Biology subjects

Ozadam, H.

Publications and source records attributed to Ozadam, H..

16 recordsLinked to original sources

bioRxiv2024

Translation efficiency covariation across cell types is a conserved organizing principle of mammalian transcriptomes

Characterizing shared patterns of RNA expression between genes across conditions has led to the discovery of regulatory networks and novel biological functions. However, it is unclear if such coordination extends to translation. Here, we uniformly analyzed 3,819 ribosome profiling datasets from 117 human and 94 mouse tissues and cell lines. We introduce the concept of translation efficiency covariation (TEC), identifying coordinated translation patterns across cell types. We nominate candidate mechanisms driving shared patterns of translation regulation. TEC is conserved across human and mouse cells and uncovers gene functions that are not evident from RNA or protein co-expression. Moreover, our observations indicate that proteins that physically interact are highly enriched for positive covariation at both translational and transcriptional levels. Our findings establish TEC as a conserved organizing principle of mammalian transcriptomes.

bioinformatics↗

bioRxiv2024

RiboGraph: An interactive visualization system for ribosome profiling data at read length resolution

SummaryRibosome 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. 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 and eliminates the need for data preprocessing. 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 ImplementationSource 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.

bioinformatics↗

bioRxiv2023

The Ribosome Assembly Factor Reh1 is Released from the Polypeptide Exit Tunnel in the Pioneer Round of Translation

Assembly of functional ribosomal subunits and successfully delivering them to the translating pool is a prerequisite for protein synthesis and cell growth. In S. cerevisiae, the ribosome assembly factor Reh1 binds to pre-60S subunits at a late stage during their cytoplasmic maturation. Previous work shows that the C-terminus of Reh1 inserts into the polypeptide exit tunnel (PET) of the pre-60S subunit. Unlike canonical assembly factors, which associate exclusively with pre-60S subunits, we observed that Reh1 sediments with polysomes in addition to free 60S subunits. We therefore investigated the intriguing possibility that Reh1 remains associated with 60S subunits after the release of the anti-association factor Tif6 and after subunit joining. Here, we show that Reh1-bound nascent 60S subunits associate with 40S subunits to form actively translating ribosomes. Using selective ribosome profiling, we found that Reh1-bound ribosomes populate open reading frames near start codons. Reh1-bound ribosomes are also strongly enriched for initiator tRNA, indicating they are associated with early elongation events. Using single particle cryo-electron microscopy to image cycloheximide-arrested Reh1-bound 80S ribosomes, we found that Reh1-bound 80S contain A site peptidyl tRNA, P site tRNA and eIF5A indicating that Reh1 does not dissociate from 60S until early stages of translation elongation. We propose that Reh1 is displaced by the elongating peptide chain. These results identify Reh1 as the last assembly factor released from the nascent 60S subunit during its pioneer round of translation.

molecular biology↗

bioRxiv2021

Single cell quantification of ribosome occupancy in early mouse development

Technological limitations precluded transcriptome-wide analyses of translation at single cell resolution. To solve this challenge, we developed a novel microfluidic isotachophoresis approach, named RIBOsome profiling via IsoTachoPhoresis (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key regulatory mechanism of genes involved in centrosome organization and N6-methyladenosine modification of RNAs. Our high coverage measurements enabled the first analysis of allele-specific ribosome engagement in early development and led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes. Finally, by integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle stage oocytes is the predominant determinant of protein abundance in the zygote. Taken together, these findings resolve the long-standing paradox of low correlation between RNA expression and protein abundance in early embryonic development. The novel Ribo-ITP approach will enable numerous applications by providing high coverage and high resolution ribosome occupancy measurements from ultra-low input samples including single cells.

developmental biology↗

bioRxiv2020

Systematic evaluation of chromosome conformation capture assays

Chromosome conformation capture (3C)-based assays are used to map chromatin interactions genome-wide. Quantitative analyses of chromatin interaction maps can lead to insights into the spatial organization of chromosomes and the mechanisms by which they fold. A number of protocols such as in situ Hi-C and Micro-C are now widely used and these differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of experimental parameters of 3C-based protocols. We find that different protocols capture different 3D genome features with different efficiencies. First, the use of cross-linkers such as DSG in addition to formaldehyde improves signal-to-noise allowing detection of thousands of additional loops and strengthens the compartment signal. Second, fragmenting chromatin to the level of nucleosomes using MNase allows detection of more loops. On the other hand, protocols that generate larger multi-kb fragments produce stronger compartmentalization signals. We confirmed our results for multiple cell types and cell cycle stages. We find that cell type-specific quantitative differences in chromosome folding are not detected or underestimated by some protocols. Based on these insights we developed Hi-C 3.0, a single protocol that can be used to both efficiently detect chromatin loops and to quantify compartmentalization. Finally, this study produced ultra-deeply sequenced reference interaction maps using conventional Hi-C, Micro-C and Hi-C 3.0 for commonly used cell lines in the 4D Nucleome Project.

genomics↗

bioRxiv2020

Loss of coordinated expression between ribosomal and mitochondrial genes revealed by comprehensive characterization of a large family with a rare mendelian disorder

Non-canonical intronic variants are a poorly characterized yet highly prevalent class of alterations associated with Mendelian disorders. Here, we report the first RNA expression and splicing analysis from a family whose members carry a non-canonical splice variant in an intron of RPL11 (c.396+3A>G). This mutation is causative for Diamond Blackfan Anemia (DBA) in this family despite incomplete penetrance and variable expressivity. Our analyses revealed a complex pattern of disruptions with many novel junctions of RPL11. These include an RPL11 transcript that is translated with a late stop codon in the 3 untranslated region (3UTR) of the main isoform. We observed that RPL11 transcript abundance is comparable among carriers regardless of symptom severity. Interestingly, both the small and large ribosomal subunit transcripts were significantly overexpressed in individuals with a history of anemia in addition to congenital abnormalities. Finally, we discovered that coordinated expression between mitochondrial components and RPL11 was lost in all carriers, which may lead to variable expressivity. Overall, this study highlights the importance of RNA splicing and expression analyses in families for molecular characterization of Mendelian diseases.

genomics↗

bioRxiv2020

Genes with 5' terminal oligopyrimidine tracts preferentially escape global suppression of translation by the SARS-CoV-2 NSP1 protein

Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a non-structural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-Seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation level. We discover a functionally-coherent subset of human genes are preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5' terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5 UTRs from TOP transcripts can drive preferential expression in the presence of NSP1. Finally, we found that LARP1, a key effector protein in the mTOR pathway may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.

molecular biology↗

bioRxiv2020

Large domains of heterochromatin direct the formation of short mitotic chromosome loops

During mitosis chromosomes reorganise into highly compact, rod-shaped forms, thought to consist of consecutive chromatin loops around a central protein scaffold. Condensin complexes are involved in chromatin compaction, but the contribution of other chromatin proteins, DNA sequence and histone modifications is less understood. A large region of fission yeast DNA inserted into a mouse chromosome was previously observed to adopt a mitotic organisation distinct from that of surrounding mouse DNA. Here we show that a similar distinct structure is common to a large subset of insertion events in both mouse and human cells and is coincident with the presence of high levels of heterochromatic H3 lysine 9 trimethylation (H3K9me3). Hi-C and microscopy indicate that the heterochromatinised fission yeast DNA is organised into smaller chromatin loops than flanking euchromatic mouse chromatin. We conclude that heterochromatin alters chromatin loop size, thus contributing to the distinct appearance of heterochromatin on mitotic chromosomes, such as at centromeres.

cell biology↗

bioRxiv2019

RiboFlow, RiboR and RiboPy: An ecosystem for analyzing ribosome profiling data at read length resolution

SummaryRibosome 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 complete software ecosystem for researchers to study translation through ribosome profiling. Availability and ImplementationFor a quickstart, please see https://ribosomeprofiling.github.io. Source code, installation instructions and links to documentation are available on GitHub: https://github.com/ribosomeprofiling

bioinformatics↗

bioRxiv2019

A chromosome folding intermediate at the condensin-to-cohesin transition during telophase

Chromosome folding is extensively modulated as cells progress through the cell cycle. During mitosis, condensin complexes fold chromosomes in helically arranged nested loop arrays. In interphase, the cohesin complex generates loops that can be stalled at CTCF sites leading to positioned loops and topologically associating domains (TADs), while a separate process of compartmentalization drives the spatial segregation of active and inactive chromatin domains. We used synchronized cell cultures to determine how the mitotic chromosome conformation is transformed into the interphase state. Using Hi-C, chromatin binding assays, and immunofluorescence we show that by telophase condensin-mediated loops are lost and a transient folding intermediate devoid of most loops forms. By late telophase, cohesin-mediated CTCF-CTCF loops and positions of TADs start to emerge rapidly. Compartment boundaries are also established in telophase, but long-range compartmentalization is a slow process and proceeds for several hours after cells enter G1. Our results reveal the kinetics and order of events by which the interphase chromosome state is formed and identify telophase as a critical transition between condensin and cohesin driven chromosome folding.

genomics↗

bioRxiv2018

The non-canonical SMC protein SmcHD1 antagonises TAD formation on the inactive X chromosome

The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), and formation of two mega-domains. In this study we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 null cells revealed the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred in an acute SmcHD1 knockout model, but in this case, independent of Xi gene de-repression. We conclude that SmcHD1 is a key factor in antagonising TAD formation on Xi.

molecular biology↗

bioRxiv2018

Higher-Order Organization Principles of Pre-translational mRNPs

Compared to noncoding RNAs (ncRNAs) such as rRNAs and ribozymes, for which high resolution structures abound, little is known about the tertiary structures of mRNAs. In eukaryotic cells, newly made mRNAs are packaged with proteins in highly compacted mRNPs, but the manner of this mRNA compaction is unknown. Here we developed and implemented RIPPLiT (RNA ImmunoPrecipitation and Proximity Ligation in Tandem), a transcriptome-wide method for probing the 3D conformations of RNAs stably-associated with defined proteins, in this case exon junction complex (EJC) core factors. EJCs multimerize with other mRNP components to form megadalton sized complexes that protect large swaths of newly synthesized mRNAs from endonuclease digestion. Unlike ncRNAs, mRNAs behave more like flexible polymers without strong locus-specific interactions. Polymer analysis of proximity ligation data for hundreds of mRNA species demonstrates that pre-translational mammalian mRNPs fold as linear rod-like structures with no strong propensity for 5 and 3 end interaction.

molecular biology↗

bioRxiv2017

Transcriptome-wide analysis of the functional intronome using spliceosome profiling

Full understanding of eukaryotic transcriptomes and how they respond to different conditions requires deep knowledge of all sites of intron excision. Although RNA-Seq provides much of this information, the low abundance of many spliced transcripts (often due to their rapid cytoplasmic decay) limits the ability of RNA-Seq alone to reveal the full repertoire of spliced species. Here we present \"spliceosome profiling\", a strategy based on deep sequencing of RNAs co-purifying with late stage spliceosomes. Spliceosome profiling allows for unambiguous mapping of intron ends to single nucleotide resolution and branchpoint identification at unprecedented depths. Our data reveal hundreds of new introns in S. pombe and numerous others that were previously misannotated. By providing a means to directly interrogate sites of spliceosome assembly and catalysis genome-wide, spliceosome profiling promises to transform our understanding of RNA processing in the nucleus much like ribosome profiling has transformed our understanding mRNA translation in the cytoplasm.

genomics↗

bioRxiv2017

The HoxD Cluster is a Dynamic and Resilient TAD Boundary Controling the Segregation of Antagonistic Regulatory Landscapes

The mammalian HoxD cluster lies between two topologically associating domains (TADs) matching distinct, enhancer-rich regulatory landscapes. During limb development, the telomeric TAD controls the early transcription of Hoxd gene in forearm cells, whereas the centromeric TAD subsequently regulates more posterior Hoxd genes in digit cells. Therefore, the TAD boundary prevents the terminal Hoxd13 gene to respond to forearm enhancers, thereby allowing proper limb patterning. To assess the nature and function of this CTCF-rich DNA region in embryo, we compared chromatin interaction profiles between proximal and distal limb bud cells isolated from mutant stocks where various parts or this boundary region were removed. The resulting progressive release in boundary effect triggered inter-TAD contacts, favored by the activity of the newly accessed enhancers. However, the boundary was highly resilient and only a 400kb large deletion including the whole gene cluster was eventually able to merge the neighboring TADs into a single structure. In this unified TAD, both proximal and distal limb enhancers nevertheless continued to work independently over a targeted transgenic reporter construct. We propose that the whole HoxD cluster is a dynamic TAD border and that the exact boundary position varies depending on both the transcriptional status and the developmental context.

developmental biology↗

bioRxiv2017

Measuring the reproducibility and quality of Hi-C data

Hi-C is currently the most widely used assay to investigate the 3D organization of the genome and to study its role in gene regulation, DNA replication, and disease. However, Hi-C experiments are costly to perform and involve multiple complex experimental steps; thus, accurate methods for measuring the quality and reproducibility of Hi-C data are essential to determine whether the output should be used further in a study. Using real and simulated data, we profile the performance of several recently proposed methods for assessing reproducibility of population Hi-C data, including HiCRep, GenomeDISCO, HiC-Spector and QuASAR-Rep. By explicitly controlling noise and sparsity through simulations, we demonstrate the deficiencies of performing simple correlation analysis on pairs of matrices, and we show that methods developed specifically for Hi-C data produce better measures of reproducibility. We also show how to use established (e.g., ratio of intra to interchromosomal interactions) and novel (e.g., QuASAR-QC) measures to identify low quality experiments. In this work, we assess reproducibility and quality measures by varying sequencing depth, resolution and noise levels in Hi-C data from 13 cell lines, with two biological replicates each, as well as 176 simulated matrices. Through this extensive validation and benchmarking of Hi-C data, we describe best practices for reproducibility and quality assessment of Hi-C experiments. We make all software publicly available at http://github.com/kundajelab/3DChromatin_ReplicateQC to facilitate adoption in the community.

genomics↗

bioRxiv2017

An Integrative Framework For Detecting Structural Variations In Cancer Genomes

Structural variants can contribute to oncogenesis through a variety of mechanisms, yet, despite their importance, the identification of structural variants in cancer genomes remains challenging. Here, we present an integrative framework for comprehensively identifying structural variation in cancer genomes. For the first time, we apply next-generation optical mapping, high-throughput chromosome conformation capture (Hi-C), and whole genome sequencing to systematically detect SVs in a variety of cancer cells.\n\nUsing this approach, we identify and characterize structural variants in up to 29 commonly used normal and cancer cell lines. We find that each method has unique strengths in identifying different classes of structural variants and at different scales, suggesting that integrative approaches are likely the only way to comprehensively identify structural variants in the genome. Studying the impact of the structural variants in cancer cell lines, we identify widespread structural variation events affecting the functions of non-coding sequences in the genome, including the deletion of distal regulatory sequences, alteration of DNA replication timing, and the creation of novel 3D chromatin structural domains.\n\nThese results underscore the importance of comprehensive structural variant identification and indicate that non-coding structural variation may be an underappreciated mutational process in cancer genomes.

genomics↗