UTurku - IsaR1

Acclimation of oxygenic photosynthesis to iron starvation is controlled by the sRNA IsaR1 in cyanobacteria
  • Organism: Synechocystis sp. PCC 6803
  • Instrument: TSQ Vantage
  • SpikeIn: No
Oxygenic photosynthesis crucially depends on proteins that possess Fe2+ or Fe/S complexes as co-factors or prosthetic groups. Here we show that the regulatory sRNA IsaR1 (Iron-stress activated RNA 1) plays a pivotal role in acclimation to low iron conditions. The IsaR1 regulon consists of more than 15 direct targets, including Fe2+-containing proteins involved in photosynthetic electron transfer, the detoxification of anion radicals, citrate cycle, and in tetrapyrrole biogenesis. IsaR1 is essential to maintain physiological levels of Fe/S cluster biogenesis proteins during iron deprivation. Consequently, IsaR1 affects the acclimation of the photosynthetic apparatus to iron starvation at three levels: (i) directly, via posttranscriptional repression of gene expression and indirectly, (ii) via the suppression of pigment biosynthesis and (iii) Fe/S cluster co-factor. Homologs of IsaR1 are widely conserved throughout the cyanobacterial phylum. We conclude that IsaR1 is a riboregulator of central importance. These findings provide a new perspective for studies of the regulation of iron homeostasis in photosynthetic organisms.
Experiment Description
The SRM assays were performed on a TSQ Vantage QQQ mass spectrometer (Thermo Fischer Scientific) equipped with a nanoelectrospray ionization source. The desalted peptides were separated on a nanoflow HPLC system (EasyNanoLC 1000; Thermo Fisher Scientific). The injected amount of each unfractionated biological triplicate was 150 ng, including the spiked-in iRT peptides (Biognosys). A 60 min non-linear gradient (5-20% B in 35 min; 20–35% B in 50min; B = acetonitrile:water, 98:5) was applied at a 300 nL/ min flow rate. Once the peptides were eluted and ionized, they were analyzed in the QQQ-MS, operated in the SRM mode, as described (Vuorijoki et al., 2016). To maintain high sensitivity of the SRM measurements, scheduled assays with a 5 min retention time window for each peptide was applied, resulting in a cycle time of 2.5 s and a dwell time of >30 ms. The protein targets and the respective SRM assay parameters were selected from the public dataset, available in Panorama Public (https://panoramaweb.org/labkey/Vuorijoki_et_al_2015.url) (Vuorijoki et al., 2016). Altogether 42 proteins were quantified for the analysis of isaR1 deletion strain and 41 proteins for IsaR1 overexpression strain. In the data-analysis, global standard normalization method with two endogenous peptides (YEAQNIEELTAEK and TPLFNIK) of the drug sensory protein A (dspA; sll0698) were used.
Sample Description
The samples were cultivated under photoautotrophic conditions on BG11 medium (Rippka et al., 1979). The IsaR1 deletion strain was cultured in reduced iron concentrations (Shcolnick et al., 2007), by supplementing iron chelator DFB (Sigma-Aldrich) to a final concentration of 100 µM. The selected timepoints for harvesting the cells were 0h,5h,24h,48h and 96h after chelator treatment. For IsaR1_OE and control strains a copper-free BG11 was used and 2 µM CuSO4 was added to induce the copper inducible petE promoter. The samples were collected at 0h, 24h and 96h timepoints after copper-induction. Protein extracts were reduced with 5 mM dithiotreitol (DTT; Sigma) and alkylated with 10 mM iodoacetamide (IAA; Sigma), followed by o/n acetone:ethanol precipitation at -20 °C. The resulting protein pellets were digested o/n in 50 mM NH4HCO3 and 5 % (v/v) acetonitrile (ACN) buffer with two additions of trypsin (Sequence grade Modified, Promega, Madison, WI, USA) with 1:100 (w/w; trypsin:protein) ratio. The samples were then desalted by Solid Phase Extraction (SPE) method using 4 mm/1 ml extraction disk cartridge (Empore C18-SD, 3M).
Created on 10/17/16, 12:42 PM
Clustergrammer Heatmap
dIsaR1_2016-10-17_12-45-32.sky.zip2016-10-17 12:41:474210710732130
IsaR1_OE_2015-10-21_12-57-41.sky.zip2016-10-17 12:41:474110410431218