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. 2017 Jul 10;68(15):4205-4217.
doi: 10.1093/jxb/erx222.

The Meiotic Regulator JASON Utilizes Alternative Translation Initiation Sites to Produce Differentially Localized Forms

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Free PMC article

The Meiotic Regulator JASON Utilizes Alternative Translation Initiation Sites to Produce Differentially Localized Forms

Simon Cabout et al. J Exp Bot. .
Free PMC article

Abstract

The JASON (JAS) protein plays an important role in maintaining an organelle band across the equator of male meiotic cells during the second division, with its loss leading to unreduced pollen in Arabidopsis. In roots cells, JAS localizes to the Golgi, tonoplast and plasma membrane. Here we explore the mechanism underlying the localization of JAS. Overall, our data show that leaky ribosom scanning and alternative translation initiation sites (TISs) likely leads to the formation of two forms of JAS: a long version with an N-terminal Golgi localization signal and a short version with a different N-terminal signal targeting the protein to the plasma membrane. The ratio of the long and short forms of JAS is developmentally regulated, with both being produced in roots but the short form being predominant and functional during meiosis. This regulation of TISs in meiocytes ensures that the short version of JAS is formed during meiosis to ensure separation of chromosome groups and the production of reduced pollen. We hypothesize that increased occurrence of unreduced pollen under stress conditions may be a consequence of altered usage of JAS TISs during stress.

Keywords: Alternative translation initiation; Golgi; JASON; leaky ribosome scanning; meiosis; plasma membrane; subcellular localization; unreduced gametes.

Figures

Fig. 1.
Fig. 1.
Schematic diagram of the constructs used to investigate JAS localization. (A) The JASg–GFP construct. The genomic region of JAS from 943 bp upstream of the predicted translation initiation ATG (+1;TAIR10) to immediately before the stop codon (2685 bp downstream of the ATG) was cloned upstream of GFP (green) between the att Gateway cloning sites of the PMDC107 vector (Curtis and Grossniklaus, 2003). The promoter region is indicated by a thin black bar, exons by rectangular boxes and introns by thin black lines. The predicted transcription start site (TSS; TAIR10) is indicated by an arrow. The region encoding the N-terminus is shaded light blue with the remainder of the coding sequence in dark blue. (B) Enlargement of the region encoding the N-terminus. The position of ATGs is marked. The AA sequence of the encoded protein is indicated below with the 28 AA N-terminal region underlined, methionine residues bold and basic AAs blue. (C) Site-directed mutagenesis was used to create constructs encoding long or short version of JAS or to alter the sequence before the first ATG. Changes to the nucleotide sequence and the encoded peptide are highlighted in red.
Fig. 2.
Fig. 2.
Subcellular localization of different forms of JAS–GFP. (A–C) CLSM images showing GFP fluorescence in whole root tips for JASg–GFP (A), longJASg–GFP (B) and shortJASg–GFP (C). (D–L) CLSM images showing colocalization of JASg–GFP (D–F), longJASg–GFP (G–I) and shortJASg–GFP (J–L) with fluorescent markers for the Golgi (D, G, J), tonoplast (E, H, K), and plasma membrane (F, I, L). The top panels show GFP fluorescence (green), the middle panels show marker fluorescence (red), and in the bottom panels colocalization can be visually assessed by the yellow color in merged images with examples highlighted with an arrowhead. Differences in marker morphology reflect differences in cell type and age due to the selection of cells being based on having both fluorescence signals that varied in location between roots. Scale bars: 10 μm (A–C); 5 μm (D–L). (M–O) Quantification of colocalization. For each combination of JAS–GFP and marker the mean (±SD) Manders’ overlap coefficient 1 value (M1) from 20 images is shown along with the mean (±SD) for the same red image with the green image randomized (M1R). **M1 value is significantly higher than the M1R value (t-test, P<0.001). Similar results were observed for two or three individual transgenic lines.
Fig. 3.
Fig. 3.
Subcellular localization of different versions of JAS-LIKE–GFP and JAS–GFP with a mutated N-terminal region. CLSM images showing colocalization of JAS-LIKE GFP (A–C), NT-JAS-LIKE–GFP (D–F) and mutlongJASg–GFP (G–I) with fluorescent markers for the Golgi (A, D, G), tonoplast (B, E, H), and plasma membrane (C, F, I) in root cells. The top panels show GFP fluorescence (green), the middle panels show marker fluorescence (red), and in the bottom panels colocalization can be visually assessed by a yellow color in merged images with examples highlighted with an arrowhead. Differences in marker morphology reflect differences in cell type and age due to the selection of cells being based on having both fluorescence signals that varied in location between roots. Scale bars: 5 μm. (J–L) Quantification of colocalization. For each combination of JAS–GFP and marker the mean (±SD) Manders’ overlap coefficient 1 value (M1) from 20 images is shown along with the mean (±SD) for the same red image with the green image randomized (M1R). **M1 value is significantly higher than the M1R value (t-test, P<0.001). Similar results were observed for two or three individual transgenic lines.
Fig. 4.
Fig. 4.
The N-terminal region of JAS-RELATED (JR) proteins in eudicots. (A) Alignment of the N-terminal region of JR proteins from throughout the eudicots. A conserved region following the M at position 29 in Arabidopsis JAS is shaded. All species have a least one long JR protein with an N-terminal extension. Basic residues in these N-terminal extensions are highlighted in red and predicted α-helices underlined. Species are as follows: Ath, Arabidopsis thaliana; Aly, Arabidopsis lyrata; Cru, Capsella rubella; Bra, Brassica rapa; Cpa, Carica papaya; Tca, Theobroma cacao; Ccl, Citrus clementine; Ptr, Populus trichocarpa; Mes, Manihot esculenta; Mtr, Medicago truncatula; Gma, Glycine max; Csa, Cucumis sativus; Ppe, Prunus persica; Vvi, Vitis vinifera; Sly, Solanum lycopersicum; Aco, Aquilegia coerulea. Gene identifiers are in Supplementary Table S2. (B, C) CLSM images of Arabidopsis root cells showing colocalization of longMtrJR1c–GFP (B) and shortMtrJR1c–GFP (C) with a fluorescent marker for the Golgi. The top panels show GFP fluorescence (green), the middle panels show Golgi marker fluorescence (red), and in the bottom panels colocalization can be visually assessed by the yellow color in merged images with an example highlighted with an arrowhead. Scale bars: 5 μm. (D, E) Quantification of colocalization. For each combination of GFP and Golgi marker the mean (±SD) Manders’ overlap coefficient 1 (M1) value from 15 images is shown along with the mean (±SD) for the same red image with the green image randomized (M1R). **M1 value is significantly higher than the M1R value (t-test, P<0.001). Similar results were observed for two individual transgenic lines.
Fig. 5.
Fig. 5.
Leaky ribosome scanning leads to the multiple forms of JAS. (A) The nucleotides surrounding the AUG at position +1 and +85 of the JAS mRNA. The Kozak sequence for strong ribosome recognition is shown on top in green with the most important nucleotides underlined. The sequence surrounding the AUGs at +1 (AUG+1) and +85 (AUG+85) is shown below with nucleotides identical to the Kozak sequence highlighted in green. (B) Transient luciferase assay using the CaMV35S promoter and the last 32 bp of the JAS 5′-UTR driving expression of the 5′-end of JAS fused to the luciferase gene lacking an ATG. Luciferase activity was measured 3–4 d after infiltration into N. benthamiana leaves and values were normalized to luciferase without an ATG. Using the unmodified (unmod) version of the 5′-end of JAS, translation could start from the ATG at position +1 or +85. Translation initiation was limited to the ATG at position +1 (ATG+1) or position +85 (ATG+85) by mutating other ATGs. Bars show the mean (±SE) of three biological replicates each with three leaves. (C–E) Images showing colocalization of CTTCJASg–GFP with fluorescent markers for the Golgi (C), tonoplast (D), and plasma membrane (E). The top panels show GFP fluorescence (green), the middle panels show marker fluorescence (red), and in the bottom panels colocalization can be visually assessed by a yellow color in merged images with examples highlighted with an arrowhead. Scale bars: 5 μm. (F) Quantification of colocalization. For each combination of GFP and marker the mean (±SD) Manders’ overlap coefficient 1 value (M1) value from 20 images is shown along with the mean (±SD) for the same red image with the green image randomized (M1R). **M1 value is significantly higher than the M1R value (t-test, P<0.001). Similar results were observed for two individual transgenic lines.
Fig. 6.
Fig. 6.
Location of long and short versions of JAS in meiotic cells. (A-C) CLSM images of longJASc–GFP (A), shortJASc–GFP (B) and control meiocytes without GFP (C) during the second meiotic division in Arabidopsis male meiocytes. Top panel shows GFP fluorescence (green), the middle panel shows DAPI staining of the chromosomes and organelle band and the bottom panel shows the merged images. Arrowheads show where longJASc–GFP is not overlapping with the organelle band. Scale bars: 10 μm.

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