Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions

Proteins usually associate with other molecules physically to execute their functions. Identifying these interactions is important for the functional analysis of proteins. Previously, we reported the parallel analysis of translated ORFs (PLATO) to couple ribosome display of full-length ORFs with affinity enrichment of mRNA/protein/ribosome complexes for the “bait” molecules, followed by the deep sequencing analysis of mRNA. However, the sample processing, from extraction of precipitated mRNA to generation of DNA libraries, includes numerous steps, which is tedious and may cause the loss of materials. Barcoded PLATO (PLATO-BC), an improved platform was further developed to test its application for protein interaction discovery. In this report, we tested the antisera-antigen interaction using serum samples from patients with inclusion body myositis (IBM). Tripartite motif containing 21 (TRIM21) was identified as a potentially new IBM autoantigen. We also expanded the application of PLATO-BC to identify protein interactions for JQ1, single ubiquitin peptide, and NS5 protein of Zika virus. From PLATO-BC analyses, we identified new protein interactions for these “bait” molecules. We demonstrate that Ewing sarcoma breakpoint region 1 (EWSR1) binds to JQ1 and their interactions may interrupt the EWSR1 association with acetylated histone H4. RIO kinase 3 (RIOK3), a newly identified ubiquitin-binding protein, is preferentially associated with K63-ubiquitin chain. We also find that Zika NS5 protein interacts with two previously unreported host proteins, par-3 family cell polarity regulator (PARD3) and chromosome 19 open reading frame 53 (C19orf53), whose attenuated expression benefits the replication of Zika virus. These results further demonstrate that PLATO-BC is capable of identifying novel protein interactions for various types of “bait” molecules. Keywords: Barcoded PLATO, Protein interaction, Ubiquitin-binding protein, Bromodomain inhibitor JQ1, Zika viru

ALTERNATIVE SPLICING REGULATES THE INNATE IMMUNE RESPONSE TO VIRAL INFECTION

Rift Valley fever virus (RVFV) is a mosquito-borne RNA virus that infects humans and livestock in sub-Saharan Africa and the Arabian peninsula, causing disease ranging from a mild flu-like illness to liver damage, blindness, hemorrhagic fever, death, and, especially in livestock animals, high rates of abortive pregnancies. There is no approved vaccine for RVFV, and as a disease with a high rate of spread that causes severe illness, it is listed as a Category A pathogen by the USA CDC. A better understanding of RVFV’s molecular virology will be instrumental to combating RVFV as climate change causes its mosquito host range to expand. RVFV infection causes global changes to host transcriptional activities, including host alternative splicing. Untangling these changes in transcription and alternative splicing will be key to a detailed understanding of viral infection and host responses to viral infection. Host cellular immunity, which is activated as soon as a viral incursion is detected, is multifaceted and complex, and many members of the innate immune response also encode alternatively spliced mRNA isoforms to regulate their activity. In this dissertation, RVFV’s wide-ranging effects on host transcription and splicing programs is described, and focus is placed on one particular alternative splicing event in the mRNA for host innate immune protein RIOK3. Chapter 1 reviews relevant aspects of cellular innate immunity and alternative splicing of innate immune genes, especially during RNA virus infection, and emphasizes recent evidence for RNA virus interference of host splicing mechanisms. Chapter 2 highlights our RNAseq work on RVFV-infected cells that shows the widespread changes in host transcription and splicing during infection, including in RIOK3. In Chapter 3, RIOK3’s antiviral role in the innate immune response to RVFV infection is demonstrated along with the observation that expression of the alternatively spliced isoform, RIOK3 X2, correlates with a diminished innate immune response, which indicates that this alternative splicing observed in RVFV infection may be important for regulation of innate immunity. Chapter 4 describes studies to more deeply characterize RIOK3’s alternatively spliced isoforms, and elucidates the importance of host splicing factor TRA2-b for the constitutive splicing of RIOK3. Of particular interest, we observed that in RVFV-infected cells TRA2-b mRNA is mostly alternatively spliced to diminish stability and translation, while conversely, TRA2-b overexpression and concomitant enhanced expression of constitutively spliced RIOK3 mRNA significantly limits RVFV replication. These data show that RVFV infection benefits from decreased RIOK3 via alternative splicing, and that RIOK3 is an essential member of the antiviral response. This work also contributes to an emerging story that mRNA splicing is vitally important for regulation of the innate immune response against viral infection

The genetics and evolution of the critically endangered Trinidad Piping Guan Pipile pipile, synonym Aburria pipile.

The Trinidad Piping Guan, Pipile pipile synonym Aburria pipile (Jaquin, 1784) is the only endemic Cracid on the island of Trinidad. The species is currently listed as Critically Endangered and is considered to be in ‘on-going decline’ by the IUCN, BirdLife International and Cracid Specialist Group. This study aims to examine aspects of genetic variation and the evolution of the mitochondrial genome in the Trinidad Piping Guan utilising, for the first time, samples collected from individuals in the wild and reference specimens of the genus Pipile sourced from museum collections. In this study the complete mitochondrial genome of the Trinidad Piping Guan was sequenced for the first time. Analysis of intra-specific variation of wild Trinidad Piping Guan individuals using single nucleotide polymorphisms demonstrates extremely limited variation within the genes of the mitochondrial genome and nuclear gene intron sequences. Limited variation within this population is consistent with both historical and contemporary contractions of populations within a restricted island system, which may have serious implications for the future of this species in terms of both genetic diversity and conservation management. Phylogenetic analysis of the complete mitochondrial genome of the Trinidad Piping Guan enabled placement of the genus Pipile within the Galliforme evolutionary tree for the first time, and subsequently places the genus within the broader context of the Aves class. Mito-genomic analysis confirms that the Cracids are one of the basal Galliforme clades, and sister taxa to the Megapodidae. Phylogenetic placement of the Pipile genus is basal to that of the Crax species within the Cracidae family, indicative of an earlier evolutionary origin of the Piping Guans. The inclusion of the Trinidad Piping Guan, in the avian evolutionary tree using the whole mitochondrial genomes expands the current genetic phylogeny of the Cracid family, yielding a better understanding of evolutionary relationships among the Galliforme order and the diversification of modern avian lineages. This study has established novel molecular techniques for the analysis of mitochondrial DNA in historical specimens of the genus Pipile from museum reference collections. The analysis of inter-specific relationships within the genus Pipile has clarified the evolutionary and biogeographic relationships between the Piping Guan species. Additionally, the Trinidad Piping Guan is genetically defined for the first time as an evolutionarily significant unit, which represents a unique evolutionary pathway within this important genus in a closed island system on the island of Trinidad

Wheat Improvement

This open-access textbook provides a comprehensive, up-to-date guide for students and practitioners wishing to access in a single volume the key disciplines and principles of wheat breeding. Wheat is a cornerstone of food security: it is the most widely grown of any crop and provides 20% of all human calories and protein. The authorship of this book includes world class researchers and breeders whose expertise spans cutting-edge academic science all the way to impacts in farmers’ fields. The book’s themes and authors were selected to provide a didactic work that considers the background to wheat improvement, current mainstream breeding approaches, and translational research and avant garde technologies that enable new breakthroughs in science to impact productivity. While the volume provides an overview for professionals interested in wheat, many of the ideas and methods presented are equally relevant to small grain cereals and crop improvement in general. The book is affordable, and because it is open access, can be readily shared and translated -- in whole or in part -- to university classes, members of breeding teams (from directors to technicians), conference participants, extension agents and farmers. Given the challenges currently faced by academia, industry and national wheat programs to produce higher crop yields --- often with less inputs and under increasingly harsher climates -- this volume is a timely addition to their toolkit