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Motivation: Ribosome profiling is a widely-used technique for measuring ribosome occupancy at nucleotide resolution. However, the need to analyze this data at nucleotide resolution introduces unique challenges in data visualization and analyses., Results: In this study, we introduce RiboGraph, a dedicated visualization tool designed to work with .ribo files, a specialized and efficient format for ribosome occupancy data. Unlike existing solutions that rely on large alignment files and time-consuming preprocessing steps, RiboGraph operates on a purpose designed compact file type. This efficiency allows for interactive, real-time visualization at ribosome-protected fragment length resolution. By providing an integrated toolset, RiboGraph empowers researchers to conduct comprehensive visual analysis of ribosome occupancy data., Availability and Implementation: Source code, step-by-step installation instructions and links to documentation are available on GitHub: https://github.com/ribosomeprofiling/ribograph. On the same page, we provide test files and a step-by-step tutorial highlighting the key features of RiboGraph., (© The Author(s) 2024. Published by Oxford University Press.)

In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Summary: Ribosome occupancy measurements enable protein abundance estimation and infer mechanisms of translation. Recent studies have revealed that sequence read lengths in ribosome profiling data are highly variable and carry critical information. Consequently, data analyses require the computation and storage of multiple metrics for a wide range of ribosome footprint lengths. We developed a software ecosystem including a new efficient binary file format named 'ribo'. Ribo files store all essential data grouped by ribosome footprint lengths. Users can assemble ribo files using our RiboFlow pipeline that processes raw ribosomal profiling sequencing data. RiboFlow is highly portable and customizable across a large number of computational environments with built-in capabilities for parallelization. We also developed interfaces for writing and reading ribo files in the R (RiboR) and Python (RiboPy) environments. Using RiboR and RiboPy, users can efficiently access ribosome profiling quality control metrics, generate essential plots and carry out analyses. Altogether, these components create a software ecosystem for researchers to study translation through ribosome profiling., Availability and Implementation: For a quickstart, please see https://ribosomeprofiling.github.io. Source code, installation instructions and links to documentation are available on GitHub: https://github.com/ribosomeprofiling., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2020. Published by Oxford University Press.)

Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2-3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)