Competition between mammalian RNAi-related gene silencing pathways is well documented. It is therefore important to identify all classes of small RNAs to determine their relationship with RNAi and how they affect each other functionally. Here, we identify two types of 59-phosphate, 39-hydroxylated human tRNA-derived small RNAs (tsRNAs). tsRNAs differ from microRNAs in being essentially restricted to the cytoplasm and in associating with Argonaute proteins, but not MOV10. The first type belongs to a previously predicted Dicer-dependent class of small RNAs that we find can modestly down-regulate target genes in trans. The 59 end of type II tsRNA was generated by RNaseZ cleavage downstream from a tRNA gene, while the 39 end resulted from transcription termination by RNA polymerase III. Consistent with their preferential association with the nonslicing Argonautes 3 and 4, canonical gene silencing activity was not observed for type II tsRNAs. The addition, however, of an oligonucleotide that was sense to the reporter gene, but antisense to an overexpressed version of the type II tsRNA, triggered robust, >80% gene silencing. This correlated with the redirection of the thus reconstituted fully duplexed double-stranded RNA into Argonaute 2, whereas Argonautes 3 and 4 were skewed toward less structured small RNAs, particularly single-strand RNAs. We observed that the modulation of tsRNA levels had minor effects on the abundance of microRNAs, but more pronounced changes in the silencing activities of both microRNAs and siRNAs. These findings support that tsRNAs are involved in the global control of small RNA silencing through differential Argonaute association, suggesting that small RNA-mediated gene regulation may be even more finely regulated than previously realized.

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The evolutionary origin of human Hepatitis Delta Virus (HDV) replication by RNA-directed transcription is unclear. Here we identify two species of 5′ capped, ∼18-25 nucleotide small RNAs. One was of antigenomic polarity corresponding to the 5′ end of Hepatitis Delta Antigen (HDAg) mRNA and interacted with HDAg and RNA POLYMERASE II (POL II), while the other mapped to a structurally analogous region on the genomic RNA hairpin. An HDAg-interaction screen uncovered MOV10, the human homologue of the A. thaliana RNA amplification factor SDE3 and D. melanogaster RISC-maturation factor Armitage. MOV10 knockdown inhibited HDV replication, but not HDAg mRNA translation supporting a role for MOV10 in RNA-directed transcription. Together, our studies define RNA hairpins as critical elements for the initiation of HDV-related RNA-directed transcription. The identification of capped small RNAs and the involvement of MOV10 in HDV replication further suggest a conserved mechanism related to RNA-directed transcription in lower eukaryotes.

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The widespread occurrence of intergenic transcription in eukaryotes is increasingly evident. Intergenic transcription in the ␤-globin gene cluster has been described in murine and human cells, and models for a role in gene and chromatin activation have been proposed. In this study, we analyze intergenic transcription and the chromatin state throughout the human ␤-globin gene cluster and find that the data are not consistent with such activation-linked models. Thus, intergenic transcript levels correlate with neither chromatin activation nor globin gene expression. Instead, we find that intergenic transcripts of the ␤-globin gene cluster are specifically upregulated in Dicer-deficient cells. This is accompanied by a shift towards more activated chromatin as indicated by changes in histone tail modifications. Our results strongly implicate RNA interference (RNAi)-related mechanisms in regulating intergenic transcription in the human ␤-globin gene cluster and further suggest that RNAi-dependent chromatin silencing in vertebrates is not restricted to the centromeres.The human ␤-globin gene cluster spans over 70 kb on chromosome 11 consisting of five ␤-globin-like genes. It is regulated by an upstream locus control region (LCR) which confers high-level and tissue-specific expression on linked globin genes in transgenic mice (18). Although the LCR autonomously opens chromatin, presumably by recruiting chromatin remodeling complexes through transcription factor association, a region upstream of the LCR has been inferred to modulate ␤-globin locus chromatin as well (21,36). This region consists of a number of repetitive elements, one of which is an endogenous retroviral (ERV) solitary long terminal repeat (LTR) (ERV solo LTR). This element has been identified as a major promoter of intergenic transcription in both erythroid cells and nonerythroid HeLa cells following treatment with the histone deacetyltransferase inhibitor trichostatin A (TSA) (35).The presence of intergenic transcription in the human ␤-globin gene cluster has given rise to various models trying to explain their existence (reviewed in reference 43). All of these models essentially positively link intergenic transcription to globin gene activity. The notion that intergenic transcription follows the same 5Ј-3Ј polarity as the structural globin genes (subsequently referred to as "sense" orientation) (2) triggered speculation that intergenic transcription may be a means of delivering polymerases to the globin gene promoters during gene activation and development (30). A variation of this tracking model explains the spatially and temporally coordinated activation of globin genes during development by arguing that intergenic transcription would be necessary for opening chromatin at the appropriate developmental stages, thus facilitating globin gene expression (16). Intergenic transcription should therefore be active at all erythropoietic stages in the LCR, consistent with its role as a constitutive globin enhancer, while it should be coupled to resident globi...