An official website of the United States government
Abstract Ribo-T is a ribosome with covalently tethered subunits where core 16S and 23S ribosomal RNAs form a single chimeric molecule. Ribo-T makes possible a functionally orthogonal ribosome–mRNA system in cells. Unfortunately, use of Ribo-T has been limited because of low activity of its original version. Here, to overcome this limitation, we use an evolutionary approach to select new tether designs that are capable of supporting faster cell growth and increased protein expression. Further, we evolve new orthogonal Ribo-T/mRNA pairs that function in parallel with, but independent of, natural ribosomes and mRNAs, increasing the efficiency of orthogonal protein expression. The Ribo-T with optimized designs is able to synthesize a diverse set of proteins, and can also incorporate multiple non-canonical amino acids into synthesized polypeptides. The enhanced Ribo-T designs should be useful for exploring poorly understood functions of the ribosome and engineering ribosomes with altered catalytic properties.
more »
« less
A custom library construction method for super-resolution ribosome profiling in Arabidopsis
Abstract BackgroundRibosome profiling, also known as Ribo-seq, is a powerful technique to study genome-wide mRNA translation. It reveals the precise positions and quantification of ribosomes on mRNAs through deep sequencing of ribosome footprints. We previously optimized the resolution of this technique in plants. However, several key reagents in our original method have been discontinued, and thus, there is an urgent need to establish an alternative protocol. ResultsHere we describe a step-by-step protocol that combines our optimized ribosome footprinting in plants with available custom library construction methods established in yeast and bacteria. We tested this protocol in 7-day-old Arabidopsis seedlings and evaluated the quality of the sequencing data regarding ribosome footprint length, mapped genomic features, and the periodic properties corresponding to actively translating ribosomes through open resource bioinformatic tools. We successfully generated high-quality Ribo-seq data comparable with our original method. ConclusionsWe established a custom library construction method for super-resolution Ribo-seq in Arabidopsis. The experimental protocol and bioinformatic pipeline should be readily applicable to other plant tissues and species.
more »
« less
- Award ID(s):
- 2051885
- PAR ID:
- 10373162
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Plant Methods
- Volume:
- 18
- Issue:
- 1
- ISSN:
- 1746-4811
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)Abstract Ribosome profiling, also known as Ribo-seq, has become a popular approach to investigate regulatory mechanisms of translation in a wide variety of biological contexts. Ribo-seq not only provides a measurement of translation efficiency based on the relative abundance of ribosomes bound to transcripts, but also has the capacity to reveal dynamic and local regulation at different stages of translation based on positional information of footprints across individual transcripts. While many computational tools exist for the analysis of Ribo-seq data, no method is currently available for rigorous testing of the pattern differences in ribosome footprints. In this work, we develop a novel approach together with an R package, RiboDiPA, for Differential Pattern Analysis of Ribo-seq data. RiboDiPA allows for quick identification of genes with statistically significant differences in ribosome occupancy patterns for model organisms ranging from yeast to mammals. We show that differential pattern analysis reveals information that is distinct and complimentary to existing methods that focus on translational efficiency analysis. Using both simulated Ribo-seq footprint data and three benchmark data sets, we illustrate that RiboDiPA can uncover meaningful pattern differences across multiple biological conditions on a global scale, and pinpoint characteristic ribosome occupancy patterns at single codon resolution.more » « less
-
Abstract Histone post‐translational modifications (PTMs) play important roles in many biological processes, including gene regulation and chromatin dynamics, and are thus of high interest across many fields of biological research. Chromatin immunoprecipitation coupled with sequencing (ChIP‐seq) is a powerful tool to profile histone PTMsin vivo. This method, however, is largely dependent on the specificity and availability of suitable commercial antibodies. While mass spectrometry (MS)–based proteomic approaches to quantitatively measure histone PTMs have been developed in mammals and several other model organisms, such methods are currently not readily available in plants. One major challenge for the implementation of such methods in plants has been the difficulty in isolating sufficient amounts of pure, high‐quality histones, a step rendered difficult by the presence of the cell wall. Here, we developed a high‐yielding histone extraction and purification method optimized forArabidopsis thalianathat can be used to obtain high‐quality histones for MS. In contrast to other methods used in plants, this approach is relatively simple, and does not require membranes or additional specialized steps, such as gel excision or chromatography, to extract highly purified histones. We also describe methods for producing MS‐ready histone peptides through chemical labeling and digestion. Finally, we describe an optimized method to quantify and analyze the resulting histone PTM data using a modified version of EpiProfile 2.0 for Arabidopsis. In all, the workflow described here can be used to measure changes to histone PTMs resulting from various treatments, stresses, and time courses, as well as in different mutant lines. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Nuclear isolation and histone acid extraction Basic Protocol 2: Peptide labeling, digestion, and desalting Basic Protocol 3: Histone HPLC‐MS/MS and data analysismore » « less
-
Visualizing Ribo-seq and other sequencing data within genes of interest is a powerful approach to studying gene expression, but its application is limited by a lack of robust tools. Here, we introduce ggRibo, a user-friendly R package for visualizing individual gene expression, integrating Ribo-seq, RNA-seq, and other genome-wide datasets with flexible scaling options. ggRibo visualizes 3-nucleotide periodicity, a hallmark of translating ribosomes, within a gene-structure context, including introns and untranslated regions, enabling the study of novel ORFs, translation of different isoforms, and mechanisms of translational regulation. ggRibo can plot multiple Ribo-seq/RNA-seq datasets from different conditions for comparison. It also contains functions for plotting single-transcript view, reading-frame decomposition, and RNA-seq coverage alone. Importantly, ggRibo supports the visualization of other omics datasets that could also be presented with single-nucleotide resolution, such as RNA degradome, transcription start sites, translation initiation sites, and epitranscriptomic modifications. We demonstrate its utility with examples of upstream ORFs, downstream ORFs, nested ORFs, and differential isoform translation in humans,Arabidopsis, tomato, and rice. We also provide examples of multiomic comparisons that reveal insights that connect the transcriptome, translatome, and degradome. In summary, ggRibo is an advanced single-gene viewer that offers a valuable resource for studying gene expression regulation through its intuitive and flexible platform.more » « less
-
Abstract A crucial step in functional genomics is identifying actively translated open reading frames (ORFs) and linking them to biological functions. The challenge lies in identifying short ORFs, as their identification is greatly influenced by data quality and depth. Here, we improved the coverage of super-resolution Ribo-seq in Arabidopsis (Arabidopsis thaliana), revealing uncharacterized translation events for nuclear, chloroplastic, and mitochondrial genes. Assisted by a transcriptome assembly, we identified 7,751 unconventional translation events, comprising 6,996 upstream ORFs (uORFs) and 209 downstream ORFs on annotated protein-coding genes, as well as 546 ORFs in presumed non-coding RNAs. Proteomics data confirmed the production of stable proteins from some of these unannotated translation events. We present evidence of active translation from primary transcripts of tasiRNAs (TAS1–4) and microRNAs (pri-MIR163, pri-MIR169), and periodic ribosome stalling supporting co-translational decay. Additionally, we developed a method for identifying extremely short uORFs, including 370 minimum uORFs (AUG-stop), and 2,921 tiny uORFs (2–10 amino acids), and 681 uORFs that overlap with each other. Remarkably, these short uORFs exhibit strong translational repression as do longer uORFs. We also systematically discovered 594 uORFs regulated by alternative splicing, suggesting widespread isoform-specific translational control. Finally, these prevalent uORFs are associated with numerous important pathways. In summary, our improved Arabidopsis translational landscape provides valuable resources to study gene expression regulation.more » « less
