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Series GSE211494 Query DataSets for GSE211494
Status Public on Aug 24, 2022
Title Retrograde Control of Cytosolic Translation Targets Synthesis of Plastid Proteins and Nuclear Responses for High-Light Acclimation.
Organism Arabidopsis thaliana
Experiment type Expression profiling by high throughput sequencing
Summary Canonical retrograde signalling comprises information transmission from organelles to the nucleus and in particular controls gene expression for organellar proteins. The need to re-assess this paradigm was suggested by discrepancies between de novo protein synthesis and transcript abundance in response to excess light. Here we uncover major components of a translation-dependent retrograde signalling pathway that first impacts translation and then transcription. The response realization depends on the kinases Mitogen-activated protein kinase 6 (MPK6) and Sucrose non-fermenting 1-related kinase (SnRK1) subunit, AKIN10. Global ribosome foot-printing revealed differential ribosome association of 951 transcripts within 10 min after transfer from low to high light. Despite predominant translational repression, 15 % of transcripts were increased in translation and enriched for chloroplast-localized photosynthetic proteins. About one third of these transcripts, including Stress associated proteins (SAP) 2 and 3, share regulatory motifs in their 5`-UTR that act as binding sites for glyceraldehyde-3-phosphate dehydrogenase (GAPC) and light responsive RNA binding proteins (RBPs). SAP2 and 3 are both translationally regulated and interact with the calcium sensor Calmodulin-like 49 (CML49), which promotes relocation to the nucleus inducing a translation-dependent nuclear stress response. Thus, translation-dependent retrograde signalling bifurcates to directly regulate a translational circuit of chloroplast proteins and simultaneously initiate a nuclear circuit synchronizing retrograde and anterograde response pathways, serving as a rapid mechanism for functional acclimation of the chloroplast.
Overall design Ribosome protected fragment (RPF)-sequencing and total RNA sequencing was performed on whole Arabidopsis rosettes from plants plants grown under low-light (LL) (8µE) or 10 min of LL to high light (HL) (800µE) treated. 3 g of pulverized plant material was used with 6 mL polysomal extraction buffer (PEB; 0.2 M Tris, pH 9.0, 0.2 M KCl, 25 mM EGTA, 35 mM MgCl, 1 % Brij, 1 % Triton X-100, 1 % Tween 20, 1 % Igepal CA630, 1% sodium deoxycholate (DOC), 1 % polyethylene-10-tridecylether (PTE), 5 mM DTT, 1 mM PMSF, 100 µg/mL cycloheximide, 100 µg/mL chloramphenicol, 100 µg/mL lincomycin). The suspension was passed twice through MiraclothTM and subsequently cleared by centrifugation. Extracts were loaded on a sucrose cushion (0.2 M Tris, pH 9.0, 0.2 M KCl, 0.025 M EGTA, 0.035 M MgCl, 1.75 M sucrose, 5 mM DTT, 50 µg/mL cycloheximide, 50 µg/mL chloramphenicol, 50 µg/mL lincomycin) and centrifuged for 18 h at 100,000 xg in a Beckmann SW71Ti rotor. The sediment was suspended in 100 µL RNase digestion buffer (20 mM Tris-HCl, pH 8.0, 140 mM KCl, 35 mM MgCl2, 50 µg/µL cycloheximide, 50 µg/µL chloramphenicol, 50 µg/µL lincomycin) and used immediately for RNase If digestion. RNase If digest was performed with 2,000 relative absorbance units OD260 of the in RNase digestion buffer suspended polysomes by adding 50 U of RNase If (New England Biolabs) in 250 µL total volume by incubation at room temperature with constant rotation for 1 h. In parallel same amounts of polysomes were treated with 5 µL SUPERase RNase inhibitor (Thermo) and kept on ice as control sample. The digestion was stopped by adding 5 µL SUPERase RNase inhibitor and the RNA precipitated by adding 1 volume of isopropanol, 300 mM NaOAc pH 5.2 and 20 µg glycogen at -80 over-night. The supernatant was discarded after centrifugation at 16,000 xg at 4 °C for 30 min and the sediment was washed twice with 75 % ethanol and resuspended in RNA loading buffer for size exclusion electrophoresis. Total RNA was isolated using Tri Reagent (Sigma-Aldrich) from extracts suspended in polysomal extraction buffer (see sucrose cushion for polysome isolation, above). Briefly, 500 µL of extract was combined with 1 mL of TriReagent, followed twice by extraction of the organic phase with 200 µL chloroform. RNA was precipitated with equal volume 100% isopropanol and incubated overnight at -20°C. The RNA was recovered by centrifugation and washed with 75% ethanol before resuspension in water. RNA quality was assessed using a LabChip GXII (Perkin-Elmer). Total RNA was DNase-treated using TURBO DNase (Invitrogen) following the manufacturer’s protocol. Preparation of total RNAseq libraries was carried out using Illumina TruSeq Stranded Total RNA with Ribo-Zero plant, scaled at half reaction volumes. Libraries of ribosome-protected fragments (RPFs) were prepared from three biological replicates per time-point and sample following RNase If digestion (above) according to Juntawong et al. (2014)
Contributor(s) Moore M, Smith A, Pogson BJ, Dietz K
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Submission date Aug 17, 2022
Last update date Aug 26, 2022
Contact name Marten Moore
Organization name The Australian National University
Department Research School of Biology
Lab Pogson
Street address 134 Linnaeus Way
State/province ACT
ZIP/Postal code 2601
Country Australia
Platforms (1)
GPL19580 Illumina NextSeq 500 (Arabidopsis thaliana)
Samples (12)
GSM6474717 RPF LL 8µE rep 1
GSM6474718 Total RNA LL 8µE rep 1
GSM6474719 RPF HL 800µE rep 1
BioProject PRJNA870499

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GSE211494_RAW.tar 4.9 Mb (http)(custom) TAR (of TXT)
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Raw data are available in SRA
Processed data provided as supplementary file

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