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Elp3) Is Required for Zebrafish Trunk Development International Journal of Molecular Sciences Article Elongator Subunit 3 (Elp3) Is Required for Zebrafish Trunk Development Diego Rojas-Benítez * and Miguel L. Allende FONDAP Center for Genome Regulation (CGR), Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Ñuñoa, Santiago 7800003, Chile; [email protected] * Correspondence: [email protected]; Tel.: +56-2-2978-7351 Received: 7 October 2019; Accepted: 6 November 2019; Published: 31 January 2020 Abstract: Transfer RNAs (tRNAs) are the most post-transcriptionally modified RNA species. Some of these modifications, especially the ones located in the anti-codon loop, are required for decoding capabilities of tRNAs. Such is the case for 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U), synthetized by the Elongator complex. Mutants for its sub-units display pleiotropic phenotypes. In this paper, we analyze the role of elp3 (Elongator catalytic sub-unit) in zebrafish development. We found that it is required for trunk development; elp3 knock-down animals presented diminished levels of mcm5s2U and sonic hedgehog (Shh) signaling activity. Activation of this pathway was sufficient to revert the phenotype caused by elp3 knockdown, indicating a functional relationship between Elongator and Shh through a yet unknown molecular mechanism. Keywords: elongator complex; tRNA modification; zebrafish 1. Introduction Most classes of RNA are post-transcriptionally modified in all cells. But transfer RNAs (tRNAs) are the most heavily modified: each one is decorated by a subset of over 90 known chemical modifications [1]. On average, ~14% of bases in a tRNA are post-transcriptionally modified [2]. Depending on their position, they play structural roles or are required for stabilizing cognate base pairing or expediting wobble base pairing, thus increasing decoding capabilities. As tRNAs, together with ribosomes, are the decoders of information held in mRNAs by recognition of consecutive triplets (codons) to add specific amino acids into the nascent polypeptide chains, modifications that affect the decoding capabilities of tRNAs will be of huge importance for translation. For instance, these types of modifications prevent frame-shifts, ensuring correct translation [3]. Mutations of tRNA-modifying enzymes display pleiotropic phenotypes in unicellular organisms and metazoans [3,4]. Many tRNA modifications are evolutionarily conserved, which has promoted the use of simple model organisms such as bacteria and yeast to understand the roles of modifications in cell physiology. Despite many years of research, there still remains much to elucidate in this field and the use of more complex models, including vertebrates, would advance our understanding of their roles. We are interested in examining a well conserved modification, 5-methoxy-carbonyl-methyl- 5 2 2-thio-uridine (mcm s U), which occurs at uridine in the wobble position (U34, Figure1A) [ 5]. This modification improves decoding of A-ending codons GAA, CAA and AAA and for its synthesis, the six-unit Elongator Complex (Elp1-6) is fundamental as it adds carboxy-methyl (cm5) moieties to uridine bases at position 34. Elp3 is the catalytic subunit of the Elongator complex [5]. In humans, Elongator complex mutations have been related to neurological disorders [6,7]. Considering the scarce research of tRNA modifications in vertebrates, we aimed to use zebrafish and a morpholino-based approach to shed light onto the potential requirements of this modification during development. We found that morphant zebrafish showed diminished levels of the mcm5s2U compared to controls and Int. J. Mol. Sci. 2020, 21, 925; doi:10.3390/ijms21030925 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 925 2 of 11 showed defects in trunk development and muscle organization. The resulting phenotype correlated with diminished sonic hedgehog signaling (Shh) activity, consistently, activation of this signaling pathwayInt. was J. Mol. su ffiSci.cient 2019, 20 to, x FOR revert PEER the REVIEW macroscopic phenotype caused by Elp3 knockdown.3 of 12 Figure 1. FigureElp3 morphants 1. Elp3 morphants present present a ventrally-curved a ventrally-curved tail. tail. (A ()A Schematic) Schematic representationrepresentation of ofthe the anti- anti-codon loop of acodon tRNA, loop indicating of a tRNA, position indicating 34 (redposition arrow) 34 (red where arrow) 5-methoxycarbonylmethyl-2-thiouridine where 5-methoxycarbonylmethyl-2- (mcm5s2U)thiouridine is located. (mcm Anti-codon5s2U) is located. is depicted Anti-codon in green.is depicted (B) in In green. situ hybridization(B) In situ hybridization of Elp3 mRNAof Elp3 in 4 cell mRNA in 4 cell (upper) and 27 hpf (lower) embryos. Scale bar 0.7 mm. (C) Saccharomyces cerevisiae (upper) and 27 hpf (lower) embryos. Scale bar 0.7 mm. (C) Saccharomyces cerevisiae mutants for ELP3 mutants for ELP3 (ΔELP3) were transformed with an expression plasmid that contained zebrafish (DELP3) wereElp3 (zElp3). transformed BY4741 withwas used an expressionas control. Image plasmid representative that contained of three zebrafishindependent Elp3 experiments. (zElp3). BY4741 was used( asD)control. Western Imageblot detecting representative Elp3 in 48 hpf of three samples independent from controlexperiments. and morphant embryos (D) Western (MO-Elp3); blot detecting Elp3 in 48β-actin hpf samples was used from as loading control control. and morphantSamples from embryos embryos (MO-Elp3);injected with βtranslation--actin was (ATG) used and as loading control. Samplessplicing-blocking from embryos (SP). (E) injectedLiquid chromatography-mass with translation- (ATG)spectrometry and splicing-blocking (LC-MS) analysis of (SP). tRNA (E ) Liquid modification. Peak area values for mcm5s2U are shown normalized to the of purified tRNA for each chromatography-mass spectrometry (LC-MS) analysis of tRNA modification. Peak area values for sample (n = 3, * p < 0.05). (F) qPCR for UPR targets BiP, Chop and Atf4. Fold change in mRNA levels 5 2 mcm s Ucomparing are shown morphant normalized embryos to to thecontrols; of purified t-test was tRNA performed for eachfor statistical sample analysis. (n = 3, *** * pp < <0.005).0.05). (G) ( F) qPCR for UPR targetsRepresentative BiP, Chop images and of control, Atf4. morphants Fold change and MO in + mRNA ELP3 mRNA levels injected comparing embryos by morphant 48 hpf (images embryos to controls;representative t-test was performed of at least six forindependent statistical experime analysis.nts, in which *** p <at least0.005). 100 embryos (G) Representative were analyzed). images of control, morphants and MO + ELP3 mRNA injected embryos by 48 hpf (images representative of at 2.2. Somite and Muscle Fiber Morphology is Affected Upon Elp3 Knockdown least six independent experiments, in which at least 100 embryos were analyzed). Morphant embryos were consistently misshapen, displaying a downward curve of the body. As 2. Resultsthe larval body is mostly composed of developing muscle in the somites, we analyzed these structures by immunohistochemistry. To analyze somite shape, we detected β-dystroglycan, a protein present 2.1. Knockdownin extracellular of Elp3 matrix Caused at Diminishedmyosepta as well tRNA as basement Modification membrane and Generated between adjacent Curved myofibers Larvae [12] that upon detection reveals the horizontal myoseptum and the borders of the somites. In morphant Elongatorfish, the complex somites present is conserved a distortion through of their eukaryotes typical chevron [8,9] shape, and Elp3 they is are its rounded catalytic and sub-unit. show a To date, in zebrafishdifferent there isangle one compared ortholog to annotated control fish [10 (Figure]. It is maternally2A,B). Morphants deposited also show and bydisruption 27 h post-fertilization of the (hpf), itshorizontal mRNA showed myoseptum a non-spatially (Figure 2A, arrowheads). restricted expressionFurthermore, pattern somite (Figurearea is diminished1B), consistent in with previousmorphants reports [ 10compared,11]. We to controls wanted (Figure to establish 2C). Although if Elp3 somite function shape and is conservedsize are abnormal, between muscle yeast and fibers are correctly differentiated, as the typical striped-pattern of myomesin (Figure 2D) consistently, zebrafish. We plated ELP3 (DELP3) mutant Saccharomyces cerevisiae alongside with control (BY4741), mutants that express either yeast ELP3 (DELP3 + ELP3) or the zebrafish cognate (DELP3 + zElp3). In both cases, rescue of the slow growth phenotype was observed (Figure1C), indicating functional conservation of the Elp3 function. Int. J. Mol. Sci. 2020, 21, 925 3 of 11 For elp3 knockdown in zebrafish, we used two previously validated morpholinos (splice- and translation-blocking) [7]. We corroborated efficient silencing by detecting endogenous Elp3. Diminished levels of Elp3 protein were detected in morphants compared to control siblings (Figure1D). To determine if the diminished Elp3 level impacted tRNA modification, we measured the levels of mcm5s2U in tRNAs from zebrafish using LC-MS (coupled liquid chromatography-mass spectrometry). In morphant animals, we detected a significant reduction in this modification compared to controls (Figure1E), indicating a conserved molecular function for this gene in zebrafish. In morphants, we also consistently observed upregulated levels of the Unfolded Protein Response (UPR)-related genes BiP, Chop and Atf4 (Figure1F). In morphological terms, we observed a consistent phenotype (65 8% of injected larvae; ± n > 100) in morphants at 48 h post-fertilization (hpf),
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