The N7-methylguanosine (m7G) cap is a unique molecular identifier that is a focal point for post-transcriptional gene regulatory pathways. The m7G cap serves as both a roadblock to enzymes that would degrade the mRNA and a landing pad for cap binding proteins that coordinate the pre-mRNA processing, nuclear export, and translation initiation of most mRNAs. Until recently, capping was thought to be exclusively nuclear, and decapping was thought to irreversibly destine the RNA to degradation. Simply stated, cytoplasmic capping is the process by which an m7G cap is returned to an uncapped mRNA in the cytoplasm. Cytoplasmic capping requires NCK1 to coordinate the sequential actions of an unknown kinase, the capping enzyme, and an RNA methyltransferase, which phosphorylate and cap the targeted mRNA and methylate the newly-added cap respectively. Although we have learned much about the biochemistry of cytoplasmic capping, many fundamental questions remain unanswered. The hypotheses driving this proposal are that: (1) Specific RNA sequence elements (or modifications) recruit and/or trigger cytoplasmic capping activity and that (2) the cytoplasmic capping of 5?-truncated mRNAs serves as a new tier of post-transcriptional gene regulation. This study will seek answers to three key questions. First, a combination of data mining and new sequencing experiments will uncover the sequences that target an mRNA to the cytoplasmic capping machinery. A bioinformatics approach integrating published data sets marking cap positions and transcription start sites (TSS) will identify non-TSS- associated caps. Oxford Nanopore direct RNA sequencing will then compare RNA harvested from cells +/- dominant negative cytoplasmic capping components to map full-length mRNA sequences. The synthesis of these studies should ascertain the 5? ends, the alternative splicing patterns, and polyadenylation site choices of cytoplasmically capped mRNAs. Second, CRISPR knockouts of mRNA decapping enzymes (Dcp2, DcpS, etc) and candidate endonucleases will identify the cellular mechanism(s) that generate uncapped ends for the cytoplasmic capping machinery. These knockouts will be paired with focused and transcriptome-wide methods to validate changes in cytoplasmic capping. Third, a combination of in vivo RNA labeling experiments and ribosome profiling will establish how cytoplasmic capping surveys mRNAs during the onset of the stress response. The generation, cytoplasmic capping, and translation of 5?-truncated mRNAs into N-terminally- shortened proteins would effectively be a new tier of post-transcriptional gene regulation with far-reaching impacts on the function(s) of the N-terminally truncated proteins. To summarize, this work will (1) identify and validate the sequences that regulate cytoplasmic capping (2) determine the mechanism(s) by which RNA substrates are generated for cytoplasmic capping, and (3) understand the in vivo function(s) of cytoplasmic capping during the onset of acute stress responses.
Several tiers of regulation ensure that genes are properly expressed in cells. Cap structures added to the beginnings of messenger RNAs (mRNAs) are critical for their regulation and represent one such mechanism. Cytoplasmic capping is an understudied process that returns a cap onto uncapped mRNAs and is likely another layer of the cell?s regulatory framework. This work?s goals are to determine how targets are generated for the cytoplasmic capping machinery, how that machinery recognizes mRNAs and the effects of cytoplasmic capping on cellular proteins in normal and stressed conditions.
