Stacking up CRISPR against RNAi for therapeutic gene inhibition.

Both RNA interference (RNAi) and clustered regularly-interspaced short palindromic repeats (CRISPR) technologies allow for the sequence-specific inhibition of gene function and therefore have the potential to be used as therapeutic modalities. By judging the current public and scientific journal interest, it would seem that CRISPR, by enabling clean, durable knockouts, will dominate therapeutic gene inhibition, also at the expense of RNAi. This review aims to look behind prevailing sentiments and to more clearly define the likely scope of the therapeutic applications of the more recently developed CRISPR technology and its relative strengths and weaknesses with regards to RNAi. It is found that largely because of their broadly overlapping delivery constraints, while CRISPR presents formidable competition for DNA-directed RNAi strategies, its impact on RNAi therapeutics triggered by synthetic oligonucleotides will likely be more moderate. Instead, RNAi and genome editing, and in particular CRISPR, are poised to jointly promote a further shift toward sequence-targeted precision medicines.

RNA interference-mediated simultaneous silencing of four genes using cross-shaped RNA.

The structural flexibility of RNA interference (RNAi)-triggering nucleic acids suggests that the design of unconventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA triggers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.

Human tRNA-derived small RNAs in the global regulation of RNA silencing.

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 5'-phosphate, 3'-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 5' end of type II tsRNA was generated by RNaseZ cleavage downstream from a tRNA gene, while the 3' 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.

Combined proteomic-RNAi screen for host factors involved in human hepatitis delta virus replication.

Human hepatitis delta virus (HDV) is the only animal virus known to replicate its RNA genome using a host polymerase because its only virally encoded proteins, the small and large hepatitis delta antigens (HDAg-S and HDAg-L), lack polymerase activity. Although this makes HDV an ideal model system to study RNA-directed transcription in mammalian cells, little is known about the host factors involved in its replication. To comprehensively identify such host factors, we created a stable cell line carrying a functional FLAG-HDAg-S. Anti-Flag immunopurification and mass spectrometry identified >100 proteins associated with FLAG-HDAg-S, many of which had predicted roles in RNA metabolism. The biological relevance of this screen was strongly supported by the identification of nine out of the 12 subunits of the RNA polymerase II complex thought to mediate HDV replication. To further investigate the significance of these factors for HDV replication, we selected 65 proteins to look for factors that would also affect the accumulation of HDV RNA following siRNA knockdown. Fifteen and three factors were found to regulate HDV RNA accumulation negatively and positively, respectively, upon RNAi knockdown. Our results provide a valuable resource for future research to advance our mechanistic understanding of HDV replication and RNA-directed transcription in mammalian cells.

Capped small RNAs and MOV10 in human hepatitis delta virus replication.

The evolutionary origin of human hepatitis delta virus (HDV) replication by RNA-directed transcription is unclear. Here we identify two species of 5'-capped, approximately 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), whereas the other mapped to a structurally analogous region on the genomic RNA hairpin. An HDAg-interaction screen indicated that HDAg interacts with MOV10, the human homolog of the Arabidopsis thaliana RNA amplification factor gene SDE3 and Drosophila melanogaster RISC-maturation factor gene Armitage (armi). MOV10 knockdown inhibited HDV replication, but not HDAg mRNA translation, further 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.

Dicer-dependent turnover of intergenic transcripts from the human beta-globin gene cluster.

The widespread occurrence of intergenic transcription in eukaryotes is increasingly evident. Intergenic transcription in the beta-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 beta-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 beta-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 beta-globin gene cluster and further suggest that RNAi-dependent chromatin silencing in vertebrates is not restricted to the centromeres.