It is demonstrated that ouORF translation regulates alternative translation initiation to control the balance between chloroplast and cytosolic protein isoforms, and establishes ouORFs as a versatile class of translational regulatory elements that coordinate both protein abundance and protein diversity.
Abstract
Upstream open reading frames (uORFs) are widespread cis-regulatory elements that modulate translation initiation of downstream main ORFs (mORFs). Among them, overlapping uORFs (ouORFs) that overlap with mORFs are predicted to exert the strongest translational repression, yet they remain largely unexplored because of the difficulty of their identification. Here, we developed complementary computational approaches to systematically identify translated ouORFs from super-resolution ribosome profiling data in Arabidopsis. We identified 965 translated ouORFs alongside 7,180 canonical non-overlapping uORFs (nuORFs). We found that ouORFs exert substantially stronger translational repression than nuORFs, and that this repression depends primarily on Kozak context rather than uORF length. In addition, genes containing ouORFs or nuORFs have weaker mORF Kozak contexts than genes without uORFs, which may further reduce mORF translation. Moreover, ouORF translation promotes initiation downstream of the annotated mORF start codon, generating N-terminally truncated protein isoforms with altered domain composition and subcellular localization. Using ATPS2 as an example, we demonstrate that ouORF translation regulates alternative translation initiation to control the balance between chloroplast and cytosolic protein isoforms. Together, our findings establish ouORFs as a versatile class of translational regulatory elements that coordinate both protein abundance and protein diversity, providing the first genome-wide characterization of translated ouORFs in plants.
A ribosome tagging and purification strategy is developed that bypasses the prolonged turnover of ribosomal proteins, enabling ribosome profiling with one-hour temporal resolution after neural stimulation, and uncovered previously unknown activity-dependent alternative translation with functional implication, such as the uORF of Egr1.
Nayan Suryawanshi, Hitoshi Uchida, R. Endo et al.· Nature Communications· 0 citations
DCP2, encoding a major mRNA decapping enzyme, is identified as a previously unrecognized ISR-induced transcript and it is shown that translational induction of DCP2 depends on an overlapping ORF whose conserved 3′ region, corresponding to a ribosome pausing site, acts as a potent inhibitory element.
There is increasing evidence that translation is not limited to annotated protein-coding genes. Ribosome profiling sequencing, mass spectrometry-based proteomics, and immunopeptidomics have identified the productive translation of non-canonical open reading frames (ORFs). This suggests that the functional proteome includes not only conserved proteins but also proteins hidden in non-coding RNAs and de novo proteins. Some of these translated products are functional peptides, while others may be non-functional, potentially arising from evolutionary events. Several non-canonical ORF-encoded peptides have been found to regulate multiple physiological and pathological functions, particularly in cancer, immunity, and inflammation, indicating that they have potential as biomarkers and novel therapeutic targets. To better understand the diversity of functional peptides and translated non-canonical ORFs based on existing data, we summarize their classification according to transcriptional features and supporting evidence, including non-canonical ORFs located in ncRNAs and canonical mRNAs. This review provides a concise summary of the origin, discovery methods, and classification of non-canonical ORFs. It offers insights into the origins and functions of non-canonical ORF-encoded peptides from an evolutionary perspective, while also exploring the biological functions and regulatory mechanisms of these non-canonical ORF-encoded hidden proteins in tumorigenesis and progression.
Jiang-Xue Li, Yanxia Qin, Zhao Li et al.· Experimental and molecular p...· 0 citations
Cells reprogram gene expression at multiple, often independently studied, regulatory layers during nutrient stress. Here we use direct RNA sequencing of polysome-fractionated transcripts that simultaneously measures transcript abundance, mRNA degradation, poly(A) tail length, and three RNA modifications (m5C, m6A, pseudouridine) to build an integrated, multi-omic view of the Saccharomyces cerevisiae response to acute glucose starvation. Lasso-penalised mixture-of-regressions clustering of stochastic translation efficiency (δSTE) against this combined feature set partitions 5,033 transcripts into five translational programs with distinct functional identities, from a strongly upregulated translation-machinery cluster to a strongly repressed glycosylation cluster. CDS sequence composition discriminates these extremes, while 5′ and 3′UTR composition contributes only marginal, largely non-significant effects after length correction. Extending this analysis, we show that the repressed glycosylation program is distinguished by a convergent stack of independent cis-regulatory signatures: weak Kozak initiation context, high upstream open reading frame burden, excess RNA secondary structure specifically over the start codon, and depletion for the targets of major stabilising and export-associated RNA-binding proteins. In other programmes, translation efficiency tunes with initiation-context strength indicating that downstream regulatory layers coregulate translation. These results provide an integrated, feature-resolved map of post-transcriptional regulation during acute nutrient stress.
O. Rossini, S. Mahmud, Nikolay E. Shirokikh et al.· bioRxiv· 0 citations
Viruses encode diverse regulatory elements, but their breadth and mechanisms remain poorly defined. To address this gap, we performed massively parallel reporter assays spanning ∼200,000 genomic segments from 297 vertebrate-infecting viral genera. We identified numerous viral elements that enhance RNA stability and translation through TENT4-mediated mixed tailing, distributed across 19 genera and grouped into six distinct subclasses, indicating extensive convergent evolution. We also found diverse TENT4-independent elements acting through alternative pathways. One such element, Pt1 from Potamipivirus, stabilizes linear mRNA to levels comparable to circular RNA, suggesting its potential for RNA therapeutics. Pt1 directly recruits canonical poly(A) polymerases (PAPγ/α)-previously thought to function exclusively in transcription-coupled nuclear pre-mRNA processing-to drive cytoplasmic polyadenylation. Together, these findings chart the rich landscape of viral regulation, extend the scope of poly(A)-tail biology, and establish the virome as a valuable source for uncovering host RNA regulatory mechanisms.
Jenny J Seo, Chemin Lee, Dongbin Lim et al.· Cell· 0 citations