Circular RNAs (circRNAs) are emerging as novel regulators of gene expression in various biological processes. CircRNAs regulate gene expression by interacting with cellular regulators such as microRNAs and RNA binding proteins (RBPs) to regulate downstream gene expression. The accumulation of high-throughput RNA–protein interaction data revealed the interaction of RBPs with the coding and noncoding RNAs, including recently discovered circRNAs. RBPs are a large family of proteins known to play a critical role in gene expression by modulating RNA splicing, nuclear export, mRNA stability, localization, and translation. However, the interaction of RBPs with circRNAs and their implications on circRNA biogenesis and function has been emerging in the last few years. Recent studies suggest that circRNA interaction with target proteins modulates the interaction of the protein with downstream target mRNAs or proteins. This review outlines the emerging mechanisms of circRNA–protein interactions and their functional role in cell physiology.

The intermolt crustacean Y-organ (YO) maintains a basal state mediated by pulsatile release of molt inhibiting hormone (MIH), a neuropeptide produced in the eyestalk ganglia, inhibiting YO ecdysteroidogenesis. Reduction of MIH results in YO activation and the animal enters premolt. In the crab, Gecarcinus lateralis, molting was induced by eyestalk ablation (ESA). ESA animals were injected with either rapamycin, an mTOR inhibitor, or DMSO vehicle at Day 0. YOs were harvested at 1, 3, and 7 days post-ESA and processed for high throughput RNA sequencing. ESA-induced increases in mRNA levels of mTOR signaling genes (e.g., mTOR, Rheb, TSC1/2, Raptor, Akt, and S6 kinase) declined following rapamycin treatment. In concert with mTOR inhibition, mRNA levels of ecdysteroid biosynthesis genes (e.g., Nvd, Spo, Sad, Dib, and Phm) were decreased and accompanied by a decrease in hemolymph ecdysteroid titer. By contrast, rapamycin increased the mRNA level of FKBP12, the rapamycin-binding protein, as well as the mRNA levels of genes associated with Wnt and insulin-like growth factor signaling pathways. Many MIH and transforming growth factor-β signaling genes were down regulated in ESA animals. These results indicate that mTOR activity either directly or indirectly controls transcription of genes that drive activation of the YO.

In the field of crustacean biology, usage of RNA-seq to study gene expression is rapidly growing. Major advances in sequencing technology have contributed to the ability to examine complex patterns of genome activity in a wide range of organisms that are extensively used for comparative physiology, ecology and evolution, environmental monitoring, and commercial aquaculture. Relative to insect and vertebrate model organisms, however, information on the organization of crustacean genomes is virtually nonexistent, making de novo transcriptome assembly, annotation and quantification problematic and challenging. We present here a summary of the methodologies and software analyses employed in 23 recent publications, which describe de novo transcriptome assembly, annotation, and differential gene expression in a variety of crustacean experimental systems. We focus on establishing a series of best practices that will allow for investigators to produce datasets that are understandable, reproducible, and of general utility for related analyses and cross-study comparisons.