Robust partitioning of microRNA targets from downstream regulatory changes.

The biological impact of microRNAs (miRNAs) is determined by their targets, and robustly identifying direct miRNA targets remains challenging. Existing methods suffer from high false-positive rates and are unable to effectively differentiate direct miRNA targets from downstream regulatory changes. Here, we present an experimental and computational framework to deconvolute post-transcriptional and transcriptional changes using a combination of RNA-seq and PRO-seq. This novel approach allows us to systematically profile the regulatory impact of a miRNA. We refer to this approach as CARP: Combined Analysis of RNA-seq and PRO-seq. We apply CARP to multiple miRNAs and show that it robustly distinguishes direct targets from downstream changes, while greatly reducing false positives. We validate our approach using Argonaute eCLIP-seq and ribosome profiling, demonstrating that CARP defines a comprehensive repertoire of targets. Using this approach, we identify miRNA-specific activity of target sites within the open reading frame. Additionally, we show that CARP facilitates the dissection of complex changes in gene regulatory networks triggered by miRNAs and identification of transcription factors that mediate downstream regulatory changes. Given the robustness of the approach, CARP would be particularly suitable for dissecting miRNA regulatory networks in vivo.

Seasonal cues induce phenotypic plasticity of Drosophila suzukii to enhance winter survival.

BACKGROUND: As global climate change and exponential human population growth intensifies pressure on agricultural systems, the need to effectively manage invasive insect pests is becoming increasingly important to global food security. Drosophila suzukii is an invasive pest that drastically expanded its global range in a very short time since 2008, spreading to most areas in North America and many countries in Europe and South America. Preliminary ecological modeling predicted a more restricted distribution and, for this reason, the invasion of D. suzukii to northern temperate regions is especially unexpected. Investigating D. suzukii phenology and seasonal adaptations can lead to a better understanding of the mechanisms through which insects express phenotypic plasticity, which likely enables invasive species to successfully colonize a wide range of environments. RESULTS: We describe seasonal phenotypic plasticity in field populations of D. suzukii. Specifically, we observed a trend of higher proportions of flies with the winter morph phenotype, characterized by darker pigmentation and longer wing length, as summer progresses to winter. A laboratory-simulated winter photoperiod and temperature (12:12 L:D and 10 °C) were sufficient to induce the winter morph phenotype in D. suzukii. This winter morph is associated with increased survival at 1 °C when compared to the summer morph, thus explaining the ability of D. suzukii to survive cold winters. We then used RNA sequencing to identify gene expression differences underlying seasonal differences in D. suzukii physiology. Winter morph gene expression is consistent with known mechanisms of cold-hardening such as adjustments to ion transport and up-regulation of carbohydrate metabolism. In addition, transcripts involved in oogenesis and DNA replication were down-regulated in the winter morph, providing the first molecular evidence of a reproductive diapause in D. suzukii. CONCLUSIONS: To date, D. suzukii cold resistance studies suggest that this species cannot overwinter in northern locations, e.g. Canada, even though they are established pests in these regions. Combining physiological investigations with RNA sequencing, we present potential mechanisms by which D. suzukii can overwinter in these regions. This work may contribute to more accurate population models that incorporate seasonal variation in physiological parameters, leading to development of better management strategies.

Functionalized nanowires for miRNA-mediated therapeutic programming of naïve T cells.

Cellular programming of naïve T cells can improve the efficacy of adoptive T-cell therapy. However, the current ex vivo engineering of T cells requires the pre-activation of T cells, which causes them to lose their naïve state. In this study, cationic-polymer-functionalized nanowires were used to pre-program the fate of primary naïve CD8+ T cells to achieve a therapeutic response in vivo. This was done by delivering single or multiple microRNAs to primary naïve mouse and human CD8+ T cells without pre-activation. The use of nanowires further allowed for the delivery of large, whole lentiviral particles with potential for long-term integration. The combination of deletion and overexpression of miR-29 and miR-130 impacted the ex vivo T-cell differentiation fate from the naïve state. The programming of CD8+ T cells using nanowire-delivered co-delivery of microRNAs resulted in the modulation of T-cell fitness by altering the T-cell proliferation, phenotypic and transcriptional regulation, and secretion of effector molecules. Moreover, the in vivo adoptive transfer of murine CD8+ T cells programmed through the nanowire-mediated dual delivery of microRNAs provided enhanced immune protection against different types of intracellular pathogen (influenza and Listeria monocytogenes). In vivo analyses demonstrated that the simultaneous alteration of miR-29 and miR-130 levels in naïve CD8+ T cells reduces the persistence of canonical memory T cells whereas increases the population of short-lived effector T cells. Nanowires could potentially be used to modulate CD8+ T-cell differentiation and achieve a therapeutic response in vivo without the need for pre-activation.