A Fly’s View of Retinoblastoma-Family Protein Conservation

By: Dhruva Kadiyala (Undergraduate Student at Michigan State University)

How do evolutionary perspectives illuminate cancer-related biochemistry? As a high school student, I was involved in a project to find targets to attack cancer cells. That project really inspired me to work on the retinoblastoma-family protein project in the Arnosti lab. I came into Dr. Arnosti’s lab in my freshmen year at Michigan State University as a Professorial Assistant from the Honors College and immediately began to learn about retinoblastoma (Rb) tumor suppressor proteins in Drosophila species.

In humans, the retinoblastoma protein is a tumor suppressor and plays an active role in cell cycle regulation. Mutations in the Rb gene or its regulatory pathway are associated with many human cancers. Rb is ancient; the gene is evolutionarily conserved in most multicellular organisms and present as a single copy gene. In mammals, however, there are three Rb paralogs: Rb, p107 and p130. Independently, in Drosophila the Rb gene duplicated about 60 million years ago, and both paralogs, Rbf1 and Rbf2, have been retained in all modern Drosophila species. This situation provides a great model system to study Rb paralog evolution and function.

To understand the evolution of Rbf1 and Rbf2, I aligned Rbf1 protein sequences from 12 Drosophila species using the Clustal Omega multiple sequence alignment tool from the European Bioinformatics Institution. I split the proteins into three different domains (N-terminus, C-terminus, and Pocket Domain) to see what part of the protein is more conserved. I found that the Rbf1 gene that most resembles the ancestral gene, based on similarity with other organisms’ Rb genes, shows a higher degree of conservation, especially in the Pocket domain important for binding to transcription factors. The derived Rbf2 gene has a higher degree of variation within Drosophila, especially in the N and C-termini.

I also aligned both Rbf1 and Rbf2 sequences from the D. melanogaster with the human Retinoblastoma-family proteins (Rb, p107, and p130). What was striking is that the more evolutionarily variable human Rb and fly Rbf2 proteins have changes especially in the C-terminus that impact a functional domain (the Instability Element IE) important for protein turnover and transcriptional regulation, an apparent case of parallel evolution.

Why do most animals outside of vertebrates make do with a single Rb gene, while Drosophila have expanded their count? To assess the structural variation in Rb genes in arthropods in general, I compared Drosophila Rbf1 sequences with those of the red flour beetle (Tribolium castaneum), eastern honey bee (Apis cerana), monarch butterfly (Danaus plexippus), western flower thrip (Frankliniella occidentalis), green peach aphid (Myzus persicae), a drywood termite (Cryptotermes secundus), a springtail (Folsomia candida), the common house spider (Parasteatoda tepidariorum), and white-legged shrimp (Penaeus vannamei).  Overall, conservation is greatest in the transcription factor binding Pocket Domain, although the internal “spacer” region within the domain is quite variable, something that may influence activity of the proteins. The C terminus was least conserved, but IE sequences are conserved. Thus, evolutionary changes in this portion of the protein seem to be restricted to cases where there are paralogous genes.

I generated a visual representation of these levels of conservation with the help of Clustal Omega. For that purpose, I turned to Jalview software, which uses the multiple sequence alignment tools Clustal Omega and MUSCLE to generate visuals for analysis. Here, I show a visual representation showing residue by residue conservation of Rb genes from arthopod species (Figure 1).

Figure 1: Multiple sequence alignment of Rbf1 of D. melanogaster and Rb genes from other arthropod species. The height and color of the bars represent percent identity and similarity. Higher bars and yellow bars are more conserved than lower brown bars. The protein skeleton is based on D. melanogaster Rbf1 protein with following denotations: Blue: cyclin fold domain, Pink: A pocket, Green: B pocket, purple: Instability element.

Overall, my work in Dr. Arnosti’s lab has been most meaningful work for my development as a researcher. I experienced firsthand how proteins that play a major role in survival and development in cancer are evolutionarily conserved and yet evolve over time among species, and thereby I have deepened my knowledge of biology and the mechanisms of evolution. I hope to continue working in the lab for the rest of my undergraduate career, discover a more disciplined researcher in myself, and contribute to science as I prepare to advance to medical studies.

Dhruva Kadiyala is a sophomore studying Neuroscience in Lyman Briggs College at Michigan State University. He is a pre-medical student also interested in biological research, and has worked with Cell and Molecular Biology Ph.D. student Rima Mouawad in the lab of David Arnosti.

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