Conserved Peptide upstream Open Reading Frames (CPuORFs): A mechanism to modulate translation in plants.

Several CPuORFs (Conserved Peptide upstream Open Reading Frames) have been shown to modulate downstream mORF (main Open Reading Frame) translation by conditionally stalling the ribosome. This thesis presents and tests a model for CPuORF function called Conditional uORF Translational Stalling (CUTS). Some CPuORFs stall the ribosome in response to a signal (repressive CUTS). The CUTS mechanism is a rapid way to modulate mORF protein levels during translation. Moreover, another subset of CPuORFs, alongside a signal, can abolish ribosome stalling and promote translation re- initiation and increase downstream mORF levels (activator CUTS). Despite partial elucidation of an Arabidopsis a rCUTS CPuORF through cryo-EM, the aCUTS mechanism is yet to be elucidated.
Employing luciferase assays, cryo-EM, and bioinformatics, this research identifies the functional CPuORFs in a rare multi-CPuORF transcript. Moreover, reporter assays provide evidence that the ribosome stalling mechanism of four aCUTS CPuORFs is abolished by the same single base substitutions of ribosome exit tunnel proteins (uL4z, uL16z and RACK1z). Consequently, suggesting that there is a common mechanistic framework for the ribosome stalling in aCUTS CPuORFs. Molecular data also emphasises the role of the full length CPuORF nascent peptide in aCUTS activity. Furthermore, I present the first low-resolution 3D model of an Arabidopsis ribosome and have developed protocols to isolate wheat germ CPuORF-stalled ribosomes. Additionally, the thesis expands a published CPuORF database and presents the eukaryotic CPuORF database (https://theeukaryoticcpuorfdatabase.github.io). Analysis of this database suggested
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that CPuORFs across taxa have been conserved in regulatory transcripts and suggesting that the CPuORF CUTS mechanism fine tunes development, signalling and metabolism in response to the changing environment.
This research contributes to the broader knowledge of translational regulation in plants, highlighting the aCUTS ribosome stalling mechanism, evolutionary conservation of CPuORFs and their mORFs, and the potential of the CPuORF CUTS peptide-switch mechanism in biotechnology.