One-pot method for preparing DNA, RNA, and protein for multiomics analysis.

Typical multiomics studies employ separate methods for DNA, RNA, and protein sample preparation, which is labor intensive, costly, and prone to sampling bias. We describe a method for preparing high-quality, sequencing-ready DNA and RNA, and either intact proteins or mass-spectrometry-ready peptides for whole proteome analysis from a single sample. This method utilizes a reversible protein tagging scheme to covalently link all proteins in a lysate to a bead-based matrix and nucleic acid precipitation and selective solubilization to yield separate pools of protein and nucleic acids.

Reversible Click Chemistry Tag for Universal Proteome Sample Preparation for Top-Down and Bottom-Up Analysis.

Successful proteome analysis requires reliable sample preparation beginning with protein solubilization and ending with a sample free of contaminants, ready for downstream analysis. Most proteome sample preparation technologies utilize precipitation or filter-based separation, both of which have significant disadvantages. None of the current technologies are able to prepare both intact proteins or digested peptides.

Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes.

Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C site of 27SB pre-rRNA. C cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors.

Insights into remodeling events during eukaryotic large ribosomal subunit assembly provided by high resolution cryo-EM structures.

Ribosomes are responsible for translating the genome, in the form of mRNA, into the proteome in all organisms. Biogenesis of ribosomes in eukaryotes is a complex process involving numerous remodeling events driven in part by the concerted actions of hundreds of protein assembly factors. A major challenge in studying eukaryotic ribosome assembly has, until recently, been a lack of structural data to facilitate understanding of the conformational and compositional changes the pre-ribosome undergoes during its construction.

The assembly factor Erb1 functions in multiple remodeling events during 60S ribosomal subunit assembly in S. cerevisiae.

A major gap in our understanding of ribosome assembly is knowing the precise function of each of the ∼200 assembly factors. The steps in subunit assembly in which these factors participate have been examined for the most part by depleting each protein from cells. Depletion of the assembly factor Erb1 prevents stable assembly of seven other interdependent assembly factors with pre-60S subunits, resulting in turnover of early preribosomes, before the ITS1 spacer can be removed from 27SA3 pre-rRNA.

Disruption of ribosome assembly in yeast blocks cotranscriptional pre-rRNA processing and affects the global hierarchy of ribosome biogenesis.

In higher eukaryotes, pre-rRNA processing occurs almost exclusively post-transcriptionally. This is not the case in rapidly dividing yeast, as the majority of nascent pre-rRNAs are processed cotranscriptionally, with cleavage at the A2 site first releasing a pre-40S ribosomal subunit followed by release of a pre-60S ribosomal subunit upon transcription termination. Ribosome assembly is driven in part by hierarchical association of assembly factors and r-proteins.