Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes.
2014; 32 (6): 562-568
RNA-protein interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited. Here we repurpose a high-throughput sequencing instrument to quantitatively measure binding and dissociation of a fluorescently labeled protein to >10(7) RNA targets generated on a flow cell surface by in situ transcription and intermolecular tethering of RNA to DNA. Studying the MS2 coat protein, we decompose the binding energy contributions from primary and secondary RNA structure, and observe that differences in affinity are often driven by sequence-specific changes in both association and dissociation rates. By analyzing the biophysical constraints and modeling mutational paths describing the molecular evolution of MS2 from low- to high-affinity hairpins, we quantify widespread molecular epistasis and a long-hypothesized, structure-dependent preference for G:U base pairs over C:A intermediates in evolutionary trajectories. Our results suggest that quantitative analysis of RNA on a massively parallel array (RNA-MaP) provides generalizable insight into the biophysical basis and evolutionary consequences of sequence-function relationships.
View details for DOI 10.1038/nbt.2880
View details for PubMedID 24727714
- Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes NATURE BIOTECHNOLOGY 2014; 32 (6): 562-?
Genetic basis of differential opsin gene expression in cichlid fishes
JOURNAL OF EVOLUTIONARY BIOLOGY
2010; 23 (4): 840-853
Visual sensitivity can be tuned by differential expression of opsin genes. Among African cichlid fishes, seven cone opsin genes are expressed in different combinations to produce diverse visual sensitivities. To determine the genetic architecture controlling these adaptive differences, we analysed genetic crosses between species expressing different complements of opsin genes. Quantitative genetic analyses suggest that expression is controlled by only a few loci with correlations among some genes. Genetic mapping identifies clear evidence of trans-acting factors in two chromosomal regions that contribute to differences in opsin expression as well as one cis-regulatory region. Therefore, both cis and trans regulation are important. The simple genetic architecture suggested by these results may explain why opsin gene expression is evolutionarily labile, and why similar patterns of expression have evolved repeatedly in different lineages.
View details for DOI 10.1111/j.1420-9101.2010.01954.x
View details for Web of Science ID 000275761400021
View details for PubMedID 20210829