TRTech-PGR: Systematic identification and characterization of noncanonical ORFs in four model species

Defining protein-coding regions in a gene, so-called open reading frames (ORFs), is essential for studying protein functions and cellular activities. Recent evidence suggests that a large number of ORFs remain undetected in plant genomes. This project will use advanced experimental techniques and computational analyses to identify and study these mysterious and potentially powerful ORFs in four plants widely used in research and agriculture: Arabidopsis, tomato, rice, and maize. Discovering and studying these hidden ORFs can reveal new ways plants use proteins and control gene activities, offering exciting opportunities to improve crop performance. This project will generate a user-friendly online tool for the broad research community, provide training opportunities to one graduate student and two postdocs in interdisciplinary research, and engage three educational activities: (1) A bioinformatics workshop analyzing translation and transcription of ORFs into proteins will be developed and held at the American Society of Plant Biology (ASPB) annual meeting. (2) Two undergraduates will participate in this project through the Plant Genomics at Michigan State University Research Experiences for Undergraduates (REU) program and receive training in both data analysis and molecular biology experiments. (3) Public educational programs will be developed in collaboration with Michigan 4H Children’s Garden, featuring model plants and plant proteins, and engaging children of all ages and adults, reaching roughly 150,000 visitors annually. Recent evidence has shown that thousands of unannotated ORFs are actively translated within plant genomes. These noncanonical ORFs include (1) upstream ORFs within the 5′ leader sequences of protein-coding genes, (2) ORFs that result from alternative translation start sites, and (3) small ORFs present in presumed non-coding RNAs. These hidden ORFs dramatically increase protein diversity and functions and represent a plethora of regulatory mechanisms in gene expression and signaling. Yet, their identification is hindered by their short length, low conservation, and the lack of experimentally validated examples. To systematically study noncanonical ORFs, this project will integrate advanced transcriptome assembly with translatome profiling and proteomic profiling to identify and validate noncanonical ORFs in four model plants widely used in research and agriculture: Arabidopsis, tomato, rice, and maize (Aim 1). Subsequently, computational analyses and experimental characterization at the global and gene-specific levels will be conducted to study upstream ORFs, ORFs arising from alternative start sites of annotated ORFs, and small ORFs translated from presumed non-coding RNAs (Aims 2 – 4). The identification and characterization of these noncanonical ORFs will enhance the understanding of their functions, conservation, and regulation across plant species, and facilitate their application in future gene engineering and synthetic biology strategies to improve plant traits. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.