Analysis of Ribosome-Associated mRNAs in Rice Reveals the Importance of Transcript Size and GC Content in Translation

Abstract

Gene expression is controlled at transcriptional and post-transcriptional levels including decoding of messenger RNA (mRNA) into polypeptides via ribosome-mediated translation. Translational regulation has been intensively studied in the model dicot plant Arabidopsis thaliana, and in this study, we assessed the translational status [proportion of steady-state mRNA associated with ribosomes] of mRNAs by Translating Ribosome Affinity Purification followed by mRNA-sequencing (TRAP-seq) in rice (Oryza sativa), a model monocot plant and the most important food crop. A survey of three tissues found that most transcribed rice genes are translated whereas few transposable elements are associated with ribosomes. Genes with short and GC-rich coding regions are overrepresented in ribosome-associated mRNAs, suggesting that the GC-richness characteristic of coding sequences in grasses may be an adaptation that favors efficient translation. Transcripts with retained introns and extended 5′ untranslated regions are underrepresented on ribosomes, and rice genes belonging to different evolutionary lineages exhibited differential enrichment on the ribosomes that was associated with GC content. Genes involved in photosynthesis and stress responses are preferentially associated with ribosomes, whereas genes in epigenetic regulation pathways are the least enriched on ribosomes. Such variation is more dramatic in rice than that in Arabidopsis and is correlated with the wide variation of GC content of transcripts in rice. Taken together, variation in the translation status of individual transcripts reflects important mechanisms of gene regulation, which may have a role in evolution and diversification.

Translational inhibition and phase separation primes the epigenetic silencing of transposons

“…1a, while the GC contents at the first and second nucleotide positions of the codons (indicated as GC1 and GC2, respectively) are similar between genes and transposons, the GC3 contents of transposons are remarkably lower than those of genes. Such divergence of codon sequence usage might contribute to weak translation of TE transcripts that was partly suggested previously for rice 35 . We paid attention particularly to translational repression of TEs because it often induces RNA cleavage through the so called No-Go RNA Decay (NGD) pathway 36–40 .…”

Innovation, conservation and repurposing of gene function in plant root cell type development

“…These steps were conducted as described in (6). In brief, cell type-specific ribosome-associated mRNAs were isolated from the frozen root tip material using TRAP (4, 6,42,44,45) and mRNA was isolated from the ribosome complexes for non-strand specific random primer-primed RNAseq library construction (46). Barcoded libraries were pooled together and sequenced on the 30 Illumina HiSeq 4000 at the UC Davis DNA Technologies Core to obtain 50-bp reads.…”

“…Nipponbare) were generated by Agrobacterium tumefaciens transformation as described by (41) or at the UC Davis Plant Transformation Facility. The Rice TRAP binary vector was constructed as described by (4) using the gateway binary vector 5 pH7WG, for hygromycin resistance, as a backbone instead of pK7WG (https://gateway.psb.ugent.be/search) and incorporating rice OsRPL18-2 as described in (42) Promoters were incorporated by LR recombination as performed for S.lycopersicum constructs to drive the expression His6-FLAG-RPL18-GFP for TRAP. The promoters used were the previously published 35Sp(4), AtSCRp (5), RSS1p (43), as well as OsCMZp (Os01g0957100) and OsSHR1p (Os07g0586900) that were amplified using primers described in Data S10.…”

Transcriptome and translatome profiling and translational network analysis during seed maturation reveals conserved transcriptional and distinct translational regulatory patterns

“…The translationally up regulated transcripts in our study have higher GC percentage in the CDS (Supplemental Figure 4B, D), and thus in our dataset GC-rich coding region is over-represented in transcripts up-regulated in PO indicating an adaptive role for translation during seed maturation. Same GC-enriched coding region has been shown for mRNAs in association with ribosome in rice (Zhao et al, 2017), indicating a conserved sequence feature for ribosome association.…”