January 7th, 2024
Recent Publications Harnessing the Power of Translatomics.
Every week we provide a digest of a small number of recent interesting papers in the field of translatomics.
In this week’s Sunday papers, Wu et al. (2023) enhance the coverage of super-resolution Ribo-seq in Arabidopsis thaliana, which allowed them to uncover translation events. Wang et al. (2023) utilized Ribo-Seq in this investigation to explore the diversity and distinct role of eukaryotic initiation factors (eIFs) in budding yeast. Lastly, Shieh et al. (2023) carry out transcriptome-wide profiling of acute stress induced changes in ribosome occupancy level using external standards.
Improved Super-Resolution Ribosome Profiling Reveals Prevalent Translation of Upstream ORFs and Small ORFs in Arabidopsis
The Plant Cell, 2023
Wu, L., Ai, Q., Teixeira, R.T., Song, G., Montes-Serey, C., Elmore, J.M., Walley, J. and Hsu, P.Y.
Identifying
translated open reading frames (ORFs) and linking them to biological functions
is a key step in functional genomics. This proves particularly challenging when
it comes to the identification of short ORFs, which is heavily influenced by
the coverage and triplet periodicity of the data. In this study, the authors
aimed to enhance the coverage of ribosome protected fragments by modifying
their Ribo-Seq protocol in Arabidopsis (Arabidopsis thaliana), which previously
allowed them to uncover translation events for nuclear, chloroplastic, and
mitochondrial genes that were previously uncharacterized (ref). By utilizing a
transcriptome assembly, they were able to identify a total of 7,751
unconventional translation events, namely 6,996 upstream ORFs (uORFs) and 209
downstream ORFs on annotated protein-coding genes, as well as 546 ORFs found in
presumed non-coding RNAs. The production of stable proteins from some of these
unannotated translation events was confirmed through proteomics data.
Furthermore, they devised a
technique to detect extremely short uORFs, comprising 370 minimum uORFs
(AUG-stop), 2,921 minuscule uORFs (2-10 codons), and 681 overlapping
uORFs. Interestingly, both longer and shorter uORFs show significant
translational repression. Additionally, they systematically identified 594
uORFs that are regulated by alternative splicing, indicating extensive
translational regulation that is specific to isoforms. Lastly, there are
several significant pathways linked to these common uORFs. In conclusion, they
claim their enhanced translational landscape of Arabidopsis offers
important tools for researching the regulation of gene expression.
Decoding the Heterogeneity and Specialized Function of Translation Machinery Through Ribosome Profiling in Yeast Mutants of Initiation Factors
Advanced Biology, 2023
Wang, J., Zhang, G., Qian, W. and Li, K.
It is generally accepted that the translation machinery, which includes ribosomes and initiation factors (IFs), remains homogeneous and translates all mRNAs encountered. Although initiation factors have been the subject of several biochemical investigations, little is known about how they function to selectively regulate different genes. Although ribosome profiling, provides a more direct measure of the translational effects of perturbing translation initiation factors, inconsistent experimental conditions and genetic backgrounds have hampered some studies, making it difficult to develop a coherent understanding of their functional specialization.
The authors utilized Ribo-Seq in this investigation to explore the diversity and distinct role of eukaryotic initiation factors (eIFs) in budding yeast. The “looping” and “scanning” groups are identified based on similarities in the ribosomal landscapes induced by perturbations of eIF4A, eIF4B, eIF4G1, CAF20, or EAP1 knockouts or knockdowns of eIF1, eIF1A, eIF4E, or PAB1. These changes in ribosomal distribution across the transcriptome are the consequence of these initiation-factor groups. Investigating the cis-regulatory sequence characteristics of genes most heavily influenced by each group, the study finds that genes more reliant on looping-group factors typically have poly(A) tails and shorter transcripts. Further identification of ribosomal heterogeneity linked to disruption of particular initiation components is made possible by the ribosomal RNA fragments found in the Ribo-Seq data, indicating their possible functions in controlling ribosomal components.
Transcriptome-wide profiling of acute stress induced changes in ribosome occupancy level using external standards
PLos one, 2023
Shieh, A.W., Bansal, S.K., Zuo, Z. and Wang, S.H.
Acute cellular stress is known to trigger a widespread reduction in mRNA translation by inhibiting cap-dependent translation. Selective translation in response to acute stress plays vital roles in regulating the stress response. However, accurately profiling transcriptome-wide translational changes in response to acute cellular stress has presented challenges. Commonly used data normalization methods assume that any systematic shifts are experimental artifacts. Therefore, when applied to profile mRNA translation changes induced by acute cellular stress, these methods are likely to yield biased estimates.
To address this challenge, the authors developed and assessed a set of 16 oligomers designed as external standards for ribosome profiling studies. Using sodium arsenite treatment-induced oxidative stress in lymphoblastoid cell lines as a model system, they incorporated spike-in oligomers as external standards. Their analysis revealed that these spike-in oligomers exhibited a robust linear correlation between observed and expected quantifications, with minimal ratio compression observed at lower concentrations.