Subfunctionalized expression drives evolutionary retention of ribosomal protein paralogs Rps27 and Rps27l in vertebrates
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eLife assessment
This study reports a cutting-edge set of experiments examining evolutionary models of paralog function differentiation for the mammalian ribosomal proteins eS27 and eS27L. No differentiated roles were identified for either paralog, but the paralogs are differentially expressed, and they preferentially associate with different transcript classes. Reciprocal switching of their coding sequences yielded no detectable phenotypes, but loss of either paralog resulted in lethality at different developmental stages, suggesting that subfunctionalized expression patterns underlie the retention of these paralogs. The work will be of interest to colleagues studying the evolution and diversification of ribosomes.
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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.
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eLife assessment
This study reports a cutting-edge set of experiments examining evolutionary models of paralog function differentiation for the mammalian ribosomal proteins eS27 and eS27L. No differentiated roles were identified for either paralog, but the paralogs are differentially expressed, and they preferentially associate with different transcript classes. Reciprocal switching of their coding sequences yielded no detectable phenotypes, but loss of either paralog resulted in lethality at different developmental stages, suggesting that subfunctionalized expression patterns underlie the retention of these paralogs. The work will be of interest to colleagues studying the evolution and diversification of ribosomes.
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Reviewer #1 (Public Review):
The authors of this study exerted a variety of laboratory experiment methods and in silico analysis of expression data, and showed the differentiated aspects of the protein functions of the product of the duplicated genes eS27 and eS27L as well as their redundant aspects. These proteins are components of the cellular machinery for translation, namely 'readout' of the genome, in eukaryotes. This study provides a valuable test case of examining why seemingly redundant genes that underwent gene duplication during evolution have been retained in the genomes of many present-day organisms.
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Reviewer #2 (Public Review):
In this manuscripts, the authors investigated differential role of two closely related proteins, S27 and S27L , which are one of the subunits of ribosome. Ribosomes containing each protein associate with a distinct set of mRNAs, suggesting that ribosomes in the cells play distinct roles depending on which subtype of S27 subunits they contain. The authors also performed functional analyses using mutant mice, and demonstrated that functions of S27-containing ribosome can be rescued by S27L-containing ribosome and vice versa. These findings provide new experimental insights into the origin of family genes fixed during the course of evolution.
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Reviewer #3 (Public Review):
A current topic in the translational control field revolves around the idea that "the ribosome" is not a singular monolith machine, but rather that there are a variety of ribosomes, some with specialized functions. The presence of evolutionarily conserved ribosomal protein gene paralogs provides a platform for testing this idea. Presumably, if a paralog is required to translate a specific mRNA or class of mRNAs in a cell or organ type specific manner, it's loss should generate an observable phenotype. In this study, Xu and colleagues exploit the evolutionarily conserved eS27 and eS27L proteins to probe this hypothesis. Technically, the work is on the cutting edge of the field. Advanced genetic engineering techniques were used to generate mice lacking either paralogous gene, to create reciprocal swaps of each …
Reviewer #3 (Public Review):
A current topic in the translational control field revolves around the idea that "the ribosome" is not a singular monolith machine, but rather that there are a variety of ribosomes, some with specialized functions. The presence of evolutionarily conserved ribosomal protein gene paralogs provides a platform for testing this idea. Presumably, if a paralog is required to translate a specific mRNA or class of mRNAs in a cell or organ type specific manner, it's loss should generate an observable phenotype. In this study, Xu and colleagues exploit the evolutionarily conserved eS27 and eS27L proteins to probe this hypothesis. Technically, the work is on the cutting edge of the field. Advanced genetic engineering techniques were used to generate mice lacking either paralogous gene, to create reciprocal swaps of each coding sequence into the other locus, and even to create genetically homogenous mice. The authors also use state of the art molecular biology methods, e.g. paralog-specific ribosome profiling, to search for differences in the mRNAs translated by ribosomes containing either of the two homologs.
Some phylogenetic evidence was presented suggesting that the paralogs first appeared during a gene duplication event in vertebrates: however, only and bird and one amphibian are represented. It is recommended that this analysis go deeper, parsing the amphibians and fish more finely. Although not identifying evidence for specialized ribosomes, they did find that it is essential that at least two copies of eS27 or eS27L are retained. Interestingly, the embryonic lethality of truncation alleles of either of the two paralogs result manifested at different stages of development, pointing to some kind of functional differences during development. The finding that eS27L containing ribosomes are more prevalent in lactating mammary gland and liver is an interesting observation, and that such ribosomes are preferentially associated with mRNAs involved in the cell cycle. From this the authors conclude that the data support subfunctionalization model of eukaryotic ribosomal protein S27 evolution rather than a specialized ribosome model. I also note that this is the most comprehensive and technologically advanced study of its kind in the translational control field and that it represents a significant contribution to the field of evolutionary biology.
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