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At least 19 recordsLinked to original sources
PubMed2021

RP-REP Ribosomal Profiling Reports: an open-source cloud-enabled framework for reproducible ribosomal profiling data processing, analysis, and result reporting.

Ribosomal profiling is an emerging experimental technology to measure protein synthesis by sequencing short mRNA fragments undergoing translation in ribosomes. Applied on the genome wide scale, this is a powerful tool to profile global protein synthesis within cell populations of interest. Such information can be utilized for biomarker discovery and detection of treatment-responsive genes. However, analysis of ribosomal profiling data requires careful preprocessing to reduce the impact of artifacts and dedicated statistical methods for visualizing and modeling the high-dimensional discrete read count data. Here we present Ribosomal Profiling Reports (RP-REP), a new open-source cloud-enabled software that allows users to execute start-to-end gene-level ribosomal profiling and RNA-Seq analysis on a pre-configured Amazon Virtual Machine Image (AMI) hosted on AWS or on the user's own Ubuntu Linux server. The software works with FASTQ files stored locally, on AWS S3, or at the Sequence Read Archive (SRA). RP-REP automatically executes a series of customizable steps including filtering of contaminant RNA, enrichment of true ribosomal footprints, reference alignment and gene translation quantification, gene body coverage, CRAM compression, reference alignment QC, data normalization, multivariate data visualization, identification of differentially translated genes, and generation of heatmaps, co-translated gene clusters, enriched pathways, and other custom visualizations. RP-REP provides functionality to contrast RNA-SEQ and ribosomal profiling results, and calculates translational efficiency per gene. The software outputs a PDF report and publication-ready table and figure files. As a use case, we provide RP-REP results for a dengue virus study that tested cytosol and endoplasmic reticulum cellular fractions of human Huh7 cells pre-infection and at 6 h, 12 h, 24 h, and 40 h post-infection. Case study results, Ubuntu installation scripts, and the most recent RP-REP source code are accessible at GitHub. The cloud-ready AMI is available at AWS (AMI ID: RPREP RSEQREP (Ribosome Profiling and RNA-Seq Reports) v2.1 (ami-00b92f52d763145d3)).

AMI↗
PubMed2022

A custom library construction method for super-resolution ribosome profiling in Arabidopsis.

BACKGROUND: Ribosome 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. RESULTS: Here 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. CONCLUSIONS: We 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.

3-nt periodicity↗
PubMed2000

Growth phase-coupled changes of the ribosome profile in natural isolates and laboratory strains of Escherichia coli.

The growth phase-dependent change in sucrose density gradient centrifugation patterns of ribosomes was analyzed for both laboratory strains of Escherichia coli and natural isolates from the ECOR collection. All of the natural isolates examined formed 100S ribosome dimers in the stationary phase, and ribosome modulation factor (RMF) was associated with the ribosome dimers in the ECOR strains as in the laboratory strain W3110. The ribosome profile (70S monomers versus 100S dimers) follows a defined pattern over time during lengthy culture in both the laboratory strains and natural isolates. There are four discrete stages: (i) formation of 100S dimers in the early stationary phase; (ii) transient decrease in the dimer level; (iii) return of dimers to the maximum level; and (iv) dissociation of 100S dimers into 70S ribosomes, which are quickly degraded into subassemblies. The total time for this cycle of ribosome profile change, however, varied from strain to strain, resulting in apparent differences in the ribosome profiles when observed at a fixed time point. A correlation was noted in all strains between the decay of 100S ribosomes and the subsequent loss of cell viability. Two types of E. coli mutants defective in ribosome dimerization were identified, both of which were unable to survive for a prolonged period in stationary phase. The W3110 mutant, with a disrupted rmf gene, has a defect in ribosome dimerization because of lack of RMF, while strain Q13 is unable to form ribosome dimers due to a ribosomal defect in binding RMF.

Escherichia coli↗
PubMed1978

Age-related changes in ribosomal profiles and in vitro protein synthesis in skeletal muscle during fasting and subsequent refeeding of rats.

Age-dependent changes in ribosomal profiles and in in vitro protein synthesis in skeletal muscle (gastrocnemius muscle) were studied in rats subjected to fasting and subsequent refeeding. Male rats of 4, 7, and 21 weeks of age were used. Incorporation of 14C-leucine into protein in vitro using skeletal muscle ribosomes from fed rats decreased with the age of the animals. Sucrose density gradient analyses of ribosomal profiles revealed that the changes in protein synthesis were accompanied by progressive declines in the concentrations of polyribosomes and total RNA in skeletal muscle. After fasting for 1 or 2 days, protein synthesis in muscle of young rats dropped markedly and this change could be attributed to the fall in the concentrations of polyribosomes. Also, the concentration and total RNA in the muscles of the young rats decreased. During refeeding of rats after fasting for 2 days, the incorporation of 14C-amino acids into protein recovered more efficiently using ribosomes of skeletal muscle of young animals compared with using those of older animals. This rapid increase in the protein synthesizing activity of skeletal muscle may be related to the efficient recovery of the ribosomes toward heavier aggregates. The results indicate that young animals adapt more efficiently to fasting and refeeding than do older animals.

Aging↗
PubMed1978

Localization of host poly(A)+ mRNA in the ribosome profile of mengovirus-infected Ehrlich ascites tumor cells.

The distribution of poly(A)+ mRNA among polysomes, monosomes, and ribosome-free supernatant fractions after mengovirus infection of Ehrlich ascites tumor (EAT) cells was investigated employing sucrose gradient centrifugation of their corresponding postnuclear supernatants. Poly(A)+ mRNA was isolated from sucrose gradient fractions and quantitated in a cell-free protein synthesizing system from uninfected EAT cells. It was also localized by annealing [3H]-poly(U) to the poly(A)-tracts of mRNA present in the sucrose gradient fractions. Both experiments revealed a gradual shift of host poly(A)+ mRNA from large to small polysomes and monosomes, respectively, with the time postinfection. The greatest part of host template RNA appears to remain ribosome-bound and only a fraction seems to be detached from the ribosomes in the course of mengovirus infection. At the end of the infectious cycle, 8 h postinfection, approximately 70% of the poly(A)+ mRNA detected in uninfected cells is still biologically active, but not translated in vivo, in agreement with data from the [3H]poly(U)hybridization experiment.

Animals↗
PubMed1991

Ribosome profiles from human skeletal muscle as a measure of protein synthesis.

The purpose of this overview is to present an outline of ribosome assessment as an estimation of in vivo protein synthesis in human skeletal muscle. The method of analysis will be described and discussed together with a variety of physiological states validating the usefulness of the technique. Applications to several clinical situations will then be described, as well as the co-variation with other techniques for estimating protein metabolism. Finally, the pros and cons of the ribosome technique will be discussed.

Editorial↗
PubMed2026

seq2ribo: structure-aware integration of machine learning and simulation to predict ribosome location profiles from RNA sequences.

MOTIVATION: Ribosome dynamics are vital in the process of protein expression. Current methods rely on ribosome profiling (Ribo-seq), RNA-seq profiles, and full genomic context. This restricts their use in de novo sequence design, like messenger RNA (mRNA) vaccines. Simulation-only approaches like the Totally Asymmetric Simple Exclusion Process (TASEP) oversimplify translation by focusing solely on codon elongation times. RESULTS: We present seq2ribo, a hybrid simulation and machine learning framework that predicts ribosome A-site locations using only an mRNA sequence as input. Our method first employs a novel structure-aware TASEP (sTASEP), which models translation using a comprehensive set of fitted parameters that include codon wait times and structural features, such as local angles, base-pairing, and discrete positional buckets. The ribosome locations generated by sTASEP are then processed by a polisher model, which learns to refine the simulated ribosome distributions. seq2ribo provides high-fidelity predictions of ribosome locations across diverse cell types (iPSC, HEK293, LCL, and RPE-1), significantly outperforming baselines. seq2ribo is the first method to achieve meaningful positional correlation with observed ribosome profiles from sequence alone, reaching transcript-level Pearson correlations up to 0.920 and within-transcript shape correlations up to 0.186, where all baselines yield near-zero values on these metrics. seq2ribo also reduces elementwise error by up to 37.7% relative to the sequence-only Translatomer baseline. By adding a task-specific head, seq2ribo achieves Pearson correlations up to 0.732 with experimental translation efficiency (TE) across several cell lines, and up to 0.903 with measured protein expression. By operating from sequence alone, seq2ribo provides a new tool for synthetic biology, enabling the rational design and optimization of mRNA sequences without the need for expression-level data or genomic context. AVAILABILITY: seq2ribo is available at https://github.com/Kingsford-Group/seq2ribo.

Machine Learning↗
PubMed2026

seq2ribo: Structure-aware integration of machine learning and simulation to predict ribosome location profiles from RNA sequences.

MOTIVATION: Ribosome dynamics are vital in the process of protein expression. Current methods rely on ribosome profiling (Ribo-seq), RNA-seq profiles, and full genomic context. This restricts their use in de novo sequence design, like messenger RNA (mRNA) vaccines. Simulation-only approaches like the Totally Asymmetric Simple Exclusion Process (TASEP) oversimplify translation by focusing solely on codon elongation times. RESULTS: We present seq2ribo, a hybrid simulation and machine learning framework that predicts ribosome A-site locations using only an mRNA sequence as input. Our method first employs a novel structure-aware TASEP (sTASEP), which models translation using a comprehensive set of fitted parameters that include codon wait times and structural features, such as local angles, base-pairing, and discrete positional buckets. The ribosome locations generated by sTASEP are then processed by a polisher model, which learns to refine the simulated ribosome distributions. seq2ribo provides high-fidelity predictions of ribosome locations across diverse cell types (iPSC, HEK293, LCL, and RPE-1), significantly outperforming baselines. seq2ribo is the first method to achieve meaningful positional correlation with observed ribosome profiles from sequence alone, reaching transcript-level Pearson correlations up to 0.920 and within-transcript shape correlations up to 0.186, where all baselines yield near-zero values on these metrics. seq2ribo also reduces elementwise error by up to 37.7% relative to the sequence-only Translatomer baseline. By adding a task-specific head, seq2ribo achieves Pearson correlations up to 0.732 with experimental translation efficiency (TE) across several cell lines, and up to 0.903 with measured protein expression. By operating from sequence alone, seq2ribo provides a new tool for synthetic biology, enabling the rational design and optimization of mRNA sequences without the need for expression-level data or genomic context.

Journal Article↗
PubMed2025

A novel regulation on the developmental checkpoint protein Sda that controls sporulation and biofilm formation in Bacillus subtilis.

UNLABELLED: Biofilm formation by Bacillus subtilis is triggered by an unusually simple environmental sensing mechanism. Certain serine codons, the four TCN codons (N for A, T, C, or G), in the gene for the biofilm repressor SinR caused lowered SinR translation and subsequent biofilm induction during transition from exponential to stationary growth. Global ribosome profiling showed that ribosomes pause when translating the four UCN (U for T on the mRNA) serine codons on mRNA, but not the two AGC/AGU serine codons. We proposed a serine codon hierarchy (AGC/AGT vs TCN) in that genes enriched in the TCN serine codons may experience reduced translation efficiency when serine is limited. In this study, we designed an algorithm to score all protein-coding genes in B. subtilis NCIB3610 based on the serine codon hierarchy. We generated a short list of 50 genes that could be subject to regulation by this novel mechanism. We further investigated one such gene from the list, sda, which encodes a developmental checkpoint protein regulating both sporulation and biofilm formation. We showed that synonymously switching the TCN serine codons to AGC in sda led to delayed biofilm formation and sporulation. This engineered strain also outgrew strains with other synonymously substituted sda alleles (TCN) in competition assays for biofilm formation and sporulation. Finally, we showed that the AGC serine codon substitutions in sda elevated the Sda protein levels. This serine codon hierarchy-based novel signaling mechanism could be exploited by bacteria in adapting to stationary phase and regulating important biological processes. IMPORTANCE: Genome-wide ribosome profiling in Bacillus subtilis shows that under serine limitation, ribosomes pause on the four TCN (N for A, C, G, and T), but not AGC/AGT serine codons, during translation at a global scale. This serine codon hierarchy (AGC/T vs TCN) differentially influences the translation efficiency of genes enriched in certain serine codons. In this study, we designed an algorithm to score all 4,000+ genes in the B. subtilis genome and generated a list of 50 genes that could be subject to this novel serine codon hierarchy-mediated regulation. We further investigated one such gene, sda, encoding a developmental checkpoint protein. We show that sda and cell developments controlled by Sda are also regulated by this novel mechanism.

Bacillus subtilis↗
PubMed1998

Optimization of ribosomal RNA profile alignments.

MOTIVATION: Large alignments of ribosomal RNA sequences are maintained at various sites. New sequences are added to these alignments using a combination of manual and automatic methods. We examine the use of profile alignment methods for rRNA alignment and try to optimize the choice of parameters and sequence weights. RESULTS: Using a large alignment of eukaryotic SSU rRNA sequences as a test case, we empirically compared the performance of various sequence weighting schemes over a range of gap penalties. We developed a new weighting scheme which gives most weight to the sequences in the profile that are most similar to the new sequence. We show that it gives the most accurate alignments when combined with a more traditional sequence weighting scheme. AVAILABILITY: The source code of all software is freely available by anonymous ftp from chah.ucc.ie in the directory /home/ftp/pub/emmet,in the compressed file PRNAA.tar: CONTACT: emmet@chah.ucc.ie, des@chah.ucc.ie

Animals↗
PubMed2026

Ribosome dynamics at the conserved PGP motif governs 2A peptide-bond-skipping efficiency.

Viral 2A oligopeptides drive an unusual ribosome recoding event in which peptide-bond formation fails at a conserved PG↓P motif, producing two discrete proteins without canonical termination. Despite decades of study, the molecular basis of 2A-mediated peptide-bond skipping remains poorly understood. Here, we combine quantitative 2A reporters with high-resolution ribosome profiling to interrogate ribosome dynamics at the core 2A sequences. We identify a pausing event at the terminal proline codon of the PGP motif that functions as a kinetic decision point: ribosome dwell time at this site inversely correlates with skipping efficiency. Increasing nascent chain flexibility by inserting linkers immediately upstream of the 2A sequence reduces ribosome occupancy at the terminal proline codon and enhances peptide-bond skipping. Strikingly, amino acid repeats positioned distally upstream also modulate 2A activity, indicating long-range coupling between nascent chain properties outside of the ribosome and the peptidyl transferase center inside the ribosome. In particular, hydrophobic residues potently suppress skipping, an effect that can be rescued by extending flexible segments within the peptide exit tunnel. Together, our findings support a model in which nascent chain features-beyond the core 2A motif-dynamically tune ribosomal recoding efficiency through co-translational feedback into the catalytic center.

Ribosomes↗
PubMed2026

Ribo-ITP enables identification of translons from limited input samples.

In the last decade, an unexpectedly large number of translated regions (translons) have been discovered using ribosome profiling and proteomics. Translons can act as regulatory elements or encode functional micropeptides. However, identification of translons has been limited to cell lines or large organs due to high input requirements for conventional ribosome profiling and mass spectrometry. Here, we address this input limitation using Ribo-ITP on difficult-to-collect samples such as microdissected hippocampal tissues and single preimplantation embryos to identify thousands of translons. To test the translational capacity of the identified translons, we engineer a translon-dependent GFP reporter system and detect expression of translons initiating at ATG and near-cognate start codons in mouse embryonic stem cells (mESCs). We identify distinct expression patterns of translons using a comparative analysis of more than a thousand ribosome profiling datasets across a wide range of cell types. Further, using a machine learning model, we predict that specific upstream translons in synaptically enriched mRNAs regulate translation efficiency of the annotated coding region. Taken together, we present a proof-of-concept study to identify non-canonical translation events from low input samples which can be applied to cell and tissue types inaccessible to conventional methods.

Animals↗
PubMedDate not supplied

Characterization of Mycobacterium fortuitum isolates from sternotomy wounds by antimicrobial susceptibilities, plasmid profiles, and ribosomal ribonucleic acid gene restriction patterns.

An outbreak of sternotomy infections due to Mycobacterium fortuitum in patients who had received cardiovascular surgery occurred in a cardiothoracic hospital in Hong Kong, and 21 such isolates from different patients had antimicrobial susceptibility studies against 14 drugs in vitro. These isolates were also studied for plasmid profiles and ribosomal ribonucleic acid gene restriction patterns. The latter method proved valuable in categorization of these isolates into two groups (comprising of nine and seven isolates, respectively) and five other sporadic strains. When the plasmid profiles and ribotyping are matched against the clinical and epidemiologic data, multisource contamination is suspected to be responsible for the outbreak. The organisms were probably derived from the environment rather than contaminated surgical equipments and materials.

Adult↗
PubMed1997

[Contribution of molecular biology to biotyping in epidemiological surveys of nosocomial infectious outbreaks].

During the period between the end of 1993 and the beginning of 1994 there was a large epidemic rush from severe nosocomial infection essentially in the form of septicemia. Authors implemented a classification of these epidemics and some strains were sent to the Institute Pasteur Paris for a further examination. These strains went under nuclear biology test (study of DNA plasmidic profiles and RNA ribosomal profiles) and passed through an identification by an acheiring biotypic system using 99 sources or carbones--biotype 99. These studies have shown that its about real epidemic, because the same strains were transmitted from one patient to another. This show the precarious condition of our hospital hygiene, involving the necessity of an efficient training of our health staff.

Adult↗
PubMed1975

Ribosomal distribution in a polyamine auxotroph of Escherichia coli.

The distribution of ribosomal particles has been studied in a polyamine-deficient mutant of Escherichia coli by sucrose gradient centrifugation analysis. Lysates from starved cells contained less 70S monomers and 30S subunits but more 50S particles than those prepared from bacteria supplemented with putrescine. The addition of the polyamine to putrescine-depleted cells induced a rapid change of the ribosomal profile. A similar effect could be obtained in vitro by equilibrium dialysis against a polyamine-containing solution. The ribosomal pattern obtained from starved bacteria was specific for polyamine deficiency. We conclude that the changes in ribosomal profiles upon restoration of putrescine levels in previously starved cells denote a shift of the equilibrium between 30S-50S couples and ribosomal subunits.

Escherichia coli↗