Identification of small RNA pathway genes using patterns of phylogenetic conservation and divergence

Tabach, Yuval; Billi, Allison C.; Hayes, Gabriel D.; Newman, Martin A.; Zuk, Or; Gabel, Harrison W.; Kamath, Ravi S.; Yacoby, Keren; Chapman, Brad; Garcia, Susana M. D. A.; Borowsky, Mark; Kim, John K.; Ruvkun, Gary

doi:10.1038/nature11779

Cited by 148 publications

(195 citation statements)

References 28 publications

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“…Together with other reports, our study reveals an evolutionarily conserved scheme in which splicing factors modulate RNAi in a manner independent of their pre-mRNA splicing activities (26)(27)(28)(29)(30). In an interesting reversal, recent studies report that small RNAs and components of the RNAi machinery modulate pre-mRNA splicing in flies and mammals (41,42).…”

Section: Discussionsupporting

confidence: 83%

“…For example, mutations in genes encoding a subset of splicing factors impair RNAi-mediated heterochromatin formation and transcriptional silencing in Schizosaccharomyces pombe (26). In addition, genome-wide RNAi screens of Caenorhabditis elegans and cultured Drosophila cells, as well as a recent analysis of phylogenetic conservation of candidate RNAi factors, indicated that specific splicing factors are required for RNA-mediated gene silencing (27)(28)(29)(30). Furthermore, SNR-3, the C. elegans homolog of SmD1, a core protein of the Drosophila small nuclear ribonucleoprotein particle (snRNP), associates with DCR-1 (31).…”

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confidence: 99%

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Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila

Xiong

Kurthkoti

Chang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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RNAi is an evolutionarily conserved gene regulatory process that operates in a wide variety of organisms. During RNAi, long doublestranded RNA precursors are processed by Dicer proteins into ∼21-nt siRNAs. Subsequently, siRNAs are incorporated into the RNAinduced silencing complexes (RISCs) that contain Argonaute-family proteins and guide RISC to target RNAs via complementary base pairing, leading to posttranscriptional gene silencing. Select premRNA splicing factors have been implicated in RNAi in fission yeast, worms, and flies, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle implicated in splicing, is required for RNAi and antiviral immunity in cultured cells and in vivo. SmD1 interacts with both Dicer-2 and dsRNA precursors and is indispensable for optimal siRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the small interfering RISC without significantly affecting the expression of major canonical siRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the small interfering RISC, including Argonaute 2, both in flies and in humans. Notably, RNAi defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the RNAi and splicing machineries are physically and functionally distinct entities. Our results suggest that Drosophila SmD1 plays a direct role in RNAi-mediated gene silencing independently of its premRNA splicing activity and indicate that the dual roles of splicing factors in posttranscriptional gene regulation may be evolutionarily widespread. RNAi is an ancient gene regulatory process (1). RNAi is triggered by ∼21-to 24-nt double-stranded siRNAs and microRNAs (miRNAs) (2-5), which are assembled into RNAinduced silencing complexes (RISCs). Once incorporated into the RISC, one strand of the siRNA or miRNA engages target mRNAs via complementary base pairing and modulates gene expression in a sequence-specific manner. RNAi generally represses gene expression posttranscriptionally by inhibiting translation and/ or promoting destabilization of target mRNAs, or transcriptionally by modulating chromatin structure at the target loci. By regulating the expression of diverse target RNAs, RNAi plays a key role in numerous biological processes, including cancer, development, homeostasis, and antiviral defense.In Drosophila melanogaster, siRNAs are generated from long dsRNA precursors that are either transcribed endogenously or introduced exogenously (6-10). Long dsRNAs are processed into duplex siRNAs by the ribonuclease III Dicer-2 (Dcr-2), which forms a stable complex with the cofactors loquacious (Loqs)-PD and R2D2 (11)(12)(13)(14). The formation of mature RISC is a multistep process. First, the Dcr-2/R2D2 heterodimer gauges the thermodynamic stability of the ends of the siRNA duplex and associates with duplex siRNAs in a polarized pattern to form the RISC-loading complex (RLC) (15, 16). Several othe...

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Section: Discussionsupporting

confidence: 83%

mentioning

confidence: 99%

Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila

Xiong

Kurthkoti

Chang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Previously, we reported the identification of several signaling kinases, including the conserved serine/threonine kinase, casein kinase II (CK2), in a genome-wide RNAi screen for candidate factors involved in small RNA-mediated silencing in C. elegans (31,32). In this study, we characterize the role of CK2 as a positive regulator of miRISC activity in C. elegans.…”

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confidence: 99%

Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1

Alessi

Khivansara

Han

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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MicroRNAs (miRNAs) play essential, conserved roles in diverse developmental processes through association with the miRNA-induced silencing complex (miRISC). Whereas fundamental insights into the mechanistic framework of miRNA biogenesis and target gene silencing have been established, posttranslational modifications that affect miRISC function are less well understood. Here we report that the conserved serine/threonine kinase, casein kinase II (CK2), promotes miRISC function in Caenorhabditis elegans. CK2 inactivation results in developmental defects that phenocopy loss of miRISC cofactors and enhances the loss of miRNA function in diverse cellular contexts. Whereas CK2 is dispensable for miRNA biogenesis and the stability of miRISC cofactors, it is required for efficient miRISC target mRNA binding and silencing. Importantly, we identify the conserved DEAD-box RNA helicase, CGH-1/DDX6, as a key CK2 substrate within miRISC and demonstrate phosphorylation of a conserved N-terminal serine is required for CGH-1 function in the miRNA pathway.

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“…RNA interference (RNAi), a sequence-specific gene silencing mechanism induced by double-stranded RNA (dsRNA), has emerged as a promising therapy for combating cancer (19,33,34). The RNAi effect can be mediated with short interfering RNA (siRNA), which is a short, double stranded RNA with 21-23 base pairs (35), or, as recently demonstrated, with short hairpin RNA (shRNA) (36). Although siRNA and shRNA elicit comparable gene silencing effects in RNAi experiments, chemically synthesized shRNA represents significant advantages over siRNA.…”

Section: Discussionmentioning

confidence: 99%

Downregulation of thymidylate synthase by RNAi molecules enhances the antitumor effect of pemetrexed in an orthotopic malignant mesothelioma xenograft mouse model

Lila

Kato

Fukushima

et al. 2016

International Journal of Oncology

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Abstract. Malignant pleural mesothelioma (MPM) is an incurable cancer with an increasing incidence. Currently, pemetrexed (PMX)-based chemotherapy is the mainstay of chemotherapy for MPM, however, the outcome of PMX-based chemotherapy in patients with MPM is dismal. RNA interference (RNAi) technology has been considered as an effective tool to substantially enhance the therapeutic efficacy of chemotherapeutic agents in many preclinical and clinical settings. In this study, therefore, we investigated whether non-viral anti-thymidylate synthase RNAi embedded liposome (TS shRNA lipoplex) would effectively guide the downregulation of TS in human malignant mesothelioma MSTO-211H cells. Consequently, it enhanced the antitumor effect of PMX both in vitro and in vivo. TS shRNA effectively enhanced the in vitro cell growth inhibition upon treatment with PMX via downregulating TS expression in the MSTO-211H cell line. In in vivo orthotopic tumor model, the combined treatment of PMX and TS shRNA lipoplex efficiently combated the progression of orthotopic thoracic tumors and as a result prolonged mouse survival, compared to each single treatment. Our findings emphasize the pivotal relevance of RNAi as an effective tool for increasing the therapeutic efficacy of PMX, a cornerstone in the treatment regimens of MPM, and thereby, raising the possibility for the development of a novel therapeutic strategy, combination therapy of TS-shRNA and PMX, that can surpass many of the currently applied, but less effective, therapeutic regimens against lethal MPM.

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Identification of small RNA pathway genes using patterns of phylogenetic conservation and divergence

Cited by 148 publications

References 28 publications

Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila

Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila

Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1

Downregulation of thymidylate synthase by RNAi molecules enhances the antitumor effect of pemetrexed in an orthotopic malignant mesothelioma xenograft mouse model

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