RNA interference of mTOR gene delays molting process in Eriocheir sinensis

Highlights

• mTOR of Chinese mitten crab is widely expressed in multiple tissues.

• mTOR expression is elevated in Eyestalk during the Intermolt.

• RNAi of mTOR leads to prolonged molt interval.

• RNAi of mTOR leads to reduced expression of upstream genes, and molting-related genes.

Abstract

mTOR is a typical and conserved serine/threonine protein kinase that regulates cell growth and metabolism of organisms. Molting is a fundamental biological process in Chinese mitten crab (Eriocheir sinensis) and is monitored by a series of genes and pathways. The structural and functional characteristics of EsmTOR was investigated to determine the role of mTOR in the molting process of. The intact CDS of EsmTOR is 7449 bp in length and encodes a polypeptide consisting of 2482 amino acids. EsmTOR was expressed in all eight tissues examined during the three molting stages (postmolt, intermolt andpremolt), with levels fluctuating significantly during the molting. RNA interference of EsmTOR significantly delayed molting, indicating that mTOR may be involved in the molting process of E. sinensis. Meanwhile, a substantial downregulation was observed for the expression of upstream genes involved in amino acid transport (EsSLC7A5 and EsVATB) and downstream genes promoting ribosomal protein synthesis (EsS6K1) in the mTOR signaling pathway, as well as typical molt-related genes (EsMIH and EsEcR) after EsmTOR RNAi treatment. In addition, EsRheb, a molecular marker for tissue growth, was also significantly down-regulated. This study suggests that EsmTOR plays a fundamental role in molting regulation through the SLC7A5-V-ATPase-mTORC1 gene network.

Introduction

The mechanistic target of rapamycin (mTOR) signaling pathway serves as a central regulator that integrates and adjusts several important aspects of cell function in response to stress, oxygen, amino acids, energy levels and growth factors (Kim and Guan, 2019; Seiliez et al., 2008; Wullschleger et al., 2006). mTOR, an atypical and conserved serine/threonine-protein kinase, belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family (Richardson et al., 2004) that controls gene expression at the transcriptional and translational levels in eukaryotic cells (Nandagopal and Roux, 2015; Saxton and Sabatini, 2017; Torres and Holz, 2021). Two distinct complexes, namely, mTOR complex1 (mTORC1) and 2 (mTORC2), control cell growth, proliferation, and survival through kinase-mediated phosphorylation process (Frias et al., 2006; Laplante and Sabatini, 2012). Nutrients promote anabolic pathways, such as protein synthesis, to increase cell size and proliferation. When nutrients are restricted, catabolic processes such as autophagy are initiated to produce energy for the cell (Melick et al., 2020). mTOR is the center of nutrient signaling and mTORC1 is linked to nutrient sensing through amino acids (AAs) (Kim and Guan, 2011, Kim and Guan, 2019; Loewith and Hall, 2011; Wullschleger et al., 2006). AAs connect to mTORC1 through transmembrane AA transporters and intracellular pathways, which include Ras-related small GTP-binding protein GTPases or Rags located on the regulator protein complex anchored to the lysosomal outer surface through a covalent bond with fatty acid and the interaction with V-ATPase (Yang et al., 2013; Zoncu et al., 2011). mTORC1 controls protein translation through the phosphorylation of two well-characterized substrates, namely, ribosomal S6 kinases (S6Ks) and eIF4E-binding proteins (4EBPs) (Jewell et al., 2013). Ras homolog enriched in brain (Rheb) is a key activator of mTOR (MacLea et al., 2012) and serves as a molecular marker for mTORC1 activity (Mykles and Medler, 2015; Mykles, 2021). Furthermore, a study of the effects of temperature on molting and gene expression in the eyestalk ganglia, YO, and heart of juvenile Metacarcinus magister has shown the result that MmRheb expression decreases with increasing temperature, and Rheb expression is highest in premolt YO at all three temperatures (Wittmann et al., 2018).

Loss of mTORC1 and mTORC2 leads nutrient starvation and cause inhibition of protein synthesis, glycogen accumulation and autophagy induction (Barbet et al., 1996; Tee, 2018). The mTOR play essential roles in diverse cell growth and metabolism in arthropods and represents a critical regulatory protein involved in autophagy, lysosomal function, and metabolic pathways in Litopenaeus vannamei (Liu et al., 2018). In Tribolium castaneum, TcmTOR silencing decreased the size of the adult appendages. Analysis of the wings and elytra revealed a decrease in cell size and number of these appendages in the TcmTOR-silenced insects. This size reduction is correlated with a decrease in the transcriptional levels of marker genes controlling the cell cycle (Lin et al., 2019). As a nutrient-sensing signaling, mTOR participates in the lifespan regulation of Drosophila melanogaster (Scialo et al., 2015). In addition to being closely related to growth, mTOR is also associated with molting in crustaceans. In Gecarcinus lateralis, rapamycin inhibits YO ecdysteroid secretion in vitro and the expression of Gl-mTOR and Gl-Akt is increased in animals induced to molt by multiple leg autotomy, suggesting that mTOR signaling isinvolved in YO activation (Abuhagr et al., 2014). mTOR activity either directly or indirectly controls the transcription of genes that drive YO activation (Shyamal et al., 2018).

Chinese mitten crab, Eriocheir sinensis, is one of the most economical freshwater species with a huge aquaculture industry and consumer demand in China and is a well-known invasive species in Europe and North America (Dittel and Epifanio, 2009; Wang et al., 2008). In China, molecular research of E. sinensis has been conducted to understand its biological characteristics. As a typical crustacean, cyclical molting is a special developmental and growth process in the lifespan of E. sinensis. Therefore, the size and growth profiles of this crab are determined by its molting characteristics. Although the mTOR plays a central role in other species, its role in the molting regulation of E. sinensis remains unknown. In this work, EsmTOR was cloned to investigate its spatiotemporal expression profiles in E. sinensis during different molting processes. Gene knockdown was conducted by using RNA interference technology. The results provided insights into the influential roles of the mTOR gene in the growth and molting process of E. sinensis and other crustaceans.