Project description:Post-transcriptional gene regulation plays a significant role in the response to oxygen deprivation. Here, we utilized advances in next-generation sequencing technology to examine changes in transcriptional control, mRNA loading on to polysome, and regulation of ribosome activity during mRNA translation in 7-day-old Arabidopsis seedlings subjected to 2 hour hypoxia treatment. 14 samples, 2 conditions (2 hr hypoxia and 2 hr normoxia), 2 bioreplicates of 3 RNA pools (total mRNA, immunopurified (TRAP) polysomal mRNA, ribosome footprints), 1 bioreplicate of 1 RNA pool (immunopurified (TRAP)-ribosome footprints).
Project description:We performed transcriptome and ribosome immunoprecipitation studies of transgenic Arabidopsis (Arabidopsis thaliana) Columbia seeds expressing a His6FLAG-tagged version of the ribosomal large subunit protein L18B (35S:HF-RPL18B)
Project description:Proteogenomics is an emerging research field yet lacking a standard method of analysis. In this article, we demonstrate the strength of proteogenomic analysis specific for N-terminal data that aims at the discovery of novel translational start sites. In summary, unidentified spectra were matched to a specific N-terminal peptide library encompassing all theoretical protein N-termini encoded in the genome. Gene prediction suggested 81 protein-coding models, of which several alternative proteoforms with unannotated protein starts. Next to the proteomic data, complementary ribosome footprinting data was generated from Arabidopsis thaliana cell cultures. Translation initiation site mapping by the ribosome footprinting data provided orthogonal evidence for 14 novel peptides identified by our proteogenomics pipeline.