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Cap1 2'-O-ribose methyltransferase (CMTR1) modifies RNA transcripts containing the 7-methylguanosine cap via 2'-O-ribose methylation of the first transcribed nucleotide, yielding cap1 structures. However, the role of CMTR1 in small RNA-mediated gene silencing remains unknown. Here, we identified and characterized a Drosophila CMTR1 gene (dCMTR1) mutation. We found that the catalytic activity of dCMTR1 was involved in the biogenesis of small interfering RNAs (siRNAs) but not microRNAs. Additionally, dCMTR1 interacted with R2D2, a key component for the assembly of the RNA-induced silencing complex (RISC) containing Argonaute 2 (Ago2). Consistent with this finding, loss of dCMTR1 function impaired RISC assembly by inhibiting the unwinding of Ago2-bound siRNA duplexes, thus preventing the retention of the guide strand. Moreover, dCMTR1 is unlikely to modify siRNAs during RISC assembly. Collectively, our data indicate that dCMTR1 is a positive regulator of the small RNA pathway associated with Ago2 with roles in both siRNA biogenesis and RISC assembly., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The steroid hormone ecdysone has a central role in the developmental transitions of insects through its control of responsive protein-coding and microRNA (miRNA) gene expression. However, the complete regulatory network controlling the expression of these genes remains to be elucidated. In this study, we performed cross-linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 (Ago1) protein, the core effector of the miRNA pathway, in Drosophila S2 cells. We found that regulatory interactions between miRNAs and their cognate targets were substantially altered by Ago1 in response to ecdysone signaling. Additionally, during the larva-to-adult metamorphosis, miR-252-5p was up-regulated via the canonical ecdysone-signaling pathway. Moreover, we provide evidence that miR-252-5p targets Abelson interacting protein ( Abi) to decrease the protein levels of cyclins A and B, controlling the cell cycle. Overall, our data suggest a potential role for the ecdysone/miR-252-5p/Abi regulatory axis partly in cell-cycle control during metamorphosis in Drosophila.-Lim, D.-H., Lee, S., Han, J. Y., Choi, M.-S., Hong, J.-S., Seong, Y., Kwon, Y.-S., Lee, Y. S. Ecdysone-responsive microR-252-5p controls the cell cycle by targeting Abi in Drosophila.

In the Drosophila RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) direct Argonaute2 (Ago2), an endonuclease, within the RNA-induced silencing complex (RISC) to cleave complementary mRNA targets. In vitro studies have shown that, for each siRNA duplex, RISC retains only one strand, the guide, and releases the other, the passenger, to form a holo-RISC complex. Here, we have isolated a new Ago2 mutant allele and provide, for the first time, in vivo evidence that endogenous Ago2 slicer activity is important to mount an RNAi response in Drosophila. We demonstrate in vivo that efficient removal of the passenger strand from RISC requires the cleavage activity of Ago2. We have also identified a new intermediate complex in the RISC assembly pathway, pre-RISC, in which Ago2 is stably bound to double-stranded siRNA.

Double-stranded RNA induces sequence-specific inhibition of gene expression at a posttranscriptional level in eukaryotes (RNAi). This natural phenomenon has been developed into a tool for studying gene function in several model organisms, including Drosophila melanogaster. Transgenes bearing inverted repeats are able to exert an RNAi effect in Drosophila, but cloning difficulties and inconsistent silencing complicate the method. We have constructed a transgene containing inverted repeats separated by a functional intron such that mRNA produced by the transgene is predicted to form loopless hairpin RNA following splicing. A single copy of the transgene effectively and uniformly silences expression of a target gene (white) in transgenic flies. We have developed a vector that is designed to produce intron-spliced hairpin RNA corresponding to any Drosophila gene. The vector is under control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. The UAS/GAL4 system allows hairpin RNA to conditionally silence gene expression in Drosophila in a tissue-specific manner. Moreover, the presence of the intron spacer greatly enhances the stability of inverted-repeat sequences in bacteria, facilitating the cloning procedure.

The Notch signaling pathway plays a central role in the development of various organisms. However, dysregulation of microRNAs (miRNAs), which are crucial regulators of gene expression, can disrupt signaling pathways at all stages of development. Although Notch signaling is involved in wing development in Drosophila, the mechanism underlying miRNA‐based regulation of the Notch signaling pathway is unclear. Here, we report that loss of Drosophila miR‐252 increases the size of adult wings, whereas the overexpression of miR‐252 in specific compartments of larval wing discs leads to patterning defects in the adult wings. The miR‐252 overexpression‐induced wing phenotypes were caused by aberrant Notch signaling with intracellular accumulation of the full‐length Notch receptor during development, which could be due to defects in intracellular Notch trafficking associated with its recycling to the plasma membrane and autophagy‐mediated degradation. Moreover, we identified Rab6 as a direct target of miR‐252‐5p; Rab6 encodes a small Ras‐like GTPase that regulates endosomal trafficking pathways. Consistent with this finding, RNAi‐mediated downregulation of Rab6 led to similar defects in both wing patterning and Notch signaling. Notably, co‐overexpression of Rab6 completely rescued the wing phenotype associated with miR‐252 overexpression, further supporting that Rab6 is a biologically relevant target of miR‐252‐5p in the context of wing development. Thus, our data indicate that the miR‐252‐5p‐Rab6 regulatory axis is involved in Drosophila wing development by controlling the Notch signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract: Small non-coding RNAs regulate gene expression in a sequence-specific manner. In Drosophila, Dicer-2 (Dcr-2) functions in the biogenesis of endogenous small interfering RNAs (endo-siRNAs). We identified 21 distinct proteins that exhibited a ⩾1.5-fold change as a consequence of loss of dcr-2 function. Most of these were metabolic genes implicated in stress resistance and aging. dcr-2 Mutants had reduced lifespan and were hypersensitive to oxidative, endoplasmic reticulum, starvation, and cold stresses. Furthermore, loss of dcr-2 function led to abnormal lipid and carbohydrate metabolism. Our results suggest roles for the endo-siRNA pathway in metabolic regulation and defense against stress and aging in Drosophila. [Copyright &y& Elsevier]

The RNase III enzyme Dicer processes RNA into si-RNAs and miRNAs, which direct a RNA-induced silencing complex (RISC) to cleave mRNA or block its translation (RNAi). We have characterized mutations in the Drosophila dicer-1 and dicer-2 genes. Mutation in dicer-1 blocks processing of miRNA precursors, Whereas dicer-2 mutants are defective for processing siRNA precursors. It has been recently found that Drosophila Dicer-1 and Dicer-2 are also components of siRNA-dependent RISC (siRISC). We find that Dicer-1 and Dicer-2 are required for siRNA-directed mRNA cleavage, though the RNase III activity of Dicer-2 is not required. Dicer-1 and Dicer-2 facilitate distinct steps in the assembly of siRISC. However, Dicer-I but not Dicer-2 is essential for miRISC-directed translation repression. Thus, siRlSCs and miRISCs are different with respect to Dicers in Drosophila. [ABSTRACT FROM AUTHOR]

We use native gel electrophoresis to characterize complexes that mediate RNA interference (RNAi) in Drosophila. Our data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. Our results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 "holo-RISC" complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi. [ABSTRACT FROM AUTHOR]

Abstract: Methionine sulfoxide reductase B3A (MsrB3A), which catalyzes the stereospecific reduction of methionine-R-sulfoxide to methionine, is localized to the endoplasmic reticulum (ER). Here, we report a critical role of the ER-targeted MsrB3 in protection against ER stress in Drosophila and in mammalian cells. Flies overexpressing human MsrB3A exhibited significantly increased resistance to ER stress induced by dithiothreitol. These flies also showed slightly enhanced resistance to tunicamycin-induced ER stress. In addition, overexpression of MsrB3A in mammalian cells increased resistance to dithiothreitol- and thapsigargin-induced ER stresses. However, MsrB3A overexpression had no effect on the resistance to tunicamycin-induced ER stress. Knockdown of MsrB3A in mammalian cells led to a significant decrease in the resistance to thapsigargin-induced ER stress, but had no effects on the resistance to either dithiothreitol- or tunicamycin-induced ER stress. Collectively, our data provide evidence that the ER-type of MsrB3 plays an important role in protection against ER stress, suggesting that MsrB3 may be involved in the regulation of ER homeostasis. [Copyright &y& Elsevier]

Abstract: The Drosophila RNase III enzyme Dicer-2 processes double-stranded RNA (dsRNA) precursors into small interfering RNAs (siRNAs). It also interacts with the siRNA product and R2D2 protein to facilitate the assembly of an RNA-induced silencing complex (RISC) that mediates RNA interference. Here, we characterized six independent missense mutations in the dicer-2 gene. Four mutations (P8S, L188F, R269W, and P365L) in the DExH helicase domain reduced dsRNA processing activity. Two mutations were located within an RNase III domain. P1496L caused a loss of dsRNA processing activity comparable to a null dicer-2 mutation. A1453T strongly reduced both dsRNA processing and RISC activity, and decreased the levels of Dicer-2 and R2D2 proteins, suggesting that this mutation destabilizes Dicer-2. We also found that the carboxyl-terminal region of R2D2 is essential for Dicer-2 binding. These results provide further insight into the structure–function relationship of Dicer, which plays a critical role in the siRNA pathway. [Copyright &y& Elsevier]