MicroRNA Regulatory Mechanisms Play Different Roles in

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MicroRNA regulatory mechanisms play different roles in Arabidopsis Rodrigo Siqueira Reis, Gene Hart-Smith, Andrew L Eamens, Marc R. Wilkins, and Peter M Waterhouse J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 21 Sep 2015 Downloaded from http://pubs.acs.org on September 24, 2015

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Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Proteome Research

TITLE: MicroRNA regulatory mechanisms play different roles in Arabidopsis

RUNNING TITLE: Proteome analysis of drb1 and drb2 mutants

Rodrigo S. Reis1,2,#,*, Gene Hart-Smith3, Andrew L. Eamens4, Marc R. Wilkins3,† and Peter M. Waterhouse1,5,*,† 1

School of Biological Sciences, University of Sydney, Macleay Building A12, Sydney, NSW 2006, Australia.

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Faculty of Agriculture and Environment, University of Sydney, Eveleigh, NSW 2015, Australia.

3

Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.

4

School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.

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Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, QLD 4001, Australia.

#

Current address: Department of Plant Molecular Biology, University of Lausanne, Lausanne, CH-1015, Switzerland.

†

Equal contribution as senior authors.

*

Corresponding authors: RSR ([email protected]; +41 2 1692 4235) and PMW ([email protected]; +61 7 3138 7793)

ACS Paragon Plus Environment

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ABSTRACT

Plant microRNAs (miRNAs) operate by guiding the cleavage or translational inhibition of mRNA targets. They act as key gene regulators for development and environmental adaptation, and Dicer-partnering proteins DRB1 and DRB2 govern which form of regulation plays the dominant role. Mutation of Drb1 impairs transcript cleavage whereas mutation of Drb2 ablates translational inhibition. Regulation of gene expression by miRNA-guided cleavage has been extensively studied but there is much less information about genes regulated through miRNA-mediated translation inhibition. Here we compared the proteomes of drb1 and drb2 mutants to gain insight into the indirect effect of the different miRNA regulatory mechanisms in Arabidopsis thaliana. Our results show that miRNAs operating through transcript cleavage regulate a broad spectrum of processes, including catabolism and anabolism, and this was particularly obvious in the fatty acid degradation pathway. Enzymes catalyzing each step of this pathway were upregulated in drb1. In contrast, DRB2-associated translational inhibition appears to be less ubiquitous and specifically aimed toward responses against abiotic or biotic stimuli.

KEYWORDS: miRNA, DRB2, DRB1, translation inhibition, Arabidopsis


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INTRODUCTION In plants and animals, miRNAs play a central role in gene expression regulation, including during development and in responses to environmental stresses 1–3. miRNAs are ~21 nucleotides in length and are produced by dicer endonucleases. In plants, DICER-LIKE1 (DCL1), assisted by a dsRNA-BINDING (DRB) protein, excise miRNAs from precursor transcripts called primary miRNAs. Once produced, a miRNA can guide an argonaute (AGO) protein to cleave or inhibit translation of highly complementary transcript sequences4. The selection of between the two mechanisms is defined by DCL1 partnering proteins, DRB1 and DRB25. DRB1 guides miRNAs to cleave their targets, whereas DRB2 represses the expression of DRB1 and promotes miRNA-guided translational inhibition of targets. Although these distinct mechanisms could play different biological roles, such differences have been largely unexplored. Many of the target genes of miRNAs, operating through cleavage, have been determined and the biological relevance of the interactions clearly established. Several important miRNAs are known to regulate their targets via translation inhibition6–9, but the implications of this control are less well known. Only two examples of miRNA-guided translational inhibition have been characterized in detail, the regulation of APETALA2 by miR172 for floral organ identity, and the control of superoxide dismutase genes for various stress responses by miR3989–11. Many of the genes required for miRNA-guided translation inhibition have been recently identified. They include KATANIN1 (KTN1)6, VARICOSE (VCS)6, ALTERED MERISTEM PROGRAM1 (AMP1)7, 'SHUTTLE' IN CHINESE (SUO)8 and DRB25. However, study of these genes has yet to uncover biologically significant processes directly influenced by translation inhibition. As DRB1 and DRB2 primarily act in the


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miRNA pathway5,12–14, and their mutants might be a functionally reciprocal pair, they represent useful resources for the comparison of miRNA-guided transcript cleavage and translational inhibition. In addition, other Arabidopsis DRBs (i.e. DRB3-5) are unlikely to play a role in the miRNA pathway5,13,15, and DRB1 and DRB2 are not expected to overlap expression as DRB2 represses DRB1 transcription5. Here we compared protein expression profiles in plants null mutants for DRB1 (drb1) to null mutants for DRB2 (drb2), relative to wild-type plants. This confirmed that cleavage and translation inhibition by miRNAs play different roles in plants, and identified many of the targeted genes and pathways. Our results show that cleavagedirecting miRNAs are crucial in maintain homeostasis while translation inhibiting miRNAs exert most of their regulation in response to stress conditions.

MATERIAL AND METHODS Plant lines and growth conditions The drb1 and drb2 T-DNA knockout insertions have been described previously15,13. Plant lines were cultivated under standard growth conditions of 16 hours (h) light/8 h dark at a constant temperature of 24°C. Wild-type and drb mutants were grown on a modified Murashige and Skoog medium containing half nitrogen concentration (0.825 g/L NH4NO3 and 0.95 g/L KNO3) supplemented with 0.4512 g/L KCl to compensate for potassium reduction. Metabolic nitrogen source with

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N labeling was achieved by replacing the

NH415NO3 and K15NO3 (Cambridge Isotope Laboratories Inc.;

>98% enriched in 15N).

Sample preparation


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Shoot apecies were sampled from 4-week-old wild-type Arabidopsis plant and drb mutants, and were mixed at an approximate 1:5 (w/w) ratio, as previously described16. Mutant label swap design was used, and each mutant was analysed as: i) a mixture of unlabeled wild-type with

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N-labeled mutant, and; ii)

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N-labeled wild-type mixed

with unlabeled mutant. Extracted proteins were separated by 1D SDS-PAGE, stained with colloidal Coomassie G-250 and gel lanes were cut into 29 pieces from low to high protein mass. Each polyacrylamide gel slice was destained, reduced and alkylated following the procedure described by Shevchenko et al.17. Per gel slice, 40 ng of trypsin (Stratagene, #204310) in 120 µL of 0.1 M NH4HCO3 was used for protein digestion via incubation at 37°C for 16 h. The resulting solutions were transferred to new microfuge tubes and gel slices treated with the following solutions sequentially for 30 minutes (min) per treatment: i) 80 µL 0.1% (v/v) formic acid / 67% (v/v) acetonitrile, followed by; 80 µL 100% acetonitrile. Pooled digest and peptide extraction solutions were dried (Savant SPD1010, Thermofisher Scientific) before resuspension in 20 µL 0.1% (v/v) formic acid.

Mass spectrometry Proteolytic peptide samples were separated by nano-LC using an UltiMate 3000 HPLC and autosampler system (Dionex, Amsterdam, Netherlands), and ionized using positive ion mode electrospray following experimental procedures described previously18. MS and MS/MS were performed using an LTQ Orbitrap Velos Pro (Thermo Electron, Bremen, Germany) hybrid linear ion trap and Orbitrap mass spectrometer. Survey scans m/z 350–2000 were acquired in the Orbitrap (resolution = 30,000 at m/z 400, with an initial expression target value of 1,000,000 ions in the linear ion trap; lock mass applied to polycyclodimethylsiloxane background ions of


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exact m/z 445.1200 and 429.0887). The instrument was set to operate in datadependent acquisition (DDA) mode, and up to the 10 most abundant ions (>5,000 counts) with charge states of >+2 were sequentially isolated and fragmented via collision-induced dissociation (CID) with an activation q = 0.25, an activation time of 30 ms, normalized collision energy of 30% and at a target value of 10,000 ions. Dynamic exclusion was enabled (exclusion duration = 45 s), and fragment ions were mass analyzed in the linear ion trap.

Sequence database searches and protein quantification Peak lists derived from LC-MS/MS were submitted to the database search program Mascot (version 2.3, Matrix Science) via Proteome Discoverer (version 1.3, Thermo Scientific). Separate searches were conducted for unlabeled and fully

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N-labeled

peptides. For unlabeled peptides, the following search parameters were employed; i) instrument type was default; ii) precursor ion and peptide fragment mass tolerances were ±5 ppm and ±0.4 Da respectively; iii) variable modifications included were acrylamide (C), carbamidomethyl (C) and oxidation (M); iv) enzyme specificity was trypsin with up to two missed cleavages, and; v) all taxonomies in the Swiss-Prot database (July 2013 release, 540,732 sequence entries) were searched. For 15N-labeled peptides, search parameters were identical to those used for unlabeled peptides, with the following fixed modifications included: 15

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N(1) (A,C,D,E,F,G,I,L,M,P,S,T,V,Y),

N(2) (K,N,Q,W), 15N(3) (H) and 15N(4) (R).

Proteome Discoverer was used to quantify peak intensities for unlabeled and

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N-

labeled peptide pairs. This was performed separately for the search outputs obtained from unlabeled and fully 15N-labeled peptide sequence database searches. These data were then combined within Proteome Discover to produce consensus quantitative


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datasets. Only peptides deemed to be statistically significant (p

MicroRNA Regulatory Mechanisms Play Different Roles in

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