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: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.
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:We previously reported that ribosome stalling at AUG-stop sequences in the 5'-UTR plays a critical role in regulating the expression of Arabidopsis thaliana NIP5;1, which encodes a boron uptake transporter, in response to boron conditions in media. Here, we conducted ribosome profiling analysis to reveal the genome-wide regulation of translation in response to boron conditions in A. thaliana. We identified 460 translationally regulated genes. Transcripts with reduced translation efficiency were rich in upstream open reading frames (uORFs), highlighting the importance of uORF-mediated translational regulation. We found that 148 uORF instances had greater ribosome density under high boron conditions. Moreover, translationally downregulated transcripts were rich in minimum uORFs (AUG-stops), suggesting that AUG-stops play a global role in the boron response. Boron increased the ribosome occupancy of stop codons, indicating that this element is involved in global translational termination processes.