Ribosome stalling position, spacing, and A-site occupancy impact translation and co-translational mRNA decay in plants

Rate limitation in translational elongation causes ribosomes to pause as they decode an open reading frame (ORF). The triggers and outcomes of these events are poorly understood in plants. Here, we size, map, and quantify footprints of individual (monosome) and closely spaced pairs (disomes) of ribosomes at single-codon resolution to capture stalling and collision events along mRNAs of Arabidopsis and maize. Ribosome footprinting was combined with 5’P-degradome-seq to examine the coincidence of pause events with co-translational decay. Our broad footprint-size selection resolves two monosome conformations and three disome configurations, including monosomes with a vacant or occupied A-site and disomes that are collided or separated by one or two codons. We find positional pausing prevalent at start, stop, and sequential Proline codons, and associated with co-translational processing. These di-Proline pauses are not associated with 5’P peaks. By contrast, brief hypoxia promotes tight stalling of A-site vacant ribosomes at Aspartate codons. This coincides with 5’P peaks on the 5’ side of the stalled ribosome, indicating that rate-limiting decoding can trigger co-translational mRNA decay. Notably, actively transcribed and translated hypoxia-response mRNAs accumulate 1- to 2-codon-separated disomes and are continuously degraded. Comparative footprinting of Arabidopsis and maize reveals that ribosome conformations and codon-specific pausing can be broadly conserved or lineage-specific, as exemplified by pausing on di-Prolines and Conserved Peptide upstream ORFs that regulate production of regulatory proteins. In sum, ribosome stalling at specific codons coupled with ribosome A-site occupancy and disome spacing modulates protein production and co-translational mRNA decay in plants.