GSE138990
Ribosome profiling in archaea reveals leaderless translation, novel translational initiation sites, and ribosome pausing at single codon resolution
Members of the third domain of life, the Archaea, are ubiquitous in all environments on Earth but remain understudied in many aspects including regulatory processes of the central dogma. Archaea present unique biology because they utilize a mosaic of molecular features from both Bacteria and Eukarya, along with unique features. The advent of a high-throughput view of the translation landscape via ribosome profiling in the Bacteria and the Eukarya has illuminated the complexity and previously underappreciated regulation of translation (i.e. translation efficiency, ribosome surveillance, etc.) that potentially has large scale effects on cellular functionality. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii and elucidated the translational landscape for the first time in the third domain of life. We coupled ribosome profiling with translation inhibitors to: (a) determine the size of the archaeal ribosome footprint, (b) systematically assign translation states of the ribosome to footprint lengths in a mostly leaderless transcriptome, (c) experimentally induce ribosome pauses and clarify the pausing landscape comprehensively, (d) identify putative novel proteins, including small open reading frames (smORFs), and (e) provide evidence that many genes initiate on putative alternative translation start sites (paTSS) around and within open reading frames (ORFs), demonstrating how a microorganism with a gene dense genome can produce proteins with distinct functions using the same gene. Overall design: H. volcanii H98 single colonies were picked and grown overnight at 42°C with shaking at 220 rpm in Hv-YPC medium supplemented with thymidine (50 µg/mL final concentration) until saturation (OD600 > 1.0). These cultures were diluted to OD600 0.02 in fresh media, grown to  OD600 0.4 and split evenly into two flasks; one flask was used as a no treatment control and the other was treated with either 1 mg/mL homo-harringtonine (CAS Number 26833-87-4), 20 mM serine hydroxamate (CAS Number 55779-32-3), or 100 µg/mL anisomycin, final concentrations. Cells were harvested by centrifugation or direct freezing of the culture in liquid nitrogen. For cells harvested by centrifugation, cultures were immediately centrifuged at 8,000 rpm for 3 minutes at room temperature, the supernatant removed, and the pellets flashed frozen in liquid nitrogen. For cell lysis, the frozen pellets were resuspended in 1 mL of 1x lysis buffer (3.4 M KCl, 500 mM MgCl2, 50 mM CaCl2, 1 M Tris pH 7.5) with an additional 100 µg/mL anisomycin, transferred to a cryomill, and pulverized with 5 cycles (1 minute of grinding at 5 Hz, 1 minute of cooling). Lysates were then thawed at room temperature, centrifuged at 10,000 x g for 5 minutes, and transferred to a new tube on ice. For direct freezing of cultures in liquid nitrogen, 100 mL of culture sprayed directly into liquid nitrogen using a serological pipette. The frozen culture form small pellets which are collected and 50g of pellets were weighed in order to add 1x lysis buffer (3.4 M KCl, 500 mM MgCl2, 50 mM CaCl2, 1 M Tris pH 7.5) and 100 µg/mL anisomycin to prevent ribosome elongation when thawed later on. The pellets were pulverized in a cryomill at 10 cycles due to the larger volume of input (1 minute of grinding at 10 Hz, 1 minute of cooling). The lysates were thawed at room temperature and ribosomes were pelleted over a 60% sucrose cushion (sucrose dissolved in lysis buffer) in an ultracentrifuge with a Ti-70 rotor at 60,000 rpm for 2 hours at 4°C. Ribosome pellets were resuspended in 200 µL lysis buffer. Cell lysates (from cells harvested by centrifugation or direct freezing) were processed by first treating 20 AU of lysate RNA (Nanodrop) with 12,000 units of MNase (Nuclease S7, Roche) for 1 hour at 25°C. After nuclease digestion, samples were loaded onto 10-50% sucrose gradients and RNA was isolated from monosome fractions as described above. Library preparation was performed as previously described (Muhammed, et al 2019). Briefly, 10 µg of RNA fragments was used to purify 15-45 nt RNA fragments by gel electrophoresis (15% TBE Urea gel); RNA fragments were treated with T4 polynucleotide kinase (NEB), then ligated to the linker (NEB Universal miRNA Cloning Linker) using T4 RNA ligase (NEB), and gel extracted from a 10% TBE Urea gel. Lastly, rRNA fragments were subtracted using the Ribo-Zero rRNA removal kit for bacteria (Illumina), then fragments reverse transcribed with SuperScript III (Invitrogen) using custom primers previously described (Muhammed, et al 2019), template RNA was degraded using NaOH and high heat, gel extracted from a 10% TBE Urea gel, circularized using CircLigase (Epicentre) and PCR amplified (8-12 cycles) with Phusion polymerase (NEB) using custom primers (Muhammed, et al 2019), and PCR products gel extracted from a 10% TBE gels. PCR products were analyzed for size and concentration using a BioAnalyzer high sensitivity DNA kit (standard protocol) before sequencing on an Illumina HiSeq 2500 at the Johns Hopkins Sequencing Core Facility (Baltimore, MD). Adapter to trim: CTGTAGGCACCATCAATAGATCGGAAGAGCACACGTCTGAACTCCAGTCA