Journal: Nature Communications

Article Title: Uncovering the small proteome of Methanosarcina mazei using Ribo-seq and peptidomics under different nitrogen conditions

doi: 10.1038/s41467-024-53008-8

Figure Lengend Snippet: A Illustrative diagram of Ribo-seq process used to map the translatome of M. mazei in two growth conditions: Translating ribosomes were initially detected on the mRNAs through the polysome fraction. MNase digestion eliminated unprotected mRNA regions, leading to the transformation of polysomes into monosomes. The translatome in specific experimental conditions was profiled by subjecting 20-34 nucleotide footprints, which were protected and isolated by 70S ribosomes, to cDNA library preparation and deep sequencing. B Fractionation of the cell lysates using sucrose gradients: To avoid the run-off of polysome, cells were harvested during the exponential growth phase using a rapid-chilling method (see Methods). Upon MNase digestion, monosomes were enriched compared to the untreated sample, as indicated by the 70S peak in the green ( + N) and red (-N) profile, contrasting with the Mock black profile. The monosomes are indicated by a shaded box. Absorbance was measured at a wavelength of 260 nm. C JBrowse genome browser screenshots show that our Ribo-seq and RNA-seq datasets distinguish between translated regions (e.g. psmB (MM_0694) encoding archaeal proteasome endopeptidase complex subunit beta) and untranslated regions like the non-coding sRNA 162 . D The operon of four TRAM domain containing small proteins shows enriched read coverage in the Ribo-seq library along their coding parts but in contrast to the RNA-seq library not in the regions (3’ and 5’ UTRs) un-protected by ribosome. Angled arrows in C and D indicate transcriptional start site (TSS).

Article Snippet: Gradients were processed using a Gradient Station (IP, Biocomp Instruments) fractionation system with continuous absorbance monitoring at 254 nm to resolve ribosomal subunit peaks.

Techniques: Transformation Assay, Isolation, cDNA Library Assay, Sequencing, Fractionation, RNA Sequencing