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 5P-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 5P peaks. By contrast, brief hypoxia promotes tight stalling of A-site vacant ribosomes at Aspartate codons. This coincides with 5P 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. Significance StatementThe translation of mRNAs into protein relies on tRNAs to deliver amino acids to ribosomes that incorporate these building blocks into the growing polypeptide chain. By monitoring ribosomes as they progress from one codon to the next, we identified ribosome "traffic jams" with different characteristics, causes, and consequences. We find that decoding of certain codons and codon pairs guides polypeptide synthesis and mRNA recycling. The environment can influence this regulation. A comparison of plant species separated by nearly 150 million years highlights deep conservation in positional ribosome stalling patterns that limit translation of mRNAs encoding a suite of regulatory proteins.