Clathrin-dependent endocytosis plays a predominant role in cellular uptake of double-stranded RNA in the red flour beetle

https://doi.org/10.1016/j.ibmb.2015.03.009Get rights and content

Highlights

  • Clathrin-dependent endocytosis inhibitors dramatically suppressed RNAi of TcLgl.

  • Clathrin-dependent endocytosis inhibitors greatly reduced cellular uptake of dsRNA.

  • RNAi of clathrin-dependent endocytosis genes significantly impaired RNAi of TcLgl.

  • Clathrin-dependent endocytosis played a major role in cellular uptake of dsRNA.

Abstract

RNA interference (RNAi) is a highly conserved gene regulatory mechanism in eukaryotic organisms; however, an understanding of mechanisms of cellular uptake of double-stranded RNA (dsRNA) in different organisms remains elusive. By using pharmacological inhibitors of different endocytic pathways in conjunction with RNAi of a marker gene (lethal giant larvae, TcLgl) in the red flour beetle (Tribolium castaneum), we demonstrated that two inhibitors (chlorpromazine and bafilomycin-A1) of clathrin-dependent endocytosis can nearly abolish or significantly diminish RNAi of TcLgl, whereas methyl-β-cyclodextrin and cytochalasin-D, known to inhibit other endocytic pathways, showed no effect on RNAi of TcLgl. By using Cy3-labeled TcLgl dsRNA, we observed significantly reduced cellular uptake of TcLgl dsRNA in midgut cells after larvae were injected with each of the two clathrin-dependent endocytosis inhibitors. By using an “RNAi of RNAi” strategy, we further demonstrated that suppression of each transcript of the four key genes encoding clathrin heavy chain (TcChc), clathrin coat assembly protein AP50 (TcAP50), vacuolar (H+)-ATPase subunit H (TcVhaSFD) and a ras-related protein (TcRab7) in clathrin-dependent endocytosis by RNAi can significantly impair RNAi of TcLgl. These results support our conclusion that clathrin-dependent endocytosis is a major mechanism in cellular uptake of dsRNA in T. castaneum. Our study also provides new insights into improving RNAi efficiency by enhancing dsRNA endosomal release.

Introduction

RNA interference (RNAi) is a highly conserved post-transcriptional gene regulatory mechanism in eukaryotic organisms, including fungi, plants, insects and mammals (Fire et al., 1998, Mello and Conte, 2004, Bellés, 2010, Zhuang and Hunter, 2011). However, the cellular uptake mechanism of exogenous double-stranded RNA (dsRNA) does not appear to be highly conserved and remains undefined in different organisms. Two pathways for exogenous dsRNA uptake have been identified or implicated, which include passive uptake via a transmembrane channel protein known as systemic RNA interference deficient-1 (SID-1) encoded by sid-1 in the nematode (Caenorhabditis elegans) (Winston et al., 2002), the honey bee (Apis mellifera) (Aronstein et al., 2006), and the fish (Siniperca chuatsi) cells (Ren et al., 2011); and endocytosis-mediated pathway in the fruit fly (Drosophila melanogaster) S2 cells (Ulvila et al., 2006, Saleh et al., 2006), the desert locust (Schistocera gregaria) (Wynant et al., 2014), and the predatory mite (Metaseiulus occidentalis) (Wu and Hoy, 2014).

Although a putative transmembrane protein encoded by AmSid-1 was found to play an essential role in dsRNA uptake in the honey bee (Aronstein et al., 2006), several studies have shown that SID-1 is unlikely to play a major role in cellular uptake of dsRNA in other insects including the red flour beetle (Tribolium castaneum) (Tomoyasu et al., 2008), the migratory locust (Locusta migratoria) (Luo et al., 2012), and the desert locust (Wynant et al., 2014). Because SID-1-dependent dsRNA uptake does not seem to be common and conserved in insects, many insects must possess additional or different genes with similar functions, or possibly even different mechanisms in cellular uptake of dsRNA (Tomoyasu et al., 2008, Zhang et al., 2010).

Ulvila et al. (2006) analyzed 2000 dsRNA fragments from a cDNA library of D. melanogaster S2 cells for their protective effect from lethality induced by RNAi against Ubi-p63E, an essential gene encoding an ubiquitin for cell viability. They identified four genes, one of which encodes clathrin heavy chain (Chc), an important component of clathrin-dependent endocytosis. When Chc transcript in S2 cells is depleted by RNAi, it protects S2 cells from the lethality induced by the Ubi-p63E dsRNA treatment, suggesting that dsRNA molecules are internalized by clathrin-dependent endocytosis. On the other hand, Saleh et al. (2006) screened a dsRNA library of D. melanogaster S2 cells and identified 23 genes likely to be involved in endocytic pathway and required for cellular uptake of dsRNA by S2 cells. Some of these genes have been known to be directly and/or indirectly involved in endocytosis as they encode proteins of the vesicle mediated transport, conserved oligomeric Golgi complex family, cytoskeleton organization and protein transport.

Results from previous studies suggest that most insects are likely to rely on different mechanisms rather than C. elegans SID-1-dependent dsRNA uptake pathway (Tomoyasu et al., 2008, Zhang et al., 2010), and endocytosis plays an important role in dsRNA uptake in D. melanogaster S2 cells and probably in many insect species such as the desert locust (Wynant et al., 2014). However, the exact mechanism of cellular dsRNA uptake in different insects remains unclear. In this study, we took the advantage of T. castaneum for its robust RNAi to examine: 1) the effect of pharmacological inhibitors of different endocytic pathways on RNAi of a marker gene (TcLgl, lethal giant larvae); 2) the effect of selective inhibitors of clathrin-dependent endocytosis on cellular uptake of Cy3-labeled TcLgl dsRNA in larval midgut; and 3) effect of RNAi targeting each of four key genes in clathrin-dependent endocytosis on RNAi of TcLgl (i.e., a “RNAi of RNAi” strategy). Our studies provided strong evidence that clathrin-dependent endocytosis plays an essential role in cellular uptake of dsRNA in T. castaneum larvae.

Section snippets

Insect culture

The Georgia-1 (GA-1) strain of T. castaneum was reared on whole-wheat flour containing 5% (by weight) of brewers' yeast at 30 °C and 65% RH in growth chamber at Kansas State University (Manhattan, KS, USA).

Subcloning and sequencing of TcChc

Total RNA was isolated from the insects by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and treated with DNase I (Fermentas, Glen Burnie, MD, USA) to remove possible genomic DNA contamination. First-strand cDNA was synthesized from 1.0 μg total RNA by using First Strand cDNA Synthesis

Effect of different endocytosis inhibitors on RNAi

We used TcLgl as a marker gene and four relatively selective pharmacological endocytosis inhibitors (CPZ, MβCD, BafA and CCD) to identify specific endocytic pathways involved in cellular uptake of TcLgl dsRNA (dsTcLgl) in T. castaneum larvae. The functions of Lgl are known to be remarkably conserved in maintenance of cell polarity and regulation of cell proliferation in various organisms. We chose TcLgl as a marker gene in this study due to its broad expression in different developmental stages

Discussion

Since the detailed mechanism of RNAi was first reported in C. elegans about 17 years ago, RNAi has rapidly emerged as a powerful tool to study gene function, regulation and interaction at the cell and whole-organism levels in various organisms (Fire et al., 1998). The current rapid paces of identification of target genes and development of new dsRNA delivery systems will also soon lead to the applications of RNAi-based technologies for human disease therapeutics (Jain et al., 2014) and pest

Acknowledgments

We thank Yoonseong Park (KSU Department of Entomology) and Joel Sanneman (KSU CVM Confocal Microscopy Core Facility) for providing confocal microscopy facilities and technical assistance. The Confocal Microscopy and Microfluorometry Core are supported by KSU-CVM. This research was supported by the Kansas Agricultural Experiment Station and the U.S. Department of Agriculture (USDA/NIFA 2014-67013-21714) to KYZ, and the China Scholarship Council to DX. This manuscript is contribution No. 15-202-J

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