Project description:Ribosome profiling has emerged as a powerful method to assess global gene translation, but methodological and analytical challenges often lead to inconsistencies across labs and model organisms. A critical issue in ribosome profiling is nuclease treatment of ribosome-mRNA complexes, as it is important to ensure both stability of ribosomal particles and complete conversion of polysomes to monosomes. We performed comparative ribosome profiling in yeast and mice with various ribonucleases including I, A, S7 and T1, characterized their cutting preferences, trinucleotide periodicity patterns, and coverage similarities across coding sequences, and showed that they yield comparable estimations of gene expression when ribosome integrity is not compromised. However, ribosome coverage patterns of individual transcripts had little in common between the ribonucleases. We further examined their potency at converting polysomes to monosomes across other commonly used model organisms, including bacteria, nematodes and fruit flies. In some cases, ribonuclease treatment completely degraded ribosome populations. Ribonuclease T1 was the only enzyme that preserved ribosomal integrity while thoroughly converting polysomes to monosomes in all examined species. This study provides a guide for ribonuclease selection in ribosome profiling experiments across most common model systems
Project description:Ribosome pauses are associated with diverse co-translational events and determine the fate of mRNAs and proteins. Thus the identification of the precise pause sites across transcriptome is a key, however, the landscape in bacterial has remained ambiguous. Here, we harnessed the multiple ribosome profiling strategies (standard, high-salt-wash, and disome) to survey the robust ribosome pause sites in E. coli. The found pause sites showed the correspondence with biochemical validation by integrated nascent chain profiling (iNP), which detects polypeptidyl-tRNA, an elongation intermediate. Among the list, ribosome pause at Asn586 of ycbZ was ensured by biochemical reporter assay, tRNA-seq, and cryo-electron microscopy. Our results provide a useful resource of ribosome stalling sites in bacteria.
Project description:We performed ribosome profiling (RIBO-seq) and transcriptome profiling (RNA-seq) to monitor RNAs associated with ribosome in the MCF-7 cell model Keywords: ribosome profiling, translation, MCF-7
