The C. elegans mRNA decapping enzyme shapes morphology of cilia

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Highlights

  • Ciliary shape mutant was identified by a forward genetic screening.

  • RNA decapping enzyme (DCAP-2) is involved in ciliary shape formation.

  • DCAP-2 acts in neurons to shape cilia.

Abstract

Cilia and flagella are evolutionarily conserved organelles that protrude from cell surfaces. Most cilia and flagella are single rod-shaped but some cilia show a variety of shapes. For example, human airway epithelial cells are multiciliated, flagella of crayfish spermatozoon are star-like shaped, and fruit fly spermatozoon extends long flagella. In Caenorhabditis elegans, cilia display morphological diversity of shapes (single, dual rod-type and wing-like and highly-branched shapes). Here we show that DCAP-1 and DCAP-2, which are the homologues of mammalian DCP1 and DCP2 mRNA decapping enzymes, respectively, are involved in formation of dual rod-type and wing-like shaped cilia in C. elegans. mRNA decapping enzyme catalyzes hydrolysis of 5′ cap structure of mRNA, which leads to degradation of mRNA. Rescue experiments showed that DCAP-2 acts not in glial cells surrounding cilia but in neurons. This is the first evidence to demonstrate that mRNA decapping is involved in ciliary shape formation.

Introduction

Cilia and flagella are evolutionarily conserved organelles that protrude from cell surfaces. Motile cilia beat and generate fluid flows, whereas non-motile cilia are thought to act as receptors for external stimuli [1]. Cilia are observed in various cell types such as neuronal cells and cells in the renal tubules, brain ventricles and airway epithelium [2]. Inside cilia, doublet microtubules are arranged in a nine-fold symmetry [1]. Along these microtubules, an intraflagellar transport (IFT) complex carries ciliary proteins to the distal tip of the cilia and returns them to the cell body [1]. Disruption of the ciliary structure or IFT often reduces ciliary length and causes ciliopathies in humans [3].

In Caenorhabditis elegans, cilia are only observed in the free endings of amphid and phasmid sensory neurons [4]. C. elegans cilia are non-motile and act as receptors for chemical compounds and temperature [4]. Cilia are shaped like a rod in most species, while C. elegans cilia display a variety of shapes including single rod-type, dual rod-type, wing-like and highly branched shapes [5]. The structure and formation mechanisms of the single rod-type cilium have been extensively studied. On the other hand, little is known about formation mechanisms of the other shape types of cilia and flagella (such as those found in the long sperm of the fruit fly and the star-like shaped sperm of crayfish) [6]. In C. elegans, amphid and phasmid sensory neurons are exposed to outside and take up lipophilic dye through cilia [4]. Shortened cilia are not exposed to the external environment, resulting in the Dyf (dye-filling defective) phenotype. Dyf mutants have been screened and many ciliary genes were identified in C. elegans [4], [7]. Glial cells surrounding cilia are also essential for ciliary shape formation in C. elegans [8], [9]. Regulation of kinesin-II motor (KAP-1) expression by forkhead transcription factor FKH-2 is important for building wing-like shaped cilia of AWB olfactory neurons [10]. Structures of AWB cilia are remodelled by sensory signalling [11]. Expression level of OIG-8, a transmembrane protein with a single immunoglobulin domain, controls branching patterns of cilia [12]. Still, little is known about the intrinsic mechanisms that regulate the expression of the variety of C. elegans ciliary shapes.

mRNA 5′ ends are capped with 7-methylguanosine (the 5′ cap) in eukaryotes [13]. The 5′ cap recruits translation initiation factors and contributes to mRNA stability [13]. The 5′ cap is hydrolysed by the DCP1–DCP2 decapping protein complex for mRNA degradation in yeasts and mammals [14], [15]. In C. elegans, the dcap-2 gene encodes the homologue of the mammalian DCP2 protein that is a member of the NUDIX (Nucleoside Diphosphate linked to some moiety X) hydrolase family [16], [17], [18]. Although mRNA decapping is proposed to serve a housekeeping function in mRNA turn over, roles for mRNA decapping in regulation of developmental process are rarely reported.

By direct observation of C. elegans cilia, we isolated a mutant that displays ciliary shape defects. Genetic mapping and rescue experiments showed that loss of dcap-2 causes the defect in ciliary shapes in the mutant. Tissue-specific rescue experiments suggest that the DCAP-2 protein acts not in glial cells surrounding cilia but in neurons. These observations suggest that mRNA decapping contributes to ciliary shape formation in C. elegans.

Section snippets

Nematode strains

C. elegans strains were cultivated at 20 °C under standard conditions [19], except that the E. coli strain NA22 was used as a food source. Strains used were N2, CB4856, CX4376 kyIs170[Psrh-220::gfp, lin-15(+)], PY3470 oyIs51[Psrh-142::gfp, lin-15(+)], CX3553 kyIs104[str-1::gfp, lin-15(+)], CX3695 kyIs140[str-2::GFP, lin-15(+)], OH3192 ntIs1[gcy-5::gfp, lin-15(+)], dcap-2(ok2023), dcap-1(ok2139) and dcap-1(tm3163).

Screening of mutants that displayed defects in ADL ciliary shape

CX4376 young adults (P generation) were soaked in M9 buffer containing 25 mM EMS

A genetic screen identified var1 mutants that display shape defects in dual- and wing-type cilia

In C. elegans, only sensory neurons possess cilia. Each sensory neuron is named after its cilium shape, such as ASE, ADF, ADL, AWB and AWC. For instance, the ASE cilium shape is a single rod-type, ADF and ADL extend dual rod-like cilia and AWB and AWC cilia spread like a wing (Fig. 1A). To elucidate the molecular mechanisms that generate the variety of shapes of cilia, we sought to obtain mutants in which the shape of ADL dual cilia was affected. We screened the mutants by using CX4376 strain

Discussion

The structural proteins within cilia are essential for cilia shape formation. Moreover, components of IFT are required for cilia shape maintenance. Disruption of these components often causes a stunted cilia phenotype. In C. elegans, stunted cilia are not exposed to the external environment, leading to show a Dyf phenotype. Although the shapes of ADL and AWB cilia are affected in dcap-2 mutants, these cilia are long enough to allow uptake of the dye. This result suggests that the components

Conflict of interest

The authors have no conflicts of interest to declare.

Acknowledgements

We thank Dr. Cornelia I. Bargmann for CX4376 strain; Dr. Piali Sengupta for PY3470 strain; Dr. Shohei Mitani at National Bioresource Project for strains; CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440) for strains; members of Izumi Lab for discussions and comments. This work was supported in part by a grant from Research Institute for Integrated Science from Kanagawa University, the Kanagawa University Grant for Joint Research and JSPS KAKENHI Grant Number

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