RESUMO
Small regulatory RNAs (sRNAs) are involved in antiviral defense and gene regulation. Although roles of RNA-dependent RNA Polymerases (RdRPs) in sRNA biology are extensively studied in nematodes, plants and fungi, understanding of RdRP homologs in other animals is still lacking. Here, we study sRNAs in the ISE6 cell line, which is derived from the black-legged tick, an important vector of human and animal pathogens. We find abundant classes of ~22nt sRNAs that require specific combinations of RdRPs and sRNA effector proteins (Argonautes or AGOs). RdRP1-dependent sRNAs possess 5'-monophosphates and are mainly derived from RNA polymerase III-transcribed genes and repetitive elements. Knockdown of some RdRP homologs misregulates genes including RNAi-related genes and the regulator of immune response Dsor1. Sensor assays demonstrate that Dsor1 is downregulated by RdRP1 through the 3'UTR that contains a target site of RdRP1-dependent repeat-derived sRNAs. Consistent with viral gene repression by the RNAi mechanism using virus-derived small interfering RNAs, viral transcripts are upregulated by AGO knockdown. On the other hand, RdRP1 knockdown unexpectedly results in downregulation of viral transcripts. This effect is dependent on Dsor1, suggesting that antiviral immunity is enhanced by RdRP1 knockdown through Dsor1 upregulation. We propose that tick sRNA pathways control multiple aspects of immune response via RNAi and regulation of signaling pathways.
Assuntos
Ixodes , Pequeno RNA não Traduzido , Animais , Humanos , Ixodes/genética , Ixodes/metabolismo , Eucariotos/genética , Sistema de Sinalização das MAP Quinases , Antivirais , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Interferência de RNA , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismoRESUMO
Mature microRNAs (miRNAs) are processed from primary transcripts (pri-miRNAs), and their expression is controlled at transcriptional and post-transcriptional levels. However, how regulation at multiple levels achieves precise control remains elusive. Using published and new datasets, we profile a time course of mature and pri-miRNAs in Drosophila embryos and reveal the dynamics of miRNA production and degradation as well as dynamic changes in pri-miRNA isoform selection. We found that 5' nucleotides influence stability of mature miRNAs. Furthermore, distinct half-lives of miRNAs from the mir-309 cluster shape their temporal expression patterns, and the importance of rapid degradation of the miRNAs in gene regulation is detected as distinct evolutionary signatures at the target sites in the transcriptome. Finally, we show that rapid degradation of miR-3/-309 may be important for regulation of the planar cell polarity pathway component Vang. Altogether, the results suggest that complex mechanisms regulate miRNA expression to support normal development.
Assuntos
Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/biossíntese , Estabilidade de RNA , Animais , Perfilação da Expressão GênicaRESUMO
Dicer is a versatile protein regulating diverse biological processes via the production of multiple classes of small regulatory RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs). In this chapter, we will discuss roles for Dicer in driving temporal changes in activity of individual small RNA classes to support oogenesis and early embryogenesis. Genetic strategies that perturb particular functions of Dicer family proteins, such as ablation of individual Dicer paralogs or their binding partners as well as introduction of point mutations to individual domains, allowed the dissection of Dicer functions in diverse small RNA pathways. Evolutionary conservation and divergence of the mechanisms highlight the importance of Dicer versatility in supporting rapid changes in gene expression during oogenesis and early development. Furthermore, we will discuss potential roles of Dicer in transgenerational inheritance of small RNA-mediated gene regulation.
Assuntos
Desenvolvimento Embrionário/genética , Oogênese/genética , Pequeno RNA não Traduzido/genética , Ribonuclease III/metabolismo , Animais , Feminino , Pequeno RNA não Traduzido/metabolismoRESUMO
In Drosophila, Dicer-1 binds Loquacious-PB (Loqs-PB) as its major co-factor. Previous analyses indicated that loqs mutants only partially impede miRNA processing, but the activity of minor isoforms or maternally deposited Loqs was not eliminated in these studies. We addressed this by generating a cell line from loqs-null embryos and found that only â¼40% of miRNAs showed clear Loqs dependence. Genome-wide comparison of the hairpin structure and Loqs dependence suggested that Loqs substrates are influenced by base-pairing status at the dicing site. Artificial alteration of base-pairing stability at this position in model miRNA hairpins resulted in predicted changes in Loqs dependence, providing evidence for this hypothesis. Finally, we found that evolutionarily young miRNA genes tended to be Loqs dependent. We propose that Loqs may have roles in assisting the de novo emergence of miRNA genes by facilitating dicing of suboptimal hairpin substrates.
