Wednesday, October 17, 2012

Genome-wide ribosome profiling reveals complex translational regulation in response to oxidative stress

Oxidative stress causes multiple gene expression changes in a cell, which include increased loading of ribosomes onto genes that can modulate gene expression.  Gerashchenko et al. created an oxidative stress condition in yeast (Saccharomyces cerevisiae) using exposure to hydrogen peroxide. Using ribosome profiling in combination with standard mRNA-Seq, quantitative expression can be determined using a simple process to quantify partial transcripts for evaluating expression level changes.  Ribosome profiling uses differential RNA isolation/RNA protection to show not only the changes in the amount of gene-specific RNAs but also the location of the protected transcripts along the length of the mRNA. Ribosomes were isolated and mRNA/ribosome complexes were treated with an RNAse to remove naked RNA in between bound ribosomes.  The existing ribosome-protected RNAs were then extracted to generate small RNA pieces that can be used to make libraries that can be sequenced, quantified and characterized. After extraction, the protected RNAs were tailed with poly-A polymerase, followed by reverse transcription with primers that contained sequencing barcode adapters. The cDNA products (~92 nucleotides) were circularized using CircLigase II ssDNA Ligase, and PCR amplified (after removing linear DNA),  using primers that contained Illumina-based Tru-Seq sequences. The 120-bp PCR products were quantified, and applied to a HiSeq flow cell and sequenced; sequence information was aligned to a S. cerevisiae reference genome, followed by transcript quantification and annotation.  This allowed the determination of the number and types of up-regulated and down-regulated genes as a result of oxidative stress.  Under stress, the ribosomes were loading onto certain open reading frames in locations 5' to the normal translation starts. This ribosomal occupancy included mRNAs that had non-AUG codon translation starts at the beginning of the ORF, and increased the duration of the protein elongation steps. Many different genes started translation along sequences in advance of the AUG start site, and this was determined to be a translation control mechanism in response to stress. Ribosomal footprinting allows analysis of only short RNAs protected by protein post- RNA isolation and these protected RNA oligos can be readily sequenced and quantified to determine the quantity and location of the ribosome loading onto given transcripts to determine the effect of oxidative stress on gene transcription and protein translation.

ResearchBlogging.orgGerashchenko MV et al. (2012). Genome-wide ribosome profiling reveals complex translational regulation in response to oxidative stress. Proceedings of the National Academy of Sciences of the United States of America PMID: 23045643

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