was identified with the spliceosomal small nuclear RNAs (snRNAs) over 30 years ago, its function and its associated protein components have remained more elusive. The U3 snoRNA is ubiquitous in eukaryotes and is required for nucleolar processing of pre-18S ribosomal RNA in all organisms where it has been tested. Biochemical and genetic analyses suggest that U3 pre-rRNA base-pairing interactions mediate endonucleolytic pre-rRNA cleavages. Here we have purified a large ribonucleoprotein (RNP) complex from Saccharomyces cerevisiae that contains the U3 snoRNA and 28 proteins. Seventeen new proteins (Utp1 17) and Rrp5 were present, as were ten known components. The Utp proteins are nucleolar and specifically associated with the U3 snoRNA. Depletion of the Utp proteins impedes production of the 18S rRNA, indicating that they are part of the active pre-rRNA processing complex. On the basis of its large size (80S; calculated relative molecular mass of at least 2,200,000) and function, this complex may correspond to the terminal knobs present at the 5' ends of nascent pre-rRNAs. We have termed this large RNP the small subunit (SSU) processome.

Pre-rRNA transcription by RNA Polymerase I (Pol I) is very robust on active rDNA repeats. Loss of yeast Topoisomerase I (Top1) generated truncated pre-rRNA fragments, which were stabilized in strains lacking TRAMP (Trf4/Trf5-Air1/Air2-Mtr4 polyadenylation complexes) or exosome degradation activities. Loss of both Top1 and Top2 blocked pre-rRNA synthesis, with pre-rRNAs truncated predominately in the 18S 59 region. Positive supercoils in front of Pol I are predicted to slow elongation, while rDNA opening in its wake might cause R-loop formation. Chromatin immunoprecipitation analysis showed substantial levels of RNA/DNA hybrids in the wild type, particularly over the 18S 59 region. The absence of RNase H1 and H2 in cells depleted of Top1 increased the accumulation of RNA/DNA hybrids and reduced pre-rRNA truncation and pre-rRNA synthesis. Hybrid accumulation over the rDNA was greatly exacerbated when Top1, Top2, and RNase H were all absent. Electron microscopy (EM) analysis revealed Pol I pileups in the wild type, particularly over the 18S. Pileups were longer and more frequent in the absence of Top1, and their frequency was exacerbated when RNase H activity was also lacking. We conclude that the loss of Top1 enhances inherent R-loop formation, particularly over the 59 region of the rDNA, imposing persistent transcription blocks when RNase H is limiting.[Keywords: rRNA synthesis; RNA polymerase I; transcription elongation; topoisomerase; RNase H; R-loops] Supplemental material is available at http://www.genesdev.org.

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Genes encoding rRNA are multicopy and thus could be regulated by changing the number of active genes or by changing the transcription rate per gene. We tested the hypothesis that the number of open genes is limiting rRNA synthesis by using an electron microscopy method that allows direct counting of the number of active genes per nucleolus and the number of polymerases per active gene. Two strains of Saccharomyces cerevisiae were analyzed during exponential growth: a control strain with a typical number of rRNA genes (ϳ143 in this case) and a strain in which the rRNA gene number was reduced to ϳ42 but which grows as well as controls. In control strains, somewhat more than half of the genes were active and the mean number of polymerases/gene was ϳ50 ؎ 20. In the 42-copy strain, all rRNA genes were active with a mean number of 100 ؎ 29 polymerases/ gene. Thus, an equivalent number of polymerases was active per nucleolus in the two strains, though the number of active genes varied by twofold, showing that overall initiation rate, and not the number of active genes, determines rRNA transcription rate during exponential growth in yeast. Results also allow an estimate of elongation rate of ϳ60 nucleotides/s for yeast Pol I and a reinitiation rate of less than 1 s on the most heavily transcribed genes.

Based on ultrastructural analysis of actively transcribing genes seen in electron micrographs, we present evidence that pre-mRNA splicing occurs with a reasonable frequency on the nascent transcripts of early Drosopbila embryo genes and that splice site selection may generally precede polyadenylation. The details of the process observed are in agreement with results from in vitro splicing systems but differ in the more rapid completion of in vivo splicing. For those introns that are removed cotranscriptionally, a series of events is initiated following 3' splice site synthesis, beginning with ribonucleoprotein (RNP) particle formation at the 3' splice site within 48 sec, intron loop formation within 2 min, and splicing within 3 min. The initiation of the process is correlated with 3' splice site synthesis but is independent of 5' splice site synthesis, the position of the intron within the transcript, and the age or length of the transcript. In some cases, introns are removed from the 5' end of a transcript before introns are synthesized at the 3' end, supporting a possible role for the order of transcription in splice site pairing. In general, our observations are consistent with the 'first-come-first-served' principle of splice site selection, although an observed example of exon skipping indicates that alternative splicing possibilities can be accommodated within this general framework.[Key Words: RNA splicing; ribonucleoprotein particles; alternative splicing; spliceosome; electron microscopy; Drosophila development] Received February 24, 1988; revised version accepted April 28, 1988.With the development of in vitro splicing systems, a great deal has been learned about the molecular bio chemistry of intron excision from eukaryotic nuclear mRNA precursors (for reviews, see Green 1986;Padgett et al. 1986;Maniatis and Reed 1987). During the first step of splicing, cleavage at the 5' splice site is coupled to lariat formation within the intron. The lariat is formed by the covalent linkage of the 5' end of the in tron to an A residue at the branch site, located 18-37 nucleotides upstream of the 3' splice site. In the second step, the two exons are ligated and the intron is released as a lariat. The existence of a bipartite intermediate not only requires that the exons be brought together and ac curately positioned, but also held together for ligation. A multicomponent ribonucleoprotein (RNP) complex termed the spliceosome serves this purpose (Brody and Abelson 1985). Conserved sequences at the intron boundaries and branch site function to assemble the spliceosome, through interactions with required factors, which are predominantly the abundant Sm class of small nuclear ribonucleoproteins (snRNPs) (Ul, U2, U5, U4/U6 snRNPs; for review, see Steitz et al. 1987). Sphceosome assembly is a stepwise process involving depo sition of proteins and snRNPs at the ends of the intron followed by the stable association of the two ends to form the splicing complex (Frendewey and Keller