Studies on the mechanism of RNAi-dependent heterochromatin assembly.

Assembly of heterochromatin at centromeric DNA regions in the fission yeast Schizosaccharomyces pombe involves an intimate interplay between chromatin modifying complexes and components of the RNAi pathway. The RNA-induced transcriptional silencing (RITS) complex, containing Chp1, Ago1, Tas3, and centromeric siRNAs, localizes to centromeric DNA repeats and is required for the assembly and maintenance of heterochromatin. RITS brings together two types of molecular recognition modules: a chromodomain protein, which binds to lysine 9 methylated histone H3 (H3K9), and Argonaute, which binds to specific sequences by siRNA-directed base-pairing interactions. The RNA-directed RNA polymerase complex (RDRC), composed of Rdp1, the Hrr1 helicase, and the Cid12 Poly(A) polymerase family member, synthesizes double-stranded RNA and creates the substrate for Dicer to generate siRNAs. RDRC physically associates with RITS, and both complexes localize to noncoding centromeric RNAs and centromeric DNA repeats, suggesting that recognition of nascent RNA transcripts may be involved in localization of these complexes to specific chromosome regions. In support of this possibility, tethering of the RITS complex to the transcript of the normally euchromatic ura4 (+) gene results in siRNA generation and RNAi- and heterochromatin-dependent silencing of the ura4 (+) gene. Finally, silencing of a subset of endogenous and transgene promoters within heterochromatic DNA domains occurs by RNAi-dependent degradation of nascent transcripts by a mechanism that we have termed co-transcriptional gene silencing (CTGS).

Common themes in mechanisms of gene silencing.

The assembly of DNA into regions of inaccessible chromatin, called silent chromatin, is involved in the regulation of gene expression and maintenance of chromosome stability in eukaryotes. Recent studies on Sir2-containing silencing complexes in budding yeast and HP1- and Swi6-containing silencing complexes in metazoans and fission yeast suggest a common mechanism for the assembly of these domains, which involves the physical coupling of histone modifying enzymes to histone binding proteins.

Net1 stimulates RNA polymerase I transcription and regulates nucleolar structure independently of controlling mitotic exit.

The budding yeast RENT complex, consisting of at least three proteins (Net1, Cdc14, Sir2), is anchored to the nucleolus by Net1. RENT controls mitotic exit, nucleolar silencing, and nucleolar localization of Nop1. Here, we report two new functions of Net1. First, Net1 directly binds Pol I and stimulates rRNA synthesis both in vitro and in vivo. Second, Net1 modulates nucleolar structure by regulating rDNA morphology and proper localization of multiple nucleolar antigens, including Pol I. Importantly, we show that the nucleolar and previously described cell cycle functions of the RENT complex can be uncoupled by a dominant mutant allele of CDC14. The independent functions of Net1 link a key event in the cell cycle to nucleolar processes that are fundamental to cell growth.

Identification of a class of small molecule inhibitors of the sirtuin family of NAD-dependent deacetylases by phenotypic screening.

The yeast transcriptional repressor Sir2p silences gene expression from the telomeric, rDNA, and silent mating-type loci and may play a role in higher order processes such as aging. Sir2p is the founding member of a large family of NAD-dependent deacetylase enzymes, named the sirtuins. These proteins are conserved from prokaryotes to eukaryotes, but most remain uncharacterized, including all seven human sirtuins. A reverse chemical genetic approach would be useful in identifying the biological function of sirtuins in a wide variety of experimental systems, but no cell-permeable small molecule inhibitors of sirtuins have been reported previously. Herein we describe a high throughput, phenotypic screen in cells that led to the discovery of a class of sirtuin inhibitors. All three compounds inhibited yeast Sir2p transcriptional silencing activity in vivo, and yeast Sir2p and human SIRT2 deacetylase activity in vitro. Such specific results demonstrate the utility and robustness of this screening methodology. Structure-activity relationship analysis of the compounds identified a key hydroxy-napthaldehyde moiety that is necessary and sufficient for inhibitory activity. Preliminary studies using one of these compounds suggest that inhibition of sirtuins interferes with body axis formation in Arabidopsis.

Enzymatic activities of Sir2 and chromatin silencing.

Heritable domains of generalized repression are a common feature of eukaryotic chromosomes and involve the assembly of DNA into a silenced chromatin structure. Sir2, a conserved protein required for silencing in yeast, has recently been shown to couple histone deacetylation to cleavage of a high-energy bond in nicotinamide adenine dinucleotide (NAD) and the synthesis of a novel product, O-acetyl-ADP-ribose. The deacetylase activity provides a direct link between Sir2 and the hypoacetylated state of silent chromatin. However, the unusual coupling of deacetylation to cleavage and synthesis of other bonds raises the possibility that deacetylation is not the only crucial function of Sir2.

Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product.

The Saccharomyces cerevisiae silencing protein Sir2 is the founding member of a universally conserved family of proteins that have been shown to possess NAD-dependent histone deacetylation and ADP-ribosylation activities. Here we show that histone deacetylation by Sir2 is coupled to cleavage of the high-energy bond that links the ADP-ribose moiety of NAD to nicotinamide. Analysis of the NAD cleavage products revealed the presence of nicotinamide, ADP-ribose, and a third product that appeared to be related to ADP-ribose. With the use of label transfer experiments, we show that the acetyl group in the histone substrate is transferred to this NAD breakdown product during deacetylation, forming a product that we conclude to be O-acetyl-ADP-ribose. Detection of this species strongly argues for obligate coupling of histone deacetylation to NAD breakdown by Sir2. We propose reaction mechanisms that could account for this coupling via acetyl-ADP-ribose formation. The unprecedented coupling of amide bond cleavage to cleavage of a high-energy bond raises the possibility that NAD breakdown by Sir2 plays an important role in silencing that is independent of its requirement for deacetylation.

Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex.

Exit from mitosis in budding yeast requires a group of essential proteins--including the GTPase Tem1 and the protein phosphatase Cdc14--that downregulate cyclin-dependent kinase activity. We identified a mutation, net1-1, that bypasses the lethality of tem1 delta. NET1 encodes a novel protein, and mass spectrometric analysis reveals that it is a key component of a multifunctional complex, denoted RENT (for regulator of nucleolar silencing and telophase), that also contains Cdc14 and the silencing regulator Sir2. From G1 through anaphase, RENT localizes to the nucleolus, and Cdc14 activity is inhibited by Net1. In late anaphase, Cdc14 dissociates from RENT, disperses throughout the cell in a Tem1-dependent manner, and ultimately triggers mitotic exit. Nucleolar sequestration may be a general mechanism for the regulation of diverse biological processes.

Net1, a Sir2-associated nucleolar protein required for rDNA silencing and nucleolar integrity.

The Sir2 protein mediates gene silencing and repression of recombination at the rDNA repeats in budding yeast. Here we show that Sir2 executes these functions as a component of a nucleolar complex designated RENT (regulator of nucleolar silencing and telophase exit). Net1, a core subunit of this complex, preferentially cross-links to the rDNA repeats, but not to silent DNA regions near telomeres or to active genes, and tethers the RENT complex to rDNA. Net1 is furthermore required for rDNA silencing and nucleolar integrity. During interphase, Net1 and Sir2 colocalize to a subdomain within the nucleous, but at the end of mitosis a fraction of Sir2 leaves the nucleolus and disperses as foci throughout the nucleus, suggesting that the structure of rDNA silent chromatin changes during the cell cycle. Our findings suggest that a protein complex shown to regulate exit from mitosis is also involved in gene silencing.

Nucleotides in 16S rRNA protected by the association of 30S and 50S ribosomal subunits.

We have studied the interaction of 16S rRNA in 30S subunits with 50S subunits using a series of chemical probes that monitor the accessibility of the RNA bases and backbone. The probes include 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT; to probe U at N-3 and G at N-1), diethylpyrocarbonate (DEPC; to probe A at N-7), dimethyl sulfate (DMS; to probe A at N-1, and C at N-3), kethoxal (to probe G at N-1 and N-2), hydroxyl radicals generated by free Fe(II)-EDTA (to probe the backbone ribose groups) and Pb(II). The sites of reaction were identified by primer extension of the probed RNA. Association of the subunits protects the bases of 11 nucleotides and the ribose groups of over 90 nucleotides of 16S rRNA. The nucleotides protected from the base-specific probes are often adjacent to one another and surrounded by sugar-phosphate backbone protections; thus, the results obtained with the different probes confirmed each other. Most of the protected nucleotides occur in five extended-stem-loop structures around positions 250, 700, 790, 900, and 1408-1495. These regions are located in the platform and bottom of the subunit in the general vicinity of inter-subunit bridges that are visible in reconstructed electron micrographs. Our results provide an extensive map of the nucleotides in 16S rRNA that are likely to be involved in subunit-subunit interactions.

Nucleotides in 23S rRNA protected by the association of 30S and 50S ribosomal subunits.

We have studied the effect of subunit association on the accessibility of nucleotides in 23S and 5S rRNA. Escherichia coli 50S subunits and 70S ribosomes were subjected to a combination of chemical probes and the sites of attack identified by primer extension. Since the ribose groups and all of the bases were probed, the present study provides a comprehensive map of the nucleotides that are likely to be involved in subunit-subunit interactions. Upon subunit association, the bases of 22 nucleotides and the ribose groups of more than 60 nucleotides are protected in 23S rRNA; no changes are seen in 5S rRNA. Interestingly, the bases of nucleotides A1866, A1891 and A1896, and G2505 become more reactive to chemical probes, indicating localized rearrangement of the structure of the 50S subunit upon association with the 30S subunit. Most of the protected nucleotides are located in four stem-loop structures around positions 715, 890, 1700, and 1920. In free 50S subunits, virtually all of the ribose groups in these four regions are strongly cleaved by hydroxyl radicals, suggesting that these stems protrude from the 50S subunit. When the 30S subunit is bound, most of the ribose groups in the 715, 890, 1700 and 1920 stem-loops are protected, as are many bases in and around the corresponding apical loops. Intriguingly, three of the protected regions of 23S rRNA are known to be linked via tertiary interactions to features of the peptidyl transferase center. Together with the juxtaposition of the subunit-protected regions of 16S rRNA with the small subunit tRNA binding sites, our findings suggest the existence of a communication pathway between the codon-anticodon binding sites of the 30S subunit with the peptidyl transferase center of the 50S subunit via rRNA-rRNA interactions.

An enzymatic activity in the yeast Sir2 protein that is essential for gene silencing.

Despite its conservation in organisms from bacteria to human and its general requirement for transcriptional silencing in yeast, the function of the Sir2 protein is unknown. Here we show that Sir2 can transfer labeled phosphate from nicotinamide adenine dinucleotide to itself and histones in vitro. A modified form of Sir2, which results from its automodification activity, is specifically recognized by anti-mono-ADP-ribose antibodies, suggesting that Sir2 is an ADP-ribosyltransferase. Mutation of a phylogenetically invariant histidine residue in Sir2 abolishes both its enzymatic activity in vitro and its silencing functions in vivo. However, the mutant protein is associated with chromatin and other silencing factors in a manner similar to wild-type Sir2. These findings suggest that Sir2 contains an ADP-ribosyltransferase activity that is essential for its silencing function.

Silent information regulator protein complexes in Saccharomyces cerevisiae: a SIR2/SIR4 complex and evidence for a regulatory domain in SIR4 that inhibits its interaction with SIR3.

The SIR2, SIR3, and SIR4 silent information regulator proteins are involved in the assembly of silent chromatin domains in the budding yeast Saccharomyces cerevisiae. Using a series of biochemical experiments, we have studied protein-protein interactions involving these proteins. We found that yeast extracts contained a SIR2/SIR4 complex that was associated with little or no SIR3. However, truncations of the N-terminal two-thirds of the SIR4 protein allowed it to efficiently associate with SIR3, suggesting that the N-terminal domain of SIR4 inhibited its interaction with SIR3. We propose that the SIR3 and SIR4 proteins interact only during the assembly of the SIR protein complex at the silencer and that an early step in assembly unmasks the SIR4 protein to allow its association with SIR3. To test whether the interactions observed in yeast extracts were direct, we tested these SIR-SIR interactions using bacterially expressed SIR proteins. We observed direct interactions between SIR4 and SIR2, SIR4 and SIR3, SIR2 and SIR3, SIR2 and SIR2, and SIR4 and SIR4, indicating that the associations observed in yeast extracts were direct.

A deubiquitinating enzyme interacts with SIR4 and regulates silencing in S. cerevisiae.

The SIR2, SIR3, and SIR4 proteins are required for silencing of transcription at the silent mating type loci and at telomeres in yeast. Using protein affinity chromatography, we show that SIR2, SIR3, and two proteins of 69 and 110 kDa tightly associate with SIR4. Surprisingly, the 110 kDa SIR4-binding protein is identical to UBP3, one of several previously described yeast enzymes that deubiquitinate target proteins. Deletion of the UBP3 gene results in markedly improved silencing of genes inserted either near a telomere or at one of the silent mating type loci, indicating that UBP3 is an inhibitor of silencing. We discuss possible roles for UBP3 in controlling the activity or assembly of the SIR protein complex.

Specific protection of 16 S rRNA by translational initiation factors.

The binding of initiation factors to 30 S ribosomal subunits protects specific sets of nucleotides in 16 S rRNA from base-specific chemical probes. Initiation factor 3 (IF-3) protects residues G700, G703 and G791 from attack by kethoxal. These protected bases are close to those in 16 S rRNA that are protected by 50 S subunits, providing a structural basis for the subunit dissociation activity of IF-3. The IF-3-dependent protections also flank bases that are protected by P-site-bound tRNA, in keeping with the possibility that IF-3 may interact with initiator tRNA, or influence the properties of the 30 S P site during initiation. IF-1 protects G530, A1492 and A1493 and causes enhanced reactivity of A1408. These bases are precisely the ones that are protected by the binding of tRNA to the ribosomal A site. This suggests that IF-1 mimics A-site-bound tRNA, and could serve to prevent premature binding of aminoacyl tRNA by blocking the 30 S A site. We were unable to detect any effect of IF-2 on the reactivity pattern of 16 S rRNA, suggesting that this factor may interact primarily through protein-protein interactions.

Maintenance of the engrailed expression pattern by Polycomb group genes in Drosophila.

The stable maintenance of expression patterns of homeotic genes depends on the function of a number of negative trans-regulators, termed the Polycomb (Pc) group of genes. We have examined the pattern of expression of the Drosophila segment polarity gene, engrailed (en), in embryos mutant for several different members of the Pc group. Here we report that embryos mutant for two or more Pc group genes show strong ectopic en expression, while only weak derepression of en occurs in embryos mutant for a single Pc group gene. This derepression is independent of two known activators of en expression: en itself and wingless. Additionally, in contrast to the strong ectopic expression of homeotic genes observed in extra sex combs- (esc-) mutant embryos, the en expression pattern is nearly normal in esc- embryos. This suggests that the esc gene product functions in a pathway independent of the other genes in the group. The data indicate that the same group of genes is required for stable restriction of en expression to a striped pattern and for the restriction of expression of homeotic genes along the anterior-posterior axis, and support a global role for the Pc group genes in stable repression of activity of developmental selector genes.

Interaction of antibiotics with A- and P-site-specific bases in 16S ribosomal RNA.

We have studied the interactions of the antibiotics apramycin, kasugamycin, myomycin, neamine and pactamycin with 16S rRNA by chemical probing of drug-ribosome complexes. Kasugamycin and pactamycin, which are believed to affect translational initiation, protect bases in common with P-site-bound tRNA. While kasugamycin protects A794 and G926, and causes enhanced reactivity of C795, pactamycin protects G693 and C795. All four of these bases were previously shown to be protected by P-site tRNA or by edeine, another P-site inhibitor. Apramycin and neamine, which both induce miscoding and inhibit translocation, protect A1408, G1419 and G1494, as was also found earlier for neomycin, gentamicin, kanamycin and paromomycin. A1408 and G1494 were previously shown to be protected by A-site tRNA. Surprisingly, myomycin fails to give strong protection of any bases in 16S rRNA, in spite of having an apparently identical target site and mode of action to streptomycin, which protects several bases in the 915 region. Instead, myomycin gives only weak protection of A1408. These results suggest that the binding site(s) of streptomycin and myomycin have yet to be identified.

Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA.

Oligonucleotide fragments derived from the 3' CCA terminus of acylated tRNA, such as CACCA-(AcPhe), UACCA-(AcLeu), and CAACCA-(fMet), bind specifically to ribosomes in the presence of sparsomycin and methanol [Monro, R. E., Celma, M. L. & Vazquez, D. (1969) Nature (London) 222, 356-358]. All three oligonucleotides protect a characteristic set of bases in 23S rRNA from chemical probes: G2252, G2253, A2439, A2451, U2506, and U2585. A2602 shows enhanced reactivity. These account for most of the same bases that are protected when peptidyl-tRNA analogues such as AcPhe-tRNA are bound to the ribosomal P site, and correspond precisely to those bases whose protection is abolished by removal of the 3'-CA end of tRNA. We conclude that most of the observed interactions between tRNA and 23S rRNA in the 50S ribosomal P site involve the conserved CCA terminus of tRNA. Sparsomycin may inhibit protein synthesis by stabilizing interaction between the peptidyl-CCA and the 23S P site, preventing formation of the intermediate A/P hybrid state.

Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA.

Transfer RNA protects a characteristic set of bases in 16 S rRNA from chemical probes when it binds to ribosomes. We used several criteria, based on construction of well-characterized in vitro ribosome-tRNA complexes, to assign these proteins to A or P-site binding. All of these approaches lead to similar conclusions. In the A site, tRNA caused protection of G529, G530, A1492 and A1493 (strongly), and A1408 and G1494 (weakly). In the P site, the protected bases are G693, A794, C795, G926 and G1401 (strong), and A532, G966, G1338 and G1339 (weak). In contrast to what is observed for 23 S rRNA, blocking the release of EF-Tu.GDP from the ribosome by kirromycin has no detectable effect on the protection of bases in 16 S rRNA.

Intermediate states in the movement of transfer RNA in the ribosome.

Direct chemical 'footprinting' shows that translocation of transfer RNA occurs in two discrete steps. During the first step, which occurs spontaneously after the formation of the peptide bond, the acceptor end of tRNA moves relative to the large ribosomal subunit resulting in 'hybrid states' of binding. During the second step, which is promoted by elongation factor EF-G, the anticodon end of tRNA, along with the messenger RNA, moves relative to the small ribosomal subunit.

Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites.

Three sets of conserved nucleotides in 23 rRNA are protected from chemical probes by binding of tRNA to the ribosomal A, P, and E sites, respectively. They are located almost exclusively in domain V, primarily in or adjacent to the loop identified with the peptidyl transferase function. Some of these sites are also protected by antibiotics such as chloramphenicol, which could explain how these drugs interfere with protein synthesis. Certain tRNA-dependent protections are abolished when the 3'-terminal A or CA or 2',3'-linked acyl group is removed, providing direct evidence for the interaction of the conserved CCA terminus of tRNA with 23S rRNA. When the EF-Tu.GTP.aminoacyl-tRNA ternary complex is bound to the ribosome, no tRNA-dependent A site protections are detected in 23S rRNA until EF-Tu is released. Thus, EF-Tu prevents interaction of the 3' terminus of the incoming aminoacyl-tRNA with the peptidyl transferase region of the ribosome during anticodon selection, thereby permitting translational proofreading.