Biology subjects

Salgania, H. K.

Publications and source records attributed to Salgania, H. K..

3 recordsLinked to original sources

bioRxiv2024

Recurrent innovation of protein-protein interactions in the Drosophila piRNA pathway

Despite being essential for fertility, genome defence pathway genes often evolve rapidly. However, little is known about the molecular basis of this adaptation. Here, we characterize the evolution of a protein interaction network within the PIWI-interacting small RNA (piRNA) genome defence pathway in Drosophila at unprecedented scale and evolutionary resolution. We uncover pervasive rapid evolution of a protein interaction network anchored at the Heterochromatin Protein 1 (HP1) paralog Rhino. Using complementary phylogenetic analysis, high-throughput yeast-two-hybrid matrix screening, and in vivo interaction analyses in cross-species transgenic flies, we characterized three distinct evolutionary protein interaction trajectories across [~]40 million years of Drosophila evolution. The data set covering 11 piRNA pathway proteins of five Drosophila species revealed several protein interactions that are fully conserved, indicating functional conservation despite overall rapid amino acid sequence change. Other interactions are preserved through co-evolution and were detected only between proteins within or from closely related species. We also identified sets of species-restricted protein interactions which, through rewiring of a Rhino-anchored transcription factor network, may preserve critical roles in enabling and adapting piRNA production from heterochromatic loci. In sum, our analyses dissected principles of interaction evolution in an adaptively evolving protein-protein interaction network uncovering evolutionary and functional insight into germline piRNA production across Drosophila species. Our work provides key experimental evidence in support of a model proposing that intermolecular interaction innovation is a major molecular mechanism of evolutionary adaptation in protein-coding genes.

molecular biology↗

bioRxiv2024

Molecular insight into the network of Drosophila cytoplasmic piRNA pathway proteins through a combination of systematic interaction screening and structural prediction

piRNA-bound PIWI proteins mediate the silencing of transposons at both the transcriptional and post-transcriptional levels, processes that are critical for genome integrity and fertility in animals. While numerous additional proteins are known to be essential for piRNA biogenesis and function in Drosophila and other animals, their molecular and mechanistic functions have remained largely unknown. To improve our molecular understanding of the Drosophila piRNA pathway, we used a cell culture-based protein-protein interaction assay called ReLo to perform a systematic pairwise interaction screen involving 22 factors operating in the cytoplasm, including PIWI proteins, Tudor domain-containing proteins (TDRDs), RNA helicases, and mitochondrial surface proteins. Through additional ReLo interaction testing and structural modeling using AlphaFold-Multimer, we have characterized six protein complexes at the molecular and structural levels. We believe that the results of this screen and our methodological approach are likely to guide future research into the molecular mechanisms underlying piRNA biogenesis and function.

biochemistry↗

bioRxiv2022

ReLo: a simple colocalization assay to identify and characterize physical protein-protein interactions

The characterization of protein-protein interactions (PPIs) is fundamental for understanding biochemical processes. Many methods have been established to identify and study direct PPIs; however, the screening and investigation of PPIs involving large or poorly soluble proteins remain challenging. As a result, we developed ReLo, a simple, rapid, and versatile cell culture-based method for detecting and investigating interactions in a cellular context. Importantly, our data strongly suggest that with ReLo specifically direct binary PPIs are detected. By applying additional bridging experiments ReLo can also be used to determine the binding topology of subunits within multiprotein complexes. Moreover, ReLo has the potential to identify protein domains that mediate complex formation, screen for interfering point mutations, study interactions that depend on conformation or protein arginine methylation, and it is sensitive to drugs that mediate or interfere with an interaction. Taken together, ReLo is a simple and quick alternative for the study of PPIs particularly when established methods fail.

cell biology↗