GSE211494
Retrograde Control of Cytosolic Translation Targets Synthesis of Plastid Proteins and Nuclear Responses for High-Light Acclimation.
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)