Ribosome Profiling Research Articles

A rapid protocol for ribosome profiling of low input samples.

Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments that are protected by ribosomes from nucleolytic digestion. While the overall principle is simple, the workflow of ribosome profiling experiments is complex and challenging, and typically requires large amounts of sample, limiting its broad applicability. Here, we present a new protocol for ultra-rapid ribosome profiling from low-input samples. It features a robust strategy for sequencing library preparation within one day that employs solid phase purification of reaction intermediates, allowing to reduce the input to as little as 0.1 pmol of ∼30 nt RNA fragments. Hence, it is particularly suited for the analyses of small samples or targeted ribosome profiling. Its high sensitivity and its ease of implementation will foster the generation of higher quality data from small samples, which opens new opportunities in applying ribosome profiling.

EEF2 diphthamide modification restrains spurious frameshifting to maintain translational fidelity.

Diphthamide (DPH), a conserved amino acid modification on eukaryotic translation elongation factor eEF2, is synthesized via a complex, multi-enzyme pathway. While DPH is non-essential for cell viability and its function has not been resolved, diphtheria and other bacterial toxins ADP-ribosylate DPH to inhibit translation. Characterizing Saccharomyces cerevisiae mutants that lack DPH or show synthetic growth defects in the absence of DPH, we show that loss of DPH increases resistance to the fungal translation inhibitor sordarin and increases -1 ribosomal frameshifting at non-programmed sites during normal translation elongation and at viral programmed frameshifting sites. Ribosome profiling of yeast and mammalian cells lacking DPH reveals increased ribosomal drop-off during elongation, and removal of out-of-frame stop codons restores ribosomal processivity on the ultralong yeast MDN1 mRNA. Finally, we show that ADP-ribosylation of DPH impairs the productive binding of eEF2 to elongating ribosomes. Our results reveal that loss of DPH impairs the fidelity of translocation during translation elongation resulting in increased rates of ribosomal frameshifting throughout elongation and leading to premature termination at out-of-frame stop codons. We propose that the costly, yet non-essential, DPH modification has been conserved through evolution to maintain translational fidelity despite being a target for inactivation by bacterial toxins.

Synthetic virology approaches to improve the safety and efficacy of oncolytic virus therapies

The large coding potential of vaccinia virus (VV) vectors is a defining feature. However, limited regulatory switches are available to control viral replication as well as timing and dosing of transgene expression in order to facilitate safe and efficacious payload delivery. Herein, we adapt drug-controlled gene switches to enable control of virally encoded transgene expression, including systems controlled by the FDA-approved rapamycin and doxycycline. Using ribosome profiling to characterize viral promoter strength, we rationally design fusions of the operator element of different drug-inducible systems with VV promoters to produce synthetic promoters yielding robust inducible expression with undetectable baseline levels. We also generate chimeric synthetic promoters facilitating additional regulatory layers for VV-encoded synthetic transgene networks. The switches are applied to enable inducible expression of fusogenic proteins, dose-controlled delivery of toxic cytokines, and chemical regulation of VV replication. This toolbox enables the precise modulation of transgene circuitry in VV-vectored oncolytic virus design.

Unraveling the influences of sequence and position on yeast uORF activity using massively parallel reporter systems and machine learning.

Upstream open-reading frames (uORFs) are potent cis-acting regulators of mRNA translation and nonsense-mediated decay (NMD). While both AUG- and non-AUG initiated uORFs are ubiquitous in ribosome profiling studies, few uORFs have been experimentally tested. Consequently, the relative influences of sequence, structural, and positional features on uORF activity have not been determined. We quantified thousands of yeast uORFs using massively parallel reporter assays in wildtype and ∆upf1 yeast. While nearly all AUG uORFs were robust repressors, most non-AUG uORFs had relatively weak impacts on expression. Machine learning regression modeling revealed that both uORF sequences and locations within transcript leaders predict their effect on gene expression. Indeed, alternative transcription start sites highly influenced uORF activity. These results define the scope of natural uORF activity, identify features associated with translational repression and NMD, and suggest that the locations of uORFs in transcript leaders are nearly as predictive as uORF sequences.

Transcriptional and translational landscape fine-tune genome annotation and explores translation control in cotton

IntroductionThe unavailability of intergenic region annotation in whole genome sequencing and pan-genomics hinders efforts to enhance crop improvement. ObjectivesDespite advances in research, the impact of post-transcriptional regulation on fiber development and translatome profiling at different stages of fiber growth in cotton (G. hirsutum) remains unexplored. MethodsWe utilized a combination of reference-guided de novo transcriptome assembly and ribosome profiling techniques to uncover the hidden mechanisms of translational control in eight distinct tissues of upland cotton. ResultsOur study identified P-site distribution at three-nucleotide periodicity and dominant ribosome footprint at 27 nucleotides. Specifically, we have detected 1,589 small open reading frames (sORFs), including 1,376 upstream ORFs (uORFs) and 213 downstream ORFs (dORFs), as well as 552 long non-coding RNAs (lncRNAs) with potential coding functions, which fine-tune the annotation of the cotton genome. Further, we have identified novel genes and lncRNAs with strong translation efficiency (TE), while sORFs were found to affect mRNA transcription levels during fiber elongation. The reliability of these findings was confirmed by the high consistency in correlation and synergetic fold change between RNA-sequencing (RNA-seq) and Ribosome-sequencing (Ribo-seq) analyses. Additionally, integrated omics analysis of the normal fiber ZM24 and short fiber pag1 cotton mutant revealed several differentially expressed genes (DEGs), and fiber-specific expressed (high/low) genes associated with sORFs (uORFs and dORFs). These findings were further supported by the overexpression and knockdown of GhKCS6, a gene associated with sORFs in cotton, and demonstrated the potential regulation of the mechanism governing fiber elongation on both the transcriptional and post-transcriptional levels. ConclusionReference-guided transcriptome assembly and the identification of novel transcripts fine-tune the annotation of the cotton genome and predicted the landscape of fiber development. Our approach provided a high-throughput method, based on multi-omics, for discovering unannotated ORFs, hidden translational control, and complex regulatory mechanisms in crop plants.

Run, Ribosome, Run: From Compromised Translation to Human Health.

Significance: Translation is an essential cellular process, and diverse signaling pathways have evolved to deal with problems arising during translation. Erroneous stalls and unresolved ribosome collisions are implicated in many pathologies, including neurodegeneration and metabolic dysregulation. Recent Advances: Many proteins involved in detection and clearance of stalled and collided ribosomes have been identified and studied in detail. Ribosome profiling techniques have revealed extensive and nonprogrammed ribosome stalling and leaky translation into the 3' untranslated regions of mRNAs. Impairment of protein synthesis has been linked to aging in yeast and mice. Critical Issues: Ribosomes act as sensors of cellular states, but the molecular mechanisms, as well as physiological relevance, remain understudied. Most of our current knowledge stems from work in yeast and simple multicellular organisms such as Caenorhabditis elegans, while we are only beginning to comprehend the role of ribosome surveillance in higher organisms. As an example, the ribotoxic stress response, a pathway responding to global translational stress, has been studied mostly in response to small translation inhibitors and ribotoxins, and has only recently been explored in physiological settings. This review focuses on ribosome-surveillance pathways and their importance for cell and tissue homeostasis upon naturally occurring insults such as oxidative stress, nutrient deprivation, and viral infections. Future Directions: A better insight into the physiological roles of ribosome-surveillance pathways and their crosstalk could lead to an improved understanding of human pathologies and aging. Antioxid. Redox Signal. 39, 336-350.

The ATR-WEE1 kinase module promotes SUPPRESSOR OF GAMMA RESPONSE 1 translation to activate replication stress responses.

DNA replication stress threatens genome stability and is a hallmark of cancer in humans. The evolutionarily conserved kinases ATR (ATM and RAD3-related) and WEE1 are essential for the activation of replication stress responses. Translational control is an important mechanism that regulates gene expression, but its role in replication stress responses is largely unknown. Here we show that ATR-WEE1 control the translation of SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a master transcription factor required for replication stress responses in Arabidopsis thaliana. Through genetic screening, we found that the loss of GENERAL CONTROL NONDEREPRESSIBLE 20 (GCN20) or GCN1, which function together to inhibit protein translation, suppressed the hypersensitivity of the atr or wee1 mutant to replication stress. Biochemically, WEE1 inhibits GCN20 by phosphorylating it; phosphorylated GCN20 is subsequently polyubiquitinated and degraded. Ribosome profiling experiments revealed that that loss of GCN20 enhanced the translation efficiency of SOG1, while overexpressing GCN20 had the opposite effect. The loss of SOG1 reduced the resistance of wee1 gcn20 to replication stress, whereas overexpressing SOG1 enhanced the resistance to atr or wee1 to replication stress. These results suggest that ATR-WEE1 inhibits GCN20-GCN1 activity to promote the translation of SOG1 during replication stress. These findings link translational control to replication stress responses in Arabidopsis.

An mRNA processing pathway suppresses metastasis by governing translational control from the nucleus

Cancer cells often co-opt post-transcriptional regulatory mechanisms to achieve pathologic expression of gene networks that drive metastasis. Translational control is a major regulatory hub in oncogenesis; however, its effects on cancer progression remain poorly understood. Here, to address this, we used ribosome profiling to compare genome-wide translation efficiencies of poorly and highly metastatic breast cancer cells and patient-derived xenografts. We developed dedicated regression-based methods to analyse ribosome profiling and alternative polyadenylation data, and identified heterogeneous nuclear ribonucleoprotein C (HNRNPC) as a translational controller of a specific mRNA regulon. We found that HNRNPC is downregulated in highly metastatic cells, which causes HNRNPC-bound mRNAs to undergo 3′ untranslated region lengthening and, subsequently, translational repression. We showed that modulating HNRNPC expression impacts the metastatic capacity of breast cancer cells in xenograft mouse models. In addition, the reduced expression of HNRNPC and its regulon is associated with the worse prognosis in breast cancer patient cohorts.

Molecular basis of translation termination at noncanonical stop codons in human mitochondria.

The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial genomes feature deviations from the standard genetic code, including the reassignment of two arginine codons to stop codons. The protein required for translation termination at these noncanonical stop codons to release the newly synthesized polypeptides is not currently known. In this study, we used gene editing and ribosomal profiling in combination with cryo-electron microscopy to establish that mitochondrial release factor 1 (mtRF1) detects noncanonical stop codons in human mitochondria by a previously unknown mechanism of codon recognition. We discovered that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual conformation in the messenger RNA in which the ribosomal RNA participates in specific recognition of the noncanonical stop codons.

Brix protein APPAN plays a role in ribosomal RNA processing in Arabidopsis

Arabidopsis APPAN is a Brix family protein that is homologous to yeast Ssf1/Ssf2 and PPan in higher eukaryotes. A previous study, mostly based on physiological experiments, revealed that APPAN plays an essential role in female gametogenesis in plants. Here, we investigated cellular functions of APPAN, which could be the molecular basis for developmental defects in snail1/appan mutants. Virus-induced gene silencing (VIGS) of APPAN in Arabidopsis resulted in abnormal shoot apices, leading to defective inflorescences and malformed flowers and leaves. APPAN is localized in the nucleolus and co-sedimented mainly with 60 S ribosome subunit. RNA gel blot analyses showed overaccumulation of processing intermediates, particularly 35 S and P-A3, and the sequences were confirmed by circular RT-PCR. These results suggested that silencing of APPAN causes defective pre-rRNA processing. Metabolic rRNA labeling showed that APPAN depletion mainly reduced 25 S rRNA synthesis. Consistently, based on the ribosome profiling, the levels of 60 S/80 S ribosomes were significantly reduced. Finally, APPAN deficiency caused nucleolar stress with abnormal nucleolar morphology and translocation of nucleolar proteins into the nucleoplasm. Collectively, these results suggest that APPAN plays a crucial role in plant rRNA processing and ribosome biogenesis, and its depletion disrupts plant growth and development.

A vast evolutionarily transient translatome contributes to phenotype and fitness.

Translation is the process by which ribosomes synthesize proteins. Ribosome profiling recently revealed that many short sequences previously thought to be noncoding are pervasively translated. To identify protein-coding genes in this noncanonical translatome, we combine an integrative framework for extremely sensitive ribosome profiling analysis, iRibo, with high-powered selection inferences tailored for short sequences. We construct a reference translatome for Saccharomyces cerevisiae comprising 5,400 canonical and almost 19,000 noncanonical translated elements. Only 14 noncanonical elements were evolving under detectable purifying selection. A representative subset of translated elements lacking signatures of selection demonstrated involvement in processes including DNA repair, stress response, and post-transcriptional regulation. Our results suggest that most translated elements are not conserved protein-coding genes and contribute to genotype-phenotype relationships through fast-evolving molecular mechanisms.

The Gut Microbiota and Metabolome and Brown Adipose Tissue Regulate the Enhanced Exercise Capacity in Mice with Disruption of the Regulator of G protein Signaling 14 (RGS14)

The Gut Microbiota and Metabolome and Brown Adipose Tissue Regulate the Enhanced Exercise Capacity in Mice with Disruption of the Regulator of G protein Signaling 14 (RGS14) Candace R. Longoria, Olufunmilola Ibironke, Lee J. Kerkhof, Marko Oydanich, Xiaoyang Su, Eric Chiles, Dorothy E. Vatner, Stephen F. Vatner, Sara C. Campbell, FACSM.Rutgers, The State University of New Jersey Disruption of the Regulator of G Protein Signaling 14 (RGS14 (knockout) KO) results in a mouse model of healthful longevity with enhanced exercise capacity. Running distance and work to exhaustion are increased by 60±9% and 54±6% respectively in the RGS14 KO mouse compared to wild type littermates (WTL). Our hypothesis was that the enhanced exercise capacity was mediated by brown adipose tissue (BAT), which was confirmed by BAT transplants from RGS14 KO mice to WTL, resulting in a reversal of phenotype with the RGS14 KO mice losing the enhanced exercise capacity and WTL gaining the enhanced exercise capacity. We further hypothesized that the mechanism mediating the enhanced exercise capacity in RGS14 KO mice involved the gut microbiota and metabolome. The gut microbiota is linked to BAT, but the mechanisms and microbes facilitating BAT function remain largely unknown. Twenty-four mice were used to identify gut microbes by ribosomal operon profiling and metabolites in BAT samples at baseline. Bacterial differential expression analysis (via DESeq2) showed significant bacterial community differences between wild-type (WT) and RGS14 KO mice. Specifically, five bacterial strains ( Acutalibacter muris KB18, Ureaplasma urealyticum 132, Ureaplasma parvum ATCC 33697, Ureaplasma parvum, and Mucispirillum schaedleri ASF457) were present in RGS14 KO mice and not in WT. RGS14 KO mice also housed two distinctive strains of A. muciniphilia (BIOML-A22 and A. muciniphilia AN78). Eleven metabolites were significantly higher in RGS14 KO BAT samples compared to WT, with significant metabolic pathways, which in turn, are related to mechanisms mediating enhanced exercise capacity. We then examined the effects of antibiotic treatment (ABX) in 8 mice. Following 1 week of ABX, the bacterial strains that were increased in RGS14 KO mice were no longer elevated and the enhanced exercise capacity in RGS14 KO mice was no longer elevated compared to WTL. Furthermore, 6 of the 7 metabolites related to glucose and amino acid metabolism were no longer increased in RGS14 KO BAT compared to WTL. Taken together, these studies show that RGS14 KO BAT, associated with specific microbes and metabolites known to be involved with BAT, mediate the increased metabolic pathways that are associated with enhanced exercise capacity in RGS14 KO mice. In conclusion, the gut microbiota appears to be linked to RGS14 KO BAT function, which mediates enhanced exercise capacity in RGS14 KO mice. ONR Grant #826640 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Translation of circHGF RNA encodes an HGF protein variant promoting glioblastoma growth through stimulation of c-MET

IntroductionHGF/c-MET signaling is a significant driver of glioblastoma (GBM) growth and disease progression. Unfortunately, c-MET targeted therapies have been found to be largely ineffective suggesting additional redundant mechanisms of c-MET activation.MethodsUtilizing RNA-sequencing (RNA-seq) and ribosome profiling analyses of circular RNAs, circ-HGF (hsa_circ_0080914) was identified as markedly upregulated in primary GBM and found to potentially encode an HGF protein variant (C-HGF) 119 amino acids in length. This candidate HGF variant was characterized and evaluated for its ability to mediate c-MET activation and regulate PDX GBM cell growth, motility and invasive potential in vitro and tumor burden in intracranial xenografts in mice.ResultsAn internal ribosome entry site (IRES) was identified within the circ-HGF RNA which mediated translation of the cross-junctional ORF encoding C-HGF and was observed to be highly expressed in GBM relative to normal brain tissue. C-HGF was also found to be secreted from GBM cells and concentrated cell culture supernatants or recombinant C-HGF activated known signaling cascades downstream of c-MET. C-HGF was shown to interact directly with the c-MET receptor resulting in its autophosphorylation and activation in PDX GBM lines. Knockdown of C-HGF resulted in suppression of c-MET signaling and marked inhibition of cell growth, motility and invasiveness, whereas overexpression of C-HGF displayed the opposite effects. Additionally, modulation of C-HGF expression regulated tumor growth in intracranial xenografted PDX GBM models.ConclusionsThese results reveal an alternative mechanism of c-MET activation via a circular RNA encoded HGF protein variant which is relevant in GBM biology. Targeting C-HGF may offer a promising approach for GBM clinical management.

Effects and translatomics characteristics of a small-molecule inhibitor of METTL3 against non-small cell lung cancer

In non-small cell lung cancer (NSCLC), the heterogeneity promotes drug resistance, and the restricted expression of programmed death-ligand 1 (PD-L1) limits the immunotherapy benefits. Based on the mechanisms related to translation regulation and the association with PD-L1 of methyltransferase-like 3 (METTL3), the novel small-molecule inhibitor STM2457 is assumed to be useful for the treatment of NSCLC. We evaluated the efficacy of STM2457 in vivo and in vitro and confirmed the effects of its inhibition on disease progression. Next, we explored the effect of STM2457 on METTL3 and revealed its effects on the inhibition of catalytic activity and upregulation of METTL3 protein expression. Importantly, we described the genome-wide characteristics of multiple omics data acquired from RNA sequencing, ribosome profiling, and methylated RNA immunoprecipitation sequencing data under STM2457 treatment or METTL3 knockout. We also constructed a model for the regulation of the translation of METTL3 and PD-L1. Finally, we found PD-L1 upregulation by STM2457 in vivo and in vitro. In conclusion, STM2457 is a potential novel suppressor based on its inhibitory effect on tumor progression and may be able to overcome the heterogeneity based on its impact on the translatome. Furthermore, it can improve the immunotherapy outcomes based on PD-L1 upregulation in NSCLC.

EIF4E phosphorylation recruits β-catenin to mRNA cap and promotes Wnt pathway translation in dentate gyrus LTP maintenance

The mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), is crucial for translation and regulated by Ser209 phosphorylation. However, the biochemical and physiological role of eIF4E phosphorylation in translational control of long-term synaptic plasticity is unknown. We demonstrate that phospho-ablated Eif4eS209A Knockin mice are profoundly impaired in dentate gyrus LTP maintenance invivo, whereas basal perforant path-evoked transmission and LTP induction are intact. mRNA cap-pulldown assays show that phosphorylation is required for synaptic activity-induced removal of translational repressors from eIF4E, allowing initiation complex formation. Using ribosome profiling, we identified selective, phospho-eIF4E-dependent translation of the Wnt signaling pathway in LTP. Surprisingly, the canonical Wnt effector, β-catenin, was massively recruited to the eIF4E cap complex following LTP induction in wild-type, but not Eif4eS209A, mice. These results demonstrate a critical role for activity-evoked eIF4E phosphorylation in dentate gyrus LTP maintenance, remodeling of the mRNA cap-binding complex, and specific translation of the Wnt pathway.

Abstract LB353: The switch from cap-dependent to cap-independent translation reshapes the immunopeptidome of lung cancer

Abstract Cancer immunosurveillance relies on the presentation of peptide antigens on human leuckocyte antigen class I (HLA-I). HLA-I molecules present a diverse array of antigens, known as the “immunopeptidome”, which are recognized by cytotoxic CD8+ T cells. Recent evidence suggests that cryptic peptide antigens derived from non-canonical open reading frames (nuORFs) contribute to the immunopeptidome and drive CD8+ T cell responses. Integrating ribosome profiling (Ribo-seq) with mass spectrometry (MS) analyses has enabled the identification of several cryptic peptides derived from nuORFs including from putative non-coding regions including the 5’UTR, 3’UTR, long non coding RNAs (lncRNAs) and retained introns. Notably, many nuORFs undergo cap-independent translation. Various physiological and pharmacological stressors arrest canonical cap-dependent translation initiation and induce cap-independent translation. However, how this shift in translational dynamics influences the immunopeptidome is unknown. Using lung cancer cell lines, we show that the eIF4A inhibitor silvestrol induces cap-independent translation and reshapes the landscape of nuORF-derived peptide antigens. Silvestrol induced changes in the immunopeptidome strongly contrast with the global translation inhibitor homoharringtonine, which dampens antigen presentation. These observations suggest that the presentation of nuORF antigens in lung cancer is dynamic and tightly controlled by translational dynamics. More broadly, our study provokes reconsideration of translation inhibitors as immunomodulatory drugs that can change the HLA-I antigen repertoire and generate peptides that can act as new targets for cancer immunotherapy. Citation Format: Anika N. Ali, Andrew D. Weeden, Alex M. Jaeger. The switch from cap-dependent to cap-independent translation reshapes the immunopeptidome of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB353.

Abstract LB351: A novel mouse model to interrogate the functional relationship between protein synthesis and antigen presentation in cancer

Abstract Antigen presentation is a fundamental component of cancer immunity. Cytotoxic CD8+ T cells, the primary mediators of cell killing, recognize peptide antigens presented in the context of Major Histocompatibility Complex Class I (MHC-I). MHC-I presents an astoundingly diverse array of peptides, collectively known as the immunopeptidome. The biosynthesis of the immunopeptidome, which integrates gene expression, protein synthesis, and proteolytic processing, is not well understood. Additionally, most studies interrogating the immunopeptidome use cultured cells in vitro, which lack physiologically relevant stimuli. In contrast, in vivo studies utilize bulk tumor or tissue lysates, in which heterogeneous cell mixtures obscure the ability to understand cell type or disease specific patterns of antigen presentation. The technical limitations have hampered our understanding of the cancer immunopeptidome in physiologically relevant tumor microenvironments in vivo. Here we present a novel genetically engineered mouse model (GEMM) of cancer, that includes KrasLox-Stop-Lox-G12D; p53fl/fl; a Cre conditionally tagged MHC-I (KbStrep), and a Cre conditionally tagged ribosome (Rpl22HA/HA), termed the “KP/RiboMHC” mouse model. The KP/RiboMHC model enables simultaneous purification of ribosomes and peptide MHC complexes to functionally interrogate translational dynamics and antigen presentation in vivo. We developed optimized workflows to perform simultaneous ribosome profiling and immunopeptidomics from lung and pancreas cancer cell lines in vitro and specifically from malignant cells in vivo. Thus, the KP/RiboMHC model provides an unprecedented opportunity to ask fundamental questions regarding the influence of physiological stimuli in the tumor microenvironment on the biosynthesis of the immunopeptidome. Insights gained using this mouse will pave the way forward for the development of context specific, peptide-centric cancer immunotherapies. Citation Format: Alex M. Jaeger, Andrew Weeden, Anika Ali. A novel mouse model to interrogate the functional relationship between protein synthesis and antigen presentation in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB351.

Disruption of Dcp1 leads to a Dcp2-dependent aberrant ribosome profiles in Aspergillus nidulans.

There are multiple RNA degradation mechanisms in eukaryotes, key among these is mRNA decapping, which requires the Dcp1-Dcp2 complex. Decapping is involved in various processes including nonsense-mediated decay (NMD), a process by which aberrant transcripts with a premature termination codon are targeted for translational repression and rapid decay. NMD is ubiquitous throughout eukaryotes and the key factors involved are highly conserved, although many differences have evolved. We investigated the role of Aspergillus nidulans decapping factors in NMD and found that they are not required, unlike Saccharomyces cerevisiae. Intriguingly, we also observed that the disruption of one of the decapping factors, Dcp1, leads to an aberrant ribosome profile. Importantly this was not shared by mutations disrupting Dcp2, the catalytic component of the decapping complex. The aberrant profile is associated with the accumulation of a high proportion of 25S rRNA degradation intermediates. We identified the location of three rRNA cleavage sites and show that a mutation targeted to disrupt the catalytic domain of Dcp2 partially suppresses the aberrant profile of Δdcp1 strains. This suggests that in the absence of Dcp1, cleaved ribosomal components accumulate and Dcp2 may be directly involved in mediating these cleavage events. We discuss the implications of this.

Rfoot-seq: Transcriptomic RNase Footprinting for Mapping Stable RNA-Protein Complexes and Rapid Ribosome Profiling.

Ribosome profiling isolates ribosome-protected fragments for sequencing and is a valuable method for studying different aspects of RNA translation. However, conventional protocols require millions of input cells and time-consuming steps to isolate translating ribosome complexes using ultracentrifugation or immunoprecipitation. These limitations have prevented their application to rare physiological samples. To address these technical barriers, we developed an RNase footprinting approach named Rfoot-seq to map stable transcriptomic RNA-protein complexes that allows rapid ribosome profiling using low-input samples (Li, Yang, Stroup, Wang, & Ji, 2022). In this assay, we treat a cell lysate with concentrated RNase without complex crosslinking and retained only RNA footprints associated with stable complexes for sequencing. The footprints in coding regions represent ribosome-protected fragments and can be used to study cytosolic and mitochondrial translation simultaneously. Rfoot-seq achieves comparable results to conventional ribosome profiling to quantify ribosome occupancy and works robustly for various cultured cells and primary tissue samples. Moreover, Rfoot-seq maps RNA fragments associated with stable non-ribosomal RNA-protein complexes in noncoding domains of small noncoding RNAs and some long noncoding RNAs. Taken together, Rfoot-seq opens an avenue to quantify transcriptomic translation and characterize functional noncoding RNA domains using low-input samples. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Harvesting and lysing adherent cells Alternate Protocol 1: Harvesting and lysing suspension cells Alternate Protocol 2: Harvesting and lysing primary tissue samples Basic Protocol 2: RNase treatment and footprint purification for low-input samples Alternate Protocol 3: RNase treatment and footprint purification for ultra-low-input samples Basic Protocol 3: Library preparation for high-throughput sequencing Support Protocol: Preparation of dsDNA markers for library size selection Basic Protocol 4: Data analysis and quality control after sequencing.

The translational landscape of bread wheat during grain development.

The dynamics of gene expression in crop grains has typically been investigated at the transcriptional level. However, this approach neglects translational regulation, a widespread mechanism that rapidly modulates gene expression to increase the plasticity of organisms. Here, we performed ribosome profiling and polysome profiling to obtain a comprehensive translatome data set of developing bread wheat (Triticum aestivum) grains. We further investigated the genome-wide translational dynamics during grain development, revealing that the translation of many functional genes is modulated in a stage-specific manner. The unbalanced translation between subgenomes is pervasive, which increases the expression flexibility of allohexaploid wheat. In addition, we uncovered widespread previously unannotated translation events, including upstream open reading frames (uORFs), downstream open reading frames (dORFs), and open reading frames (ORFs) in long noncoding RNAs, and characterized the temporal expression dynamics of small ORFs. We demonstrated that uORFs act as cis-regulatory elements that can repress or even enhance the translation of mRNAs. Gene translation may be combinatorially modulated by uORFs, dORFs, and microRNAs. In summary, our study presents a translatomic resource that provides a comprehensive and detailed overview of the translational regulation in developing bread wheat grains. This resource will facilitate future crop improvements for optimal yield and quality.