Krishnaswamy, M. | bioRxiv Science

bioRxiv2025

Biophysical constraints on mRNA decay rates shape macroevolutionary divergence in steady-state abundances

Interspecific comparisons of cell-type-specific gene expression levels can provide information about the evolutionary processes that drove divergence between species. From these comparisons, it is now evident that the predominant mode of gene expression evolution has been stabilizing selection, both on the steady-state (mean) protein levels as well as on the mRNA levels with additional lineage-specific shifts resulting from directional selection. However, as all previous work has used bulk RNA measurements, it has been impossible to determine which of the many cellular processes that contribute to mean abundances are highly constrained and which are more evolutionary labile. Assessing this is further complicated by the expectation that components of complex systems will evolve over time independent of changes in the selective regime so long as the net output of a system (i.e., mean expression) remains near the evolutionary optima. This process, known as evolutionary systems drift (ESD), has been frequently invoked as a non-adaptive explanation for changes in cellular phenotypes but has never been quantitatively tested or accounted for in any statistical test for selective constraints. Here, we develop a new paradigm that addresses both of these open problems simultaneously. Using single-cell expression data and biophysical models, we estimate mRNA transcriptional bursting rates, splicing rates, and decay rates across multiple vertebrate species. We then derive new mathematical results that describe how these various biophysical parameters are expected to co-evolve under ESD and then test whether we need additional evolutionary constraints to explain the divergences in these parameters. We find evidence that the biophysical parameters are indeed evolving in a coordinated manner as predicted by ESD and that there are additional strong constraints on transcriptional bursting, likely as a consequence of selection to reduce noise in expression. More broadly, this work opens up a whole new approach for studying the evolutionary dynamics of complex cellular systems.

evolutionary biology↗

bioRxiv2025

The evolution of function in the DNA binding domain of the CRP/FNR family

Transcriptional regulation, facilitated by transcription factors (TFs), contributes to bacterial response to environmental and cellular perturbations. How have TFs diversified and gained their functions? We use the CRP/FNR family to study this question across [~]6000 prokaryotic genomes. Characterized by homology to TFs CRP (cyclic AMP Receptor Protein) and FNR (Regulator of Fumarate and Nitrate Reductase) in Escherichia coli, the two functions of this family - sequence-specific DNA binding and transcriptional activation by direct contacts to RNA polymerase - are conferred by the helix-turn-helix containing DNA binding domain. We constructed a rooted phylogeny of this domain and performed residue conservation analysis on extant and in-silico reconstructed ancestral sequences of CRP/FNR family members. Residues that confer DNA binding specificity likely emerged in the overall ancestor of the sequence family. This ancestor does not fall into classes containing either E. coli CRP (Eco-CRP) or E. coli FNR (Eco-FNR) clades, but instead a large and diverse class that we call "CRP-like". Residues key for base-specific DNA binding remain largely conserved in the family. Residues (together called AR1) which make direct contacts with CTD of RNA Polymerase are conserved only in the Eco-CRP class and restricted to Gammaproteobacteria. The corresponding 287 determinant interface on CTD is again fully conserved only in Gammaproteobacteria, which contain Eco-CRP members, following step-wise evolution initiated at the Proteobacterial common ancestor. These suggest suggesting co-emergence of the interface residues in Eco-CRP. Our analysis hence shows that Class I transcriptional activation through AR1 on CRP and the determinant of CTD, as described in E. coli CRP, is phylogenetically restricted, while base-specific DNA binding has been present in the CRP/FNR family throughout its evolutionary history.

bioinformatics↗