In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a double-stranded RNA (dsRNA) corresponding to exon sequences in the mRNA (refs 1,2). The use of this "RNA interference" (RNAi) in mammalian studies had lagged well behind its utility in lower animals because uninterrupted RNA duplexes longer than 30 base pairs trigger generalized cellular responses through activation of dsRNA-dependent protein kinases. Recently it was demonstrated that RNAi can be made to work in cultured human cells by introducing shorter, synthetic duplex RNAs (approximately 20 base pairs) through liposome transfection. We have explored several strategies for expressing similar short interfering RNA (siRNA) duplexes within cells from recombinant DNA constructs, because this might allow long-term target-gene suppression in cells, and potentially in whole organisms. Effective suppression of target gene product levels is achieved by using a human U6 small nuclear RNA (snRNA) promoter to drive nuclear expression of a single RNA transcript. The siRNA-like parts of the transcript consists of a 19 base pair siRNA stem with the two strands joined by a tightly structured loop and a U1-4 3' overhang at the end of the antisense strand. The simplicity of the U6 expression cassette and its widespread transcription in human cell types suggest that this mode of siRNA delivery could be useful for suppressing expression of a wide range of genes.

Early transfer RNA (tRNA) processing events in Saccharomyces cerevisiae are coordinated in the nucleolus, the site normally associated with ribosome biosynthesis. To test whether spatial organization of the tRNA pathway begins with nucleolar clustering of the genes, we have probed the subnuclear location of five different tRNA gene families. The results show that tRNA genes, though dispersed in the linear genome, colocalize with 5S ribosomal DNA and U14 small nucleolar RNA at the nucleolus. Nucleolar localization requires tRNA gene transcription-complex formation, because inactivation of the promoter at a single locus removes its nucleolar association. This organization of tRNA genes must profoundly affect the spatial packaging of the genome and raises the question of whether gene types might be coordinated in three dimensions to regulate transcription.

The 274 tRNA genes in Saccharomyces cerevisiae are scattered throughout the linear maps of the 16 chromosomes, but the genes are clustered at the nucleolus when compacted in the nucleus. This clustering is dependent on intact nucleolar organization and contributes to tRNA gene-mediated (tgm) silencing of RNA polymerase II transcription near tRNA genes. After examination of the localization mechanism, we find that the chromosome-condensing complex, condensin, is involved in the clustering of tRNA genes. Conditionally defective mutations in all five subunits of condensin, which we confirm is bound to active tRNA genes in the yeast genome, lead to loss of both pol II transcriptional silencing near tRNA genes and nucleolar clustering of the genes. Furthermore, we show that condensin physically associates with a subcomplex of RNA polymerase III transcription factors on the tRNA genes. Clustering of tRNA genes by condensin appears to be a separate mechanism from their nucleolar localization, as microtubule disruption releases tRNA gene clusters from the nucleolus, but does not disperse the clusters. These observations suggest a widespread role for condensin in gene organization and packaging of the interphase yeast nucleus.[Keywords: tRNA gene; condensin; microtubules; nuclear organization; nucleolus] Supplemental material is available at http://www.genesdev.org.

Effective intracellular expression of small RNA therapeutics inclusion of specific U6 snRNA sequences from positions depends on a number of factors. The RNA, whether anti-+19 to +27. In situ localization of the transcripts shows that sense, ribozyme, or RNA aptamer, must be efficiently tranboth tRNA and U6 promoter transcripts give primarily puncscribed, stabilized against rapid degradation, folded cortate nuclear patterns, and that capping of transcripts is not rectly, and directed to the part of the cell where it can be required for nuclear retention. Several different insert most effective. To overcome a number of these problems RNAs directed against HIV-1 were tested by cotransfection we have been testing expression cassettes based on the with HIV-1 provirus and assay for subsequent viral reverse human tRNA met and U6 snRNA promoters, in which trantranscriptase production. These include antisense RNA, scripts encoding small RNA inserts are protected against hairpin and hammerhead ribozymes, and RNA ligands attack from the 3′ end. Transient expression in cultured (aptamers) for Tat and Rev RNA binding proteins. Results cells results in 10 3 -2 × 10 7 full-length transcripts per cell,show that Rev-binding RNAs efficiently block HIV-1 gene depending partially on the promoter construct used but expression, whereas other RNAs have little or no effect also on the nature of the insert RNA. 5′ ␥-Phosphate when expressed in these cassettes. methylation (capping) depended, as expected, on the

Transcription by RNA polymerase II is antagonized by the presence of a nearby tRNA gene in Saccharomyces cerevisiae. To test hypotheses concerning the mechanism of this tRNA gene-mediated (tgm) silencing, the effects of specific gene deletions were determined. The results show that the mechanism of silencing near tRNA genes is fundamentally different from other forms of transcriptional silencing in yeast. Rather, tgm silencing is dependent on the ability to cluster the dispersed tRNA genes in or near the nucleolus, constituting a form of three-dimensional gene control.Chromatin-mediated transcriptional silencing has been extensively studied in Saccharomyces cerevisiae: at the two silent mating type loci, near telomeres, and in the single cluster of tandemly repeated rRNA genes (1). Mutations affecting these silencing forms affect chromatin structure by altering histone modifications and remodeling. Unlike other eukaryotes, S. cerevisiae appears to lack RNA-mediated forms of silencing (2).Actively transcribed tRNA genes can suppress transcription of nearby genes by RNA polymerase II (pol II) 1 in yeast (3,4). This phenomenon, termed either tRNA gene position effect (5) or tRNA gene-mediated (tgm) silencing (6), is independent of the tRNA gene orientation and does not involve simple steric blockage of RNA pol II upstream activator sites (6). It is dependent on transcription of the tRNA gene, since mutations in the pol III promoters and conditional mutations in RNA polymerase III (pol III) alleviate tgm silencing. The degree to which this effect suppresses nearby pol II transcription varies depending the pol II promoter (6).Unlike other silencing elements, tRNA genes are scattered throughout the genome in large numbers and could potentially influence neighboring genes, although pol II promoters are underrepresented near tRNA genes (5). Notable exceptions to this are the Ty retrotransposons (5,7,8), which appear to have adapted to the environment and preferentially insert near tRNA genes. The mechanism of tgm silencing is unknown, but genetic and cytological data suggest that it might be linked to spatial organization of the tRNA genes in the nucleus. The early pre-tRNA processing pathway and most tRNA genes associate with the nucleolus in yeast (9, 10), and tgm silencing is released by a mutation affecting nucleolar rRNA processing (6).To explore the mechanism of tgm silencing we have examined its relationship to other silencing forms and its dependence on nucleolar localization. MATERIALS AND METHODSYeast Strains and Genetic Manipulations-The strains used for screening gene deletions is BY4741 (MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0 GAL4 GAL80) and its derivatives, ResGen Invitrogen Corp. (Carlsbad, CA). Deletions affecting tgm silencing were confirmed by PCR. Growth on selective media was performed by standard methods except that the G418 concentration in kanamycin selections was doubled (11).Identification of Silencing Suppressors-The deleted gene strains with plasmid pSUP4o (4) were plated on four different syntheti...