Global analysis of ribosome-associated noncoding RNAs unveils new modes of translational regulation

Significance

Noncoding RNAs are an underexplored reservoir of regulatory molecules in eukaryotes. We analyzed the environmental response of roots to phosphorus (Pi) nutrition to understand how a change in availability of an essential element is managed. Pi availability influenced translational regulation mediated by small upstream ORFs on protein-coding mRNAs. Discovery, classification, and evaluation of long noncoding RNAs (lncRNAs) associated with translating ribosomes uncovered diverse new examples of translational regulation. These included Pi-regulated small peptide synthesis, ribosome-coupled phased small interfering RNA production, and the translational regulation of natural antisense RNAs and other regulatory RNAs. This study demonstrates that translational control contributes to the stability and activity of regulatory RNAs, providing an avenue for manipulation of traits.

Abstract

Eukaryotic transcriptomes contain a major non–protein-coding component that includes precursors of small RNAs as well as long noncoding RNA (lncRNAs). Here, we utilized the mapping of ribosome footprints on RNAs to explore translational regulation of coding and noncoding RNAs in roots of Arabidopsis thaliana shifted from replete to deficient phosphorous (Pi) nutrition. Homodirectional changes in steady-state mRNA abundance and translation were observed for all but 265 annotated protein-coding genes. Of the translationally regulated mRNAs, 30% had one or more upstream ORF (uORF) that influenced the number of ribosomes on the principal protein-coding region. Nearly one-half of the 2,382 lncRNAs detected had ribosome footprints, including 56 with significantly altered translation under Pi-limited nutrition. The prediction of translated small ORFs (sORFs) by quantitation of translation termination and peptidic analysis identified lncRNAs that produce peptides, including several deeply evolutionarily conserved and significantly Pi-regulated lncRNAs. Furthermore, we discovered that natural antisense transcripts (NATs) frequently have actively translated sORFs, including five with low-Pi up-regulation that correlated with enhanced translation of the sense protein-coding mRNA. The data also confirmed translation of miRNA target mimics and lncRNAs that produce trans-acting or phased small-interfering RNA ( tasiRNA/phasiRNAs). Mutational analyses of the positionally conserved sORF of TAS3a linked its translation with tasiRNA biogenesis. Altogether, this systematic analysis of ribosome-associated mRNAs and lncRNAs demonstrates that nutrient availability and translational regulation controls protein and small peptide-encoding mRNAs as well as a diverse cadre of regulatory RNAs.