Biology subjects

Vierstra, R. D.

Publications and source records attributed to Vierstra, R. D..

3 recordsLinked to original sources

bioRxiv2021

Variation in upstream open reading frames contributes to allelic diversity in protein abundance

The 5 untranslated region (UTR) sequence of eukaryotic mRNAs may contain upstream open reading frames (uORFs), which can regulate translation of the main open reading frame (mORF). The current model of translational regulation by uORFs posits that when a ribosome scans an mRNA and encounters a uORF, translation of that uORF can prevent ribosomes from reaching the mORF and cause decreased mORF translation. In this study, we first observed that rare variants in the 5 UTR dysregulate protein abundance. Upon further investigation, we found that rare variants near the start codon of uORFs can repress or derepress mORF translation, causing allelic changes in protein abundance. This finding holds for common variants as well, and common variants that modify uORF start codons also contribute disproportionately to metabolic and whole-plant phenotypes, suggesting that translational regulation by uORFs serves an adaptive function. These results provide evidence for the mechanisms by which natural sequence variation modulates gene expression, and ultimately, phenotype.

genomics↗

bioRxiv2021

A Trio of Ubiquitin Ligases Sequentially Drive Ubiquitylation and Autophagic Degradation of Dysfunctional Yeast Proteasomes

As central effectors of ubiquitin (Ub)-mediated proteolysis, proteasomes are regulated at multiple levels, including degradation of unwanted or dysfunctional particles via autophagy (termed proteaphagy). In yeast, inactive proteasomes are exported from the nucleus, sequestered into cytoplasmic aggresomes via the Hsp42 chaperone, extensively ubiquitylated, and then tethered to the expanding phagophore by the autophagy receptor Cue5. Here, we demonstrate the need for ubiquitylation driven by the trio of Ub ligases (E3s) San1, Rsp5 and Hul5, which, together with their corresponding E2s, work sequentially to promote nuclear export and Cue5 recognition. Whereas San1 functions prior to nuclear export, Rsp5 and Hul5 likely decorate aggresome-localized proteasomes in concert. Ultimately, topologically complex Ub chain(s) containing both K48 and K63 Ub-Ub linkages are assembled, mainly on the regulatory particle, to generate autophagy-competent substrates. As San1, Rsp5, Hul5, Hsp42, and Cue5 also participate in general proteostasis, proteaphagy likely engages an essential mechanism for eliminating inactive/misfolded proteins. HIGHLIGHTSO_LIUbiquitylation is essential for the autophagic turnover of dysfunctional proteasomes. C_LIO_LIThe San1, Rsp5 and Hul5 E3s act sequentially to drive proteaphagy. C_LIO_LIThe E2s Ubc1, Ubc4 and Ubc5 are collectively required. C_LIO_LIBoth K48- and K63-mediated Ub-Ub linkages are assembled for efficient proteaphagy. C_LI

cell biology↗

bioRxiv2020

Ubiquitylome Analysis Reveals a Central Role for the Ubiquitin-Proteasome System in Plant Innate Immunity

Protein ubiquitylation profoundly expands proteome functionality and diversifies cellular signaling processes, with recent studies providing ample evidence for its importance to plant immunity. To gain a proteome-wide appreciation of ubiquitylome dynamics during immune recognition, we employed a two-step affinity enrichment protocol based on a 6His-tagged ubiquitin (Ub) variant coupled with high sensitivity mass spectrometry to identify Arabidopsis proteins rapidly ubiquitylated upon plant perception of the microbe-associated molecular pattern (MAMP) peptide flg22. The catalog from two-week-old seedlings treated for only 30 minutes with flg22 contained nearly 1,000 conjugates, 150 Ub footprints, and all seven types of Ub linkages, and included previously uncharacterized conjugates of immune components, such as RECEPTOR-LIKE KINASE 1 (RKL1) shown to negatively regulate plant immunity. In vivo ubiquitylation assays confirmed modification of several candidates upon immune elicitation, and revealed distinct modification patterns and dynamics for key immune components, including poly- and monoubiquitylation, as well as induced or reduced levels of ubiquitylation. Gene ontology and network analyses of the collection also uncovered rapid modification of the Ub-proteasome system itself, suggesting a critical auto-regulatory loop necessary for an effective MAMP-triggered immune response and subsequent disease resistance. Included targets were UBIQUITIN-CONJUGATING ENZYME 13 (UBC13) and proteasome component REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b), whose subsequent biochemical and genetic analyses implied negative roles in immune elicitation. Collectively, our proteomic analyses further strengthened the connection between ubiquitylation and flg22-based immune signaling, identified novel components and pathways regulating plant immunity, and increased the database of ubiquitylated substrates in plants. One-sentence summaryProteome-wide catalogs of ubiquitylated proteins revealed a rapid engagement of the ubiquitin-proteasome system in Arabidopsis innate immunity.

plant biology↗