Ribosome profiling in mitochondria from flowers of wildtype Arabidopsis thaliana and DWEORG1 (At3g15590) mutant

As one of the largest protein family in landplants pentatricopeptide repeat (PPR) proteins offer a wide research array. They are mostly involved in the RNA processing steps in Chloroplasts and Mitochondria. Recently it has been shown, that they are not only involved in the RNA processing steps, but are also part of the mitoribosome (rPPRs) as regulators of translation and ribosome stabilizers. This experiment focuses on a non-previously reported rPPR protein and the ribosome profiling experiment was aimed to analyze its impact on mitochondrial translation. Arabidopsis mutants are compared to the wild type using duplicates for the Ribosome Profiling experiment. RNASEQ (determining the transcript abundance) and RIBOSEQ ( for detecting ribosome occupancy on each transcript) of wild type and mutants were conducted to determine the translational efficiency of mitochondrial transcripts in the mutant and wild type. The plants were grown in the Greenhouse under long day conditions. Flowers were harvested simultaneously for all genotypes, snap-frozen in liquid nitrogen and stored at -80°C until use.Total ribosome footprints were prepared from a mix of flower buds and open flowers.For total RNA extraction from flowers Trizol was used. Total RNA were treated with Dnase and depleted of rRNAs using the RiboMinusTM Plant Kit for RNA-Seq (invitrogen), following the manufacturer's instructions.The sequencing libraries of ribosomal footprints of two biological repeats per genotype were prepared using the NEXTflex Small RNA-Seq Kit v3 (Bioo Scientific) following the manufacturer's instructions. For sequencing Illumina NextSeq 500 75 bp single reads were used. The reads were than mapped to the Col-0 mitochondrial genome of Arabidopsis thaliana acccession JF729201.