Small interfering RNAs (siRNAs) are short, double-stranded RNAs that mediate efficient gene silencing in a sequence-specific manner by utilizing the endogenous RNA interference (RNAi) pathway. The current standard synthetic siRNA structure harbors a 19-base-pair duplex region with 3' overhangs of 2 nucleotides (the so-called 19+2 form). However, the synthetic 19+2 siRNA structure exhibits several sequence-independent, nonspecific effects, which has posed challenges to the development of RNAi therapeutics and specific silencing of genes in research. In this study, we report on the identification of truncated siRNA backbone structures with duplex regions shorter than 19 bp (referred to as asymmetric shorter-duplex siRNAs or asiRNAs) that can efficiently trigger gene silencing in human cell lines. Importantly, this asiRNA structure significantly reduces nonspecific effects triggered by conventional 19+2 siRNA scaffold, such as sense-strand-mediated off-target gene silencing and saturation of the cellular RNAi machinery. Our results suggest that this asiRNA structure is an important alternative to conventional siRNAs for both functional genomics studies and therapeutic applications.
Ser-5 phosphorylation of the RNA polymerase II (Pol II) C-terminal domain by TFIIH kinase has been implicated in critical steps in mRNA synthesis, such as Pol II promoter escape and mRNA 5 -capping. However, the general requirement and precise role of TFIIH kinase in Pol II transcription still remain elusive. Here we use a chemical genetics approach to show that, for a majority of budding-yeast genes, specific inhibition of the yeast TFIIH kinase results in a dramatic reduction in both mRNA level and Ser-5 C-terminal domain phosphorylation. Surprisingly, inhibition of TFIIH kinase activity only partially affected both Pol II density and Ser-2 phosphorylation level. The discrepancy between mRNA level and Pol II density is attributed to the defective 5 -capping, which results in the destabilization of mRNAs. Therefore, contrary to the current belief, our study points strongly toward a minor role of TFIIH kinase in Pol II transcription, and a more significant role in mRNA capping in budding yeast.T he C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) contains a series of YSPTSPS heptad repeats that are multiply-phosphorylated during the eukaryotic transcription cycle. Phosphorylation of the Ser-5 residue of the CTD heptad repeats has been implicated in multiple aspects of mRNA synthesis, such as promoter clearance (for transition from initiation to early elongation) and 5Ј-end capping of pre-mRNA (1). CTD Ser-2 phosphorylation has been implicated in productive elongation and the 3Ј-end processing of the transcript (2). However, it remains unclear whether CTD phosphorylation is required for transcription in general or functions in a promoter-and activatorspecific manner. Moreover, the requirement of Ser-5 phosphorylation for subsequent Ser-2 phosphorylation and transcriptional elongation remains controversial. An earlier study suggested that the CTD Ser-5 phosphorylation by Kin28 does not affect the Ser-2 phosphorylation level (3). In contrast, a recent study suggests that the CTD Ser-5 phosphorylation stimulates Ser-2 phosphorylation by BUR1/BUR2 kinase (4). These issues must be resolved to gain a clear understanding of the role of CTD phosphorylation in Pol II transcription in vivo.Temperature-sensitive mutants of CTD kinases have been used to study the functions of these enzymes in vivo (5). A genome-wide expression analysis using a temperature-sensitive mutant of KIN28, the Saccharomyces cerevisiae TFIIH kinase gene, showed that the loss of Kin28 function resulted in global shutdown of Pol II transcription (6). However, it has been demonstrated that the same mutation also disrupts other subunits in the TFIIH complex upon temperature shift, which makes the unambiguous functional analysis of this kinase difficult (7). Inhibition of CTD kinases with conventional pharmacological inhibitors is also problematic because these agents can nonspecifically inhibit other kinases (8). To overcome these obstacles, we used the ''analog-sensitive'' kinase-mutant strategy to dissect the unique roles of...
The data obtained in the present study provide an example of synthetic multi-functional RNA structures that enable multiple gene interference in mammalian cells, which could become powerful tools for an efficient combinatorial iRNA strategy.
The gene-silencing activity of a small interfering RNA (siRNA) is determined by various factors. Considering that RNA interference (RNAi) is an unparalleled technology in both basic research and therapeutic applications, thorough understanding of the factors determining RNAi activity is critical. This report presents observations that siRNAs targeting KRT7 show cell-line-dependent activity, which correlates with the expression level of KRT7 mRNA. By modulating the target mRNA level, it was confirmed that highly expressed genes are more susceptible to siRNA-mediated gene silencing. Finally, several genes that show different expression levels in a cell-line dependent manner were tested, which verified the expression-level-dependent siRNA activities. These results strongly suggest that the abundance of target mRNA is a critical factor that determines the efficiency of the siRNA-mediated gene silencing in a given cellular context. This report should provide practical guidelines for designing RNAi experiments and for selecting targetable genes in RNAi therapeutics studies.
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