The marriage of molecular biology to informatics brought about a revolution in broad scale eukaryotic evolutionary biology. To date, interpretations of how organisms themselves have evolved, along with their key cellular and metabolic features, have relied almost exclusively on sequence-based phylogenetic reconstruction. At the same time, genome-level comparisons continue to reveal new sources of bias that can mislead tree-building computations. As phylogenomic investigations converge on a single framework for understanding eukaryotic diversity, a universal "Tree of Life", it essential to develop new, experimental methodologies to test and extend computation-based hypotheses. This project develops an innovative approach to address one of the most vexing problems in comparative evolution, genomics and proteomics; that is, the origin of green plants and, specifically, whether they share a sister relationship with red algae. Comparative sets of proteins that bind the RNA polymerase II C-terminal domain (CTD), a key organizing platform for complex gene expression, will be identified experimentally in respective green plant and red algal models. Phospho-CTD associated proteins (PCAPs) will be isolated and identified from the rhodophyte Cyanidioschyzon merolae and chlorophyte Chlamydomonas rheinhardii; both of these unicellular algae have complete and well-annotated genomes, permitting direct identification of specific isolated proteins through mass spectrometry. They represent, at present, the best system for experimental comparative proteomics of CTD-based transcription in the red and green lineages.
Broader impacts: As an example of how empirical approaches can be used to test computational hypotheses directly, this research can be transformative in the broader fields of evolutionary proteomics and genomics. The work fosters development of interdisciplinary collaborative research, supports training of a Masters-level student, and provides research opportunities for multiple undergraduates. The research will integrate with ongoing collaborations within the Porphyra (Rhodophyta) Genome Research Coordinating Network, and raise public awareness of science through a high school outreach program and public symposia that accompany annual meetings. The goals and preliminary results from this project are scheduled to be highlighted for developing network collaborations, both at a Porphyra RCN symposium at the 2009 joint meeting of the American Society of Plant Biologists and the Phycological Society of America, and at the subsequent RCN meeting scheduled at East Carolina University for spring 2010.
Intellectual merit. The advent and marriage of molecular biology and informatics have resulted in revolutionary new ideas about broad scale eukaryotic relationships. Most interpretations of organismal evolution, as well as of key cellular and metabolic features, have relied almost exclusively on sequence-based phylogenetic reconstructions and related computational approaches. As these investigations converge on a single framework for understanding eukaryotic diversity, a presumed "Tree of Life", it essential to develop new, experimental methodologies to test and extend computationally-based hypotheses. This project built upon previous computational investigations of the co-evolution of the RNA polymerase II (RNAP II) C-terminal domain (CTD) with its myriad of associated proteins, specifically comparing proteins that bind the domain in the unicellular red alga Cyanidioschyzon merolae and green alga Chlamydomonas reinhardtii. The question was investigated experimentally through isolation of proteins from both organisms that bind the the CTD when it is phosphorylated (phospho-CTD associated proteins or PCAPs), and their identification using high-performance liquid chromatography with tandem mass spectrometry (LC/MS/MS). Unlike more derived, multicellular red algae, in which the CTD is largely disorganized, Cyanidioschyzon contains two sets of tandemly repeated motifs; one of seven amino acid repeats with the sequence YSPSSPS, the other nine residue repeats of YSPSSPNVA. In contrast, and similar to more complex, multicellular green plants, Chlamydomonas has a 'normal' CTD composed of tandemly repeated seven-residue repeats of the consensus sequence shown above. Although the methods employed had been used previously to characterize scores of PCAPs in animals and yeast, no prior experimental work on the role the CTD had been undertaken in either red or green algae. Through this project, hundreds of individual proteins that bind to synthetic phospho-CTD repeats from Cyanidioschyzon and Chlamydomonas were isolated and identified. This includes a number of proteins that bind differentially to the two types of repeats found in Cyanidioschyzon, the first experimental evidence of specific CTD-protein interactions in any red alga. Because of the exploratory nature of this work, LC/MS/MS analyses were completed only at the end of the funding period, and comparative analyses of these results are ongoing at the time of submission of this report. Two research publications are in preparation, with expected submissions before the end of 2013. One will detail an expanded investigation of the comparative structure and evolution of the CTD across the eukaryotic tree of life, which serves as a framework for interpreting experimental work on CTD function from diverse organisms; the other will detail the experimental results of PCAP biochemical analyses, both within and between Cyanidioschyzon and Chlamydomonas. Broader impacts. The project provided extensive training for a doctoral candidate in experimental protein biochemistry, specific protein interactions with the RNAP II CTD, as well as LC/MS/MS protein identification and analyses. It also afforded him the opportunity to perform updated and thorough evolutionary analyses, which will serve as the framework for interpreting ongoing and future comparative functional investigations. In an age of increasing specialization, researchers with this kind of broad training, and an ability to make bridges across scientific disciplines, become increasing important for developing integrative biological research. The project also provided a platform to involve students generally underrepresented in biological sciences, primarily through two semesters of training for a female African-American undergraduate researcher, who gained experience in laboratory techniques, algal culturing and growth measurements, and protein biochemistry. The work further enhanced the PI's broader research interactions; this included involvement in the Porphyra Genome Project working group and the Algal Genomics Research Coordination Network, leading to two major papers on Porphyra transcriptomics, and helped to foster collaborative research that led to a publication on horizontal transfer of CTD-related proteins into apicomplexan parasites, a group that includes the causative agent of malaria. Work on Cyanidioschyzon and Chlamydomonas was highlighted in the PI’s public outreach and education work, in particular through laboratory tours and public displays as part of the annual Science Fair and Biology Open House sponsored by the East Carolina Center for Biodiversity. In April 2013, the event drew over two hundred participants, including several bus loads of young people from the Boys and Girls club of Greenville, who are primarily from groups underrepresented in the biological sciences.
