Subfunctionalized expression drives evolutionary retention of ribosomal protein paralogs Rps27 and Rps27l in vertebrates
- Stanford University, United States
- University of California, Santa Cruz, United States
- University of California, Los Angeles, United States
- University of California, San Francisco, United States
Abstract
The formation of paralogs through gene duplication is a core evolutionary process. For paralogs that encode components of protein complexes such as the ribosome, a central question is whether they encode functionally distinct proteins, or whether they exist to maintain appropriate total expression of equivalent proteins. Here, we systematically tested evolutionary models of paralog function using the ribosomal protein paralogs Rps27 (eS27) and Rps27l (eS27L) as a case study. Evolutionary analysis suggests that Rps27 and Rps27l likely arose during whole-genome duplication(s) in a common vertebrate ancestor. We show that Rps27 and Rps27l have inversely correlated mRNA abundance across mouse cell types, with the highest Rps27 in lymphocytes and the highest Rps27l in mammary alveolar cells and hepatocytes. By endogenously tagging the Rps27 and Rps27l proteins, we demonstrate that Rps27- and Rps27l-ribosomes associate preferentially with different transcripts. Furthermore, murine Rps27 and Rps27l loss-of-function alleles are homozygous lethal at different developmental stages. However, strikingly, expressing Rps27 protein from the endogenous Rps27l locus or vice versa completely rescues loss-of-function lethality and yields mice with no detectable deficits. Together, these findings suggest that Rps27 and Rps27l are evolutionarily retained because their subfunctionalized expression patterns render both genes necessary to achieve the requisite total expression of two equivalent proteins across cell types. Our work represents the most in-depth characterization of a mammalian ribosomal protein paralog to date and highlights the importance of considering both protein function and expression when investigating paralogs.
Data availability
Ribosome profiling sequencing data have been deposited in GEO under accession code GSE201845. Other data generated in this study are provided in the supplementary materials and Source Data files. Code used for data analysis is available at https://gitfront.io/r/adelefxu/f94QE89EJwyp/eS27-paralogs/.
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Subfunctionalized expression drives evolutionary retention of ribosomal protein paralogs in vertebratesNCBI Gene Expression Omnibus, GSE201845.
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MCA DGE DataFigshare, doi:10.6084/m9.figshare.5435866.v8.
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Single-cell RNA-seq data from Smart-seq2 sequencing of FACS sorted cells (v2)Figshare, doi:10.6084/m9.figshare.5829687.v8.
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Differentiation dynamics of mammary epithelial cells revealed by single-cell RNA-sequencingNCBI Gene Expression Omnibus, GSE106273.
Article and author information
Author details
Jonathan K Pritchard
Funding
National Institutes of Health (F30HD100123)
- Adele Francis Xu
Stanford Bio-X
- Adele Francis Xu
National Institutes of Health (5R01HG008140)
- Jonathan K Pritchard
New York Stem Cell Foundation (NYSCF-R-I36)
- Maria Barna
National Institutes of Health (R01HD086634)
- Maria Barna
Alfred P. Sloan Foundation
- Maria Barna
Pew Charitable Trusts
- Maria Barna
National Institutes of Health (R01HD098722)
- Lindsay Hinck
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal work was reviewed and approved by the Stanford Administrative Panel on Laboratory Animal Care (APLAC, protocol #27463). The Stanford APLAC is accredited by the American Association for the Accreditation of Laboratory Animal Care. All mice used in the study were housed at Stanford University except where otherwise noted. CRISPR-edited mouse lines were generated at the Gladstone Institute Transgenic Gene Targeting Core (San Francisco, CA). All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of California, San Francisco (protocol #AN180952-01B).
Copyright
© 2023, Xu et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Subfunctionalized expression drives evolutionary retention of ribosomal protein paralogs Rps27 and Rps27l in vertebrates
eLife 12:e78695.
https://doi.org/10.7554/eLife.78695
Further reading
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- Evolutionary Biology
Gene duplication drives evolution by providing raw material for proteins with novel functions. An influential hypothesis by Ohno (1970) posits that gene duplication helps genes tolerate new mutations and thus facilitates the evolution of new phenotypes. Competing hypotheses argue that deleterious mutations will usually inactivate gene duplicates too rapidly for Ohno’s hypothesis to work. We experimentally tested Ohno’s hypothesis by evolving one or exactly two copies of a gene encoding a fluorescent protein in Escherichia coli through several rounds of mutation and selection. We analyzed the genotypic and phenotypic evolutionary dynamics of the evolving populations through high-throughput DNA sequencing, biochemical assays, and engineering of selected variants. In support of Ohno’s hypothesis, populations carrying two gene copies displayed higher mutational robustness than those carrying a single gene copy. Consequently, the double-copy populations experienced relaxed purifying selection, evolved higher phenotypic and genetic diversity, carried more mutations and accumulated combinations of key beneficial mutations earlier. However, their phenotypic evolution was not accelerated, possibly because one gene copy rapidly became inactivated by deleterious mutations. Our work provides an experimental platform to test models of evolution by gene duplication, and it supports alternatives to Ohno’s hypothesis that point to the importance of gene dosage.
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- Cell Biology
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Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion of mreB from Pseudomonas fluorescens SBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis, we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for evolutionary rescue with genetic reconstructions demonstrating causality. Compensatory pbp1A mutations that targeted transpeptidase activity enhanced homogeneity of cell wall synthesis on lateral surfaces and restored cell size homeostasis. Mechanistic explanations require enhanced understanding of why deletion of mreB causes heterogeneity in cell wall synthesis. We conclude by presenting two testable hypotheses, one of which posits that heterogeneity stems from non-functional cell wall synthesis machinery, while the second posits that the machinery is functional, albeit stalled. Overall, our data provide support for the second hypothesis and draw attention to the importance of balance between transpeptidase and glycosyltransferase functions of peptidoglycan building enzymes for cell shape determination.
