Methodology for Ribosome Profiling of Key Stages of the Caulobacter crescentus Cell Cycle

Abstract

Bacterial cell division is the result of a productive round of the cell cycle to yield two daughter cells. The cell cycle is highly coordinated in Caulobacter crescentus where it is driven by a cell cycle gene-regulatory network that coordinates gene expression with the major cell cycle events such as chromosome replication and cell division. Recent ribosomes profiling data showed that 484 genes undergo changes in translation efficiency during the cell cycle, suggesting a broad role for translational control in cell cycle-regulation. In this chapter, we focus on how to perform ribosome profiling to measure the translation efficiency across cellular mRNAs at key stages in the Caulobacter cell cycle. This methodology relies on the high-yield ludox gradient synchronization of Caulobacter cells followed by ribosome profiling to measure ribosome density and total-RNA-seq to measure mRNA levels.

Absolute Measurements of mRNA Translation in Caulobacter crescentus Reveal Important Fitness Costs of Vitamin B 12 Scavenging

“…Next, to reduce the impact of fast- and slow-moving ribosomes in the ribosome occupancy profiles along ORFs on the quantitative level of translation, we used winsorization to correct the average ribosome footprint density of each ORF (see Table S1 in the supplemental material). Start codon and stop codon regions were omitted from the analysis to avoid biases in slow-moving ribosomes that are initiating or terminating (13, 16).…”

“…Ribosome profiling was performed similarly to procedures in references 8 and 36, except that contaminating rRNA fragments generated during micrococcal nuclease (MNase) digestion were depleted to allow deeper quantitation of resulting mRNA translation similarly to reference 2. For a detailed protocol for the procedure, see reference 16. Five hundred milliliters of NA1000 cells were grown in M2G medium to an OD 600 of 0.5, treated with 100 μg/ml chloramphenicol for 2 min, and then harvested by centrifugation and flash-frozen in liquid nitrogen.…”

“…After footprinting, MNase was quenched with EGTA, and samples were separated by sucrose gradient fractionation. 70S peaks were purified, phenol chloroform extracted, and ethanol precipitated (16). Resulting mRNA fragments were size selected by 10% acrylamide-1× TBE-7 M urea PAGE, end repaired, 3′ adapter ligated, reverse transcribed, circularized, and depleted of rRNA fragments (2, 14, 16).…”

BR-bodies provide selectively permeable condensates that stimulate mRNA decay and prevent release of decay intermediates

“…The pellet was air dried and resuspended in 10mM Tris pH 7.0. RNA-seq library construction was performed as described in (Aretakis et al, 2018) using 1.0 µg of total RNA. Raw sequencing data is available in the NCBI GEO database with accession number GSE133522.…”

“…The RNA samples were spun at 20,000 × g at 4 °C for 1 hour, pellets were washed with 80% ethanol for 10 min, air dried, and resuspended in 10 mM Tris-HCl (pH 7.0). The RNA-Seq libraries were made using 5µg of total RNA samples, rRNA was removed by ribozero gram negative kit, and library construction was performed according to protocol (Aretakis et al, 2018). Raw sequencing data is available in the NCBI GEO database with accession number GSE133532.…”