Grad-seq establishes an RNA/protein complexome

Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading bacterial organism with respect to global protein-protein networks. Yet, there has been no global census of RNA/protein complexes in this model species of microbiology.

University of Würzburg researchers have performed Grad-seq to establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ∼85% of all E. coli transcripts and ∼49% of the proteins. These include the majority of small noncoding RNAs (sRNAs) detectable in this bacterium as well as the general sRNA-binding proteins, CsrA, Hfq and ProQ. In presenting use cases for utilization of these RNA and protein maps, the researchers show that a stable association of RyeG with 30S ribosomes gives this seemingly noncoding RNA of prophage origin away as an mRNA of a toxic small protein. Similarly, they show that the broadly conserved uncharacterized protein YggL is a 50S subunit factor in assembled 70S ribosomes.

Grad-seq reveals the E. coli RNA/protein complexome

(A) Overview of the Grad-seq workflow. (B) A_{260 nm} profile of the gradient. Low‐molecular‐weight complexes (bulk peak) and ribosomal subunits (30S, 50S) are highlighted. Particles larger than the 50S subunit were pelleted. (C) Ethidium bromide‐stained RNA gel. Bands corresponding to abundant housekeeping RNAs are indicated. (D) Coomassie‐stained SDS-PAGE. Bands corresponding to abundant housekeeping proteins are indicated. (E) Western blot. The β-subunit of RNAP (RpoB) and the major σ factor σ⁷⁰ (RpoD) co-migrate. (F) Heat map of digital in‐gradient distributions of known RNA-protein complexes derived from RNA‐seq and LC–MS/MS data. For each molecule, the spike-in-normalized sedimentation profiles are normalized to the range from 0 to 1 by dividing the values of each fraction by the maximum value of the corresponding molecule. M, size marker. L, lysate (input control). P, pellet fraction. (G) Sucrose polysome gradient of a wild-type lysate followed by northern blotting. 6S RNA, ChiX and CsrB are only present in the bulk peak, whereas GcvB and Spot 42 show additional abundances around the polysomes (compare to (F)). The lpp mRNA is only present in ribosomal fractions.

Overall, this study crucially extends our knowledge about the cellular interactome of the primary model bacterium E. coli through providing global RNA/protein complexome information and should facilitate functional discovery in this and related species.

Availability – To facilitate data accessibility und usability, the researchers set up an online browser (https://helmholtz-hiri.de/en/datasets/gradseqec/) for interactive exploration of these datasets