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.

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 ~40% 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 the 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.

Small regulatory RNAs (sRNAs) are involved in anti-viral defense and gene regulation. Although RNA-dependent RNA Polymerases (RdRPs) are known to produce sRNA in nematodes, plants and fungi, whether they play roles in sRNA biogenesis in other animals remains controversial. In this study, 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 identify abundant classes of ~22nt sRNAs that require specific combinations of RdRPs and sRNA effector proteins (Argonautes or AGOs). RdRP-dependent sRNAs are mainly derived from sense and antisense strands of RNA polymerase III-transcribed genes and repetitive elements. Unlike C. elegans sRNA pathways, 5′-tri-phosphorylated sRNAs are not detected, suggesting that the tick pathways are distinct from the pathways known in worms. Knockdown of one of the RdRPs unexpectedly results in downregulation of a subset of viral transcripts, in contrast to their upregulation by AGO knockdown. Furthermore, we show that knockdown of AGO/RdRP causes misregulation of protein-coding genes including RNAi-related genes, suggesting feedback regulation. Luciferase assays demonstrate that one of the RdRP-regulated genes, the MEK1 ortholog IscDsor1 is regulated through its 3′UTR, where a putative sRNA target site resides. These results provide evidence that arachnid RdRPs are important sRNA biogenesis factors, and the discovery of novel pathways underscores the importance of characterizing sRNA biogenesis in various organisms to understand virus-vector interactions and to exploit RNAi for pest control.

MiRNAs are a class of small non-coding RNAs that play important roles in post-transcriptional regulation of complementary target mRNAs. Loquacious (Loqs) is a dsRNA-binding protein that binds Dicer-1 to cleave pre-miRNAs into short duplexes. The maternally deposited Loqs protein in Drosophila could potentially complicate their study in zygotic mutants. Here, Loqs function was investigated in the complete absence of maternal and zygotic contribution by using a cell line established from loqs-null fly embryos. Genome-wide analysis of pre-miRNA structures showed that Loqs enhances processing of sub-optimal substrates with unstable base-pairing at the dicing site. Post-transcriptional mechanisms that regulate miRNA expression during fly embryogenesis are not well understood. Here, distinct half-lives of individual miRNAs and identity of terminal 5' nucleotides were found to influence miRNA expression profiles. Overall, the results illustrate roles for dsRNA-binding proteins and miRNA stability in regulation of miRNA expression, demonstrating the complexity that underlies these processes.

No abstract

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.

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.