DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function.

DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA.

Rpd3- and spt16-mediated nucleosome assembly and transcriptional regulation on yeast ribosomal DNA genes.

Ribosomal DNA (rDNA) genes in eukaryotes are organized into multicopy tandem arrays and transcribed by RNA polymerase I. During cell proliferation, ∼50% of these genes are active and have a relatively open chromatin structure characterized by elevated accessibility to psoralen cross-linking. In Saccharomyces cerevisiae, transcription of rDNA genes becomes repressed and chromatin structure closes when cells enter the diauxic shift and growth dramatically slows. In this study, we found that nucleosomes are massively depleted from the active rDNA genes during log phase and reassembled during the diauxic shift, largely accounting for the differences in psoralen accessibility between active and inactive genes. The Rpd3L histone deacetylase complex was required for diauxic shift-induced H4 and H2B deposition onto rDNA genes, suggesting involvement in assembly or stabilization of the entire nucleosome. The Spt16 subunit of FACT, however, was specifically required for H2B deposition, suggesting specificity for the H2A/H2B dimer. Miller chromatin spreads were used for electron microscopic visualization of rDNA genes in an spt16 mutant, which was found to be deficient in the assembly of normal nucleosomes on inactive genes and the disruption of nucleosomes on active genes, consistent with an inability to fully reactivate polymerase I (Pol I) transcription when cells exit stationary phase.

Identification of novel proteins associated with yeast snR30 small nucleolar RNA.

H/ACA small nucleolar RNPs (snoRNPs) that guide pseudouridylation reactions are comprised of one small nucleolar RNA (snoRNA) and four common proteins (Cbf5, Gar1, Nhp2 and Nop10). Unlike other H/ACA snoRNPs, snR30 is essential for the early processing reactions that lead to the production of 18S ribosomal RNA in the yeast Saccharomyces cerevisiae. To determine whether snR30 RNP contains specific proteins that contribute to its unique functional properties, we devised an affinity purification strategy using TAP-tagged Gar1 and an RNA aptamer inserted in snR30 snoRNA to selectively purify the RNP. Northern blotting and pCp labeling experiments showed that S1-tagged snR30 snoRNA can be selectively purified with streptavidin beads. Protein analysis revealed that aptamer-tagged snR30 RNA was associated with the four H/ACA proteins and a number of additional proteins: Nop6, ribosomal proteins S9 and S18 and histones H2B and H4. Using antibodies raised against Nop6 we show that endogenous Nop6 localizes to the nucleolus and that it cosediments with snR30 snoRNA in sucrose density gradients. We demonstrate through primer extension experiments that snR30 snoRNA is required for cleavages at site A0, A1 and A2, and that the absence of Nop6 decreases the efficiency of cleavage at site A2. Finally, electron microscopy analyses of chromatin spreads from cells depleted of snR30 snoRNA show that it is required for SSU processome assembly.

Distinguishing the roles of Topoisomerases I and II in relief of transcription-induced torsional stress in yeast rRNA genes.

To better understand the role of topoisomerase activity in relieving transcription-induced supercoiling, yeast genes encoding rRNA were visualized in cells deficient for either or both of the two major topoisomerases. In the absence of both topoisomerase I (Top1) and topoisomerase II (Top2) activity, processivity was severely impaired and polymerases were unable to transcribe through the 6.7-kb gene. Loss of Top1 resulted in increased negative superhelical density (two to six times the normal value) in a significant subset of rRNA genes, as manifested by regions of DNA template melting. The observed DNA bubbles were not R-loops and did not block polymerase movement, since genes with DNA template melting showed no evidence of slowed elongation. Inactivation of Top2, however, resulted in characteristic signs of slowed elongation in rRNA genes, suggesting that Top2 alleviates transcription-induced positive supercoiling. Together, the data indicate that torsion in front of and behind transcribing polymerase I has different consequences and different resolution. Positive torsion in front of the polymerase induces supercoiling (writhe) and is largely resolved by Top2. Negative torsion behind the polymerase induces DNA strand separation and is largely resolved by Top1.

Mrd1p is required for release of base-paired U3 snoRNA within the preribosomal complex.

In eukaryotes, ribosomes are made from precursor rRNA (pre-rRNA) and ribosomal proteins in a maturation process that requires a large number of snoRNPs and processing factors. A fundamental problem is how the coordinated and productive folding of the pre-rRNA and assembly of successive pre-rRNA-protein complexes is achieved cotranscriptionally. The conserved protein Mrd1p, which contains five RNA binding domains (RBDs), is essential for processing events leading to small ribosomal subunit synthesis. We show that full function of Mrd1p requires all five RBDs and that the RBDs are functionally distinct and needed during different steps in processing. Mrd1p mutations trap U3 snoRNA in pre-rRNP complexes both in base-paired and non-base-paired interactions. A single essential RBD, RBD5, is involved in both types of interactions, but its conserved RNP1 motif is not needed for releasing the base-paired interactions. RBD5 is also required for the late pre-rRNP compaction preceding A(2) cleavage. Our results suggest that Mrd1p modulates successive conformational rearrangements within the pre-rRNP that influence snoRNA-pre-rRNA contacts and couple U3 snoRNA-pre-rRNA remodeling and late steps in pre-rRNP compaction that are essential for cleavage at A(0) to A(2). Mrd1p therefore coordinates key events in biosynthesis of small ribosome subunits.

Electron microscope visualization of RNA transcription and processing in Saccharomyces cerevisiae by Miller chromatin spreading.

The Miller chromatin spreading technique for electron microscopic visualization of gently dispersed interphase chromatin has proven extremely valuable for analysis of genetic activities in vivo. It provides a unique view of transcription and RNA processing at the level of individual active genes. The budding yeast Saccharomyces cerevisiae has also been an invaluable model system for geneticists and molecular biologists. In this chapter, we describe methods for applying the Miller chromatin-spreading method to Saccharomyces cerevisiae. This allows one to use electron microscopic visualization of a gene of interest to study effects of specific mutations on gene activity. We are applying the method to study transcription and processing of ribosomal RNA.

TOR regulates the subcellular distribution of DIM2, a KH domain protein required for cotranscriptional ribosome assembly and pre-40S ribosome export.

Eukaryotic ribosome synthesis is a highly dynamic process that involves the transient association of scores of trans-acting factors to nascent pre-ribosomes. Many ribosome synthesis factors are nucleocytoplasmic shuttling proteins that engage the assembly pathway at early nucleolar stages and escort pre-ribosomes to the nucleoplasm and/or the cytoplasm. Here, we report that two 40S ribosome synthesis factors, the KH-domain protein DIM2 and the HEAT-repeats/Armadillo-domain and export factor RRP12, are nucleolar restricted upon nutritional, osmotic, and oxidative stress. Nucleolar entrapment of DIM2 and RRP12 was triggered by rapamycin treatment and was under the strict control of the target of rapamycin (TOR) signaling cascade. DIM2 binds pre-rRNAs directly through its KH domain at the 5'-end of ITS1 (D-A(2) segment) and, consistent with its requirements in early nucleolar pre-rRNA processing, is required for efficient cotranscriptional ribosome assembly. The substitution of a single and highly conserved amino acid (G207A) within the KH motif is sufficient to inhibit pre-rRNA processing in a fashion similar to genetic depletion of DIM2. DIM2 carries an evolutionarily conserved putative nuclear export sequence (NES) at its carboxyl-terminal end that is required for efficient pre-40S ribosome export. Strikingly, DIM2 and RRP12 are both involved in the nucleocytoplasmic translocation of pre-ribosomes, suggesting that this step in the ribosome assembly pathway has been selected as a regulatory target for the TOR pathway.

Mrd1p binds to pre-rRNA early during transcription independent of U3 snoRNA and is required for compaction of the pre-rRNA into small subunit processomes.

In Saccharomyces cerevisiae, synthesis of the small ribosomal subunit requires assembly of the 35S pre-rRNA into a 90S preribosomal complex. SnoRNAs, including U3 snoRNA, and many trans-acting proteins are required for the ordered assembly and function of the 90S preribosomal complex. Here, we show that the conserved protein Mrd1p binds to the pre-rRNA early during transcription and is required for compaction of the pre-18S rRNA into SSU processome particles. We have exploited the fact that an Mrd1p-GFP fusion protein is incorporated into the 90S preribosomal complex, where it acts as a partial loss-of-function mutation. When associated with the pre-rRNA, Mrd1p-GFP functionally interacts with the essential Pwp2, Mpp10 and U3 snoRNP subcomplexes that are functionally interconnected in the 90S preribosomal complex. The fusion protein can partially support 90S preribosome-mediated cleavages at the A(0)-A(2) sites. At the same time, on a substantial fraction of transcripts, the composition and/or structure of the 90S preribosomal complex is perturbed by the fusion protein in such a way that cleavage of the 35S pre-rRNA is either blocked or shifted to aberrant sites. These results show that Mrd1p is required for establishing productive structures within the 90S preribosomal complex.

Visual analysis of the yeast 5S rRNA gene transcriptome: regulation and role of La protein.

5S rRNA genes from Saccharomyces cerevisiae were examined by Miller chromatin spreading, representing the first quantitative analysis of RNA polymerase III genes in situ by electron microscopy. These very short genes, approximately 132 nucleotides (nt), were engaged by one to three RNA polymerases. Analysis in different growth conditions and in strains with a fourfold range in gene copy number revealed regulation at two levels: number of active genes and polymerase loading per gene. Repressive growth conditions (presence of rapamycin or postexponential growth) led first to fewer active genes, followed by lower polymerase loading per active gene. The polymerase III elongation rate was estimated to be in the range of 60 to 75 nt/s, with a reinitiation interval of approximately 1.2 s. The yeast La protein, Lhp1, was associated with 5S genes. Its absence had no discernible effect on the amount or size of 5S RNA produced yet resulted in more polymerases per gene on average, consistent with a non-rate-limiting role for Lhp1 in a process such as polymerase release/recycling upon transcription termination.

Transcription elongation by RNA polymerase I is linked to efficient rRNA processing and ribosome assembly.

The synthesis of ribosomes in eukaryotic cells is a complex process involving many nonribosomal protein factors and snoRNAs. In general, the processes of rRNA transcription and ribosome assembly are treated as temporally or spatially distinct. Here, we describe the identification of a point mutation in the second largest subunit of RNA polymerase I near the active center of the enzyme that results in an elongation-defective enzyme in the yeast Saccharomyces cerevisiae. In vivo, this mutant shows significant defects in rRNA processing and ribosome assembly. Taken together, these data suggest that transcription of rRNA by RNA polymerase I is linked to rRNA processing and maturation. Thus, RNA polymerase I, elongation factors, and rRNA sequence elements appear to function together to optimize transcription elongation, coordinating cotranscriptional interactions of many factors/snoRNAs with pre-rRNA for correct rRNA processing and ribosome assembly.

The PINc domain protein Utp24, a putative nuclease, is required for the early cleavage steps in 18S rRNA maturation.

Ribosome biogenesis is a complex process that requires >150 transacting factors, many of which form macromolecular assemblies as big and complex as the ribosome itself. One of those complexes, the SSU processome, is required for pre-18S rRNA maturation. Although many of its components have been identified, the endonucleases that cleave the pre-18S rRNA have remained mysterious. Here we examine the role of four previously uncharacterized PINc domain proteins, which are predicted to function as nucleases, in yeast ribosome biogenesis. We also included Utp23, a protein homologous to the PINc domain protein Utp24, in our analysis. Our results demonstrate that Utp23 and Utp24 are essential nucleolar proteins and previously undescribed components of the SSU processome. In that sense, both Utp23 and Utp24 are required for the first three cleavage steps in 18S rRNA maturation. In addition, single-point mutations in the conserved putative active site of Utp24 but not Utp23 abrogate its function in ribosome biogenesis. Our results suggest that Utp24 might be the elusive endonuclease that cleaves the pre-rRNA at sites A(1) and/or A(2.).

Esf2p, a U3-associated factor required for small-subunit processome assembly and compaction.

Esf2p is the Saccharomyces cerevisiae homolog of mouse ABT1, a protein previously identified as a putative partner of the TATA-element binding protein. However, large-scale studies have indicated that Esf2p is primarily localized to the nucleolus and that it physically associates with pre-rRNA processing factors. Here, we show that Esf2p-depleted cells are defective for pre-rRNA processing at the early nucleolar cleavage sites A0 through A2 and consequently are inhibited for 18S rRNA synthesis. Esf2p was stably associated with the 5' external transcribed spacer (ETS) and the box C+D snoRNA U3, as well as additional box C+D snoRNAs and proteins enriched within the small-subunit (SSU) processome/90S preribosomes. Esf2p colocalized on glycerol gradients with 90S preribosomes and slower migrating particles containing 5' ETS fragments. Strikingly, upon Esf2p depletion, chromatin spreads revealed that SSU processome assembly and compaction are inhibited and glycerol gradient analysis showed that U3 remains associated within 90S preribosomes. This suggests that in the absence of proper SSU processome assembly, early pre-rRNA processing is inhibited and U3 is not properly released from the 35S pre-rRNAs. The identification of ABT1 in a large-scale analysis of the human nucleolar proteome indicates that its role may also be conserved in mammals.

Pre-18S ribosomal RNA is structurally compacted into the SSU processome prior to being cleaved from nascent transcripts in Saccharomyces cerevisiae.

Recent studies have revealed multiple dynamic complexes that are precursors to eukaryotic ribosomes. EM visualization of nascent rRNA transcripts provides in vivo temporal and structural context for these events. In exponentially growing S. cerevisiae, pre-18S rRNA is dramatically compacted into a large particle (SSU processome) within seconds of completion of its transcription and is released cotranscriptionally by cleavage in ITS1. After cleavage, a new terminal knob is formed on the nascent large subunit rRNA, compacting it progressively in a 5'-3' direction. Depletion of individual components shows that cotranscriptional SSU processome formation is a sensitive indicator of the occurrence or timing of the early A0-A2 cleavages and depends on factors not isolated in preribosome complexes, as well as on favorable growth conditions. The results show that the approximately 40 components of the SSU processome/90S preribosome can complete their tasks within approximately 85 s in optimal conditions.

Adenovirus protein VII condenses DNA, represses transcription, and associates with transcriptional activator E1A.

Adenovirus protein VII is the major protein component of the viral nucleoprotein core. It is highly basic, and an estimated 1070 copies associate with each viral genome, forming a tightly condensed DNA-protein complex. We have investigated DNA condensation, transcriptional repression, and specific protein binding by protein VII. Xenopus oocytes were microinjected with mRNA encoding HA-tagged protein VII and prepared for visualization of lampbrush chromosomes. Immunostaining revealed that protein VII associated in a uniform manner across entire chromosomes. Furthermore, the chromosomes were significantly condensed and transcriptionally silenced, as judged by the dramatic disappearance of transcription loops characteristic of lampbrush chromosomes. During infection, the protein VII-DNA complex may be the initial substrate for transcriptional activation by cellular factors and the viral E1A protein. To investigate this possibility, mRNAs encoding E1A and protein VII were comicroinjected into Xenopus oocytes. Interestingly, whereas E1A did not associate with chromosomes in the absence of protein VII, expression of both proteins together resulted in significant association of E1A with lampbrush chromosomes. Binding studies with proteins produced in bacteria or human cells or by in vitro translation showed that E1A and protein VII can interact in vitro. Structure-function analysis revealed that an N-terminal region of E1A is responsible for binding to protein VII. These studies define the in vivo functions of protein VII in DNA binding, condensation, and transcriptional repression and indicate a role in E1A-mediated transcriptional activation of viral genes.

Transcriptional termination by RNA polymerase I requires the small subunit Rpa12p.

We identify Rpa12p of RNA polymerase I (Pol I) as a termination factor. Combined analyses using transcription run-on, electron microscopy-visualized chromatin spreading and RT-PCR have been applied to the rRNA-encoding genes of Saccharomyces cerevisiae. These confirm that Pol I termination occurs close to the Reb1p-dependent terminator in wild-type strains. However, deletion mutants for the 3' end-processing enzyme Rnt1p or the Rpa12p subunit of Pol I both show Pol I transcription in the spacer. For Deltarpa12, these spacer polymerases are devoid of nascent transcripts, suggesting they are immediately degraded. The homology of Rpa12p to the small subunit Rpb9p of Pol II and Rpc11p of Pol III, both implicated in transcriptional termination, points to a common termination mechanism for all three classes of RNA polymerase.

In exponentially growing Saccharomyces cerevisiae cells, rRNA synthesis is determined by the summed RNA polymerase I loading rate rather than by the number of active genes.

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 ( approximately 143 in this case) and a strain in which the rRNA gene number was reduced to approximately 42 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 approximately 50 +/- 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 approximately 60 nucleotides/s for yeast Pol I and a reinitiation rate of less than 1 s on the most heavily transcribed genes.

EM visualization of Pol II genes in Drosophila: most genes terminate without prior 3' end cleavage of nascent transcripts.

Transcription termination by RNA polymerase II (Pol II) on most mRNA-encoding genes is dependent on transcription through a functional poly(A) signal. One model to explain this dependence predicts co-trancriptional cleavage of RNA at the poly(A) site. Electron microscopic (EM) visualization was used to investigate the in vivo frequency of transcript cleavage prior to termination. Over 100 unidentified Drosophila Pol II-transcribed genes were analyzed. Although some genes exhibited cleaved transcripts near their 3' ends, and some had a lower polymerase density at their 3' end relative to the rest of the gene, the majority of genes (64%) had uncleaved transcripts and no change in polymerase density at the 3' end, consistent with release of full-length transcripts at a discrete site. Thus, in Drosophila, cleavage at the poly(A) site sometimes occurs co-transcriptionally, but does not appear to be a prerequisite to termination. Next, two components of the polyadenylation complex were immunolocalized on polytene chromosomes and were found to differ in distribution both qualitatively and quantitatively. The EM results indicate that co-transcriptional recognition of the poly(A) signal, which is required for termination, does not equate with co-transcriptional cleavage, and the immunofluorescence results suggest that this may be due to incomplete or nonstoichiometric assembly of the polyadenylation machinery on nascent transcripts.