Biology subjects

Barna, M.

Publications and source records attributed to Barna, M..

9 recordsLinked to original sources

bioRxiv2026

Human organoid screen links transient translational modulation to progenitor progression in corticogenesis

Human pluripotent stem cell-derived models are increasingly used to recapitulate aspects of human corticogenesis, including the emergence of diverse cortical cell types and their progression through developmental states. These systems offer an opportunity to dissect the molecular mechanisms regulating transitions between cell states, but methods for perturbing them systematically at scale remain limited. Here, we developed a semi-automated culture and high-throughput arrayed screening platform for human cortical organoids. Using a library of 1,280 small molecules designed to systematically target major signaling and regulatory pathways, we screened for modulators of EOMES, which encodes for the transcription factor TBR2, and identified Emetine, an inhibitor of translation elongation, as the top hit. The effect of Emetine on cortical cell-state progression was reproduced across human pluripotent stem cell lines, non-human primate cortical organoids, and primary human cortical tissue but not broadly across translation inhibitors. Moreover, transient exposure resulted in a shift from dividing radial glia towards cortical glutamatergic neurons. Mechanistically, nascent-proteome profiling revealed an early and coordinated remodeling of newly translated proteins that preceded detectable transcriptional changes, placing translational regulation upstream of the ensuing cell-state transition. Together, these findings highlight a role for specific translational inhibitors in promoting cortical cell-state progression and establish a scalable platform for systematic perturbation and discovery in human neural organoids.

neuroscience↗

bioRxiv2023

RAPIDASH: A tag-free enrichment of ribosome-associated proteins reveals compositional dynamics in embryonic tissues and stimulated macrophages

Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translational control. However, a lack of technologies to enrich RAPs across many sample types has prevented systematic analysis of RAP number, dynamics, and functions. Here, we have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including DHX30 and LLPH, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development that is linked to the translation of genes with long coding sequences. Finally, we characterized ribosome composition remodeling during immune activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs ranging from those with neuroregulatory functions to those activated by immune stimuli, thereby providing critical insights into how ribosomes are remodeled.

molecular biology↗

bioRxiv2023

A comprehensive rRNA variation atlas in health and disease

Ribosomal DNA and RNA (rDNA and rRNA) sequences are usually discarded from sequencing analyses. But with hundreds of copies of rDNA genes it is unknown whether they possess sequence variations that form different types of ribosomes that affect human physiology and disease. Here, we developed an algorithm for variant-calling between paralog genes (termed RGA) and compared rDNA variations found in short- and long-read sequencing data from the 1,000 Genomes Project (1KGP) and Genome In A Bottle (GIAB). We additionally developed a novel protocol for long-read sequencing full-length rRNA (RIBO-RT) from actively translating ribosomes. Our analyses identified hundreds of rDNA variants, most of which, surprisingly, are short insertion-deletions (indels) and dozens of highly abundant rRNA variants that are incorporated into translationally active ribosomes. To visualize variant ribosomes at the single cell level, we developed an in-situ rRNA sequencing method (SWITCH-seq) which revealed that variants are co-expressed within individual cells. Strikingly, by analyzing rDNA, we found that variants assemble into distinct ribosome subtypes. We discovered that these subtypes acquire different rRNA structures by successfully employing dimethyl sulfate (DMS) probing of full length rRNA. With this atlas we investigated rRNA variation changes across human tissues and cancer types. This revealed tissue-specific rRNA subtype expression in endoderm/ectoderm-derived tissues. In cancer, low abundant rRNA variants can become highly expressed, which suggests the presence of cancer-specific ribosomes. Together, this study identifies and comprehensively characterizes the diversity of ribosomes at the level of rRNA variants which is dominated by indel variants, their chromosomal location and unique structure as well as the association of ribosome variation with tissue-specific biology and cancer.

genetics↗

bioRxiv2022

Subfunctionalized expression drives evolutionary retention of ribosomal protein paralogs in vertebrates

The formation of paralogs through gene duplication is a core evolutionary process. For paralogs that encode components of protein complexes such as the ribosome, a central question is whether they encode functionally distinct proteins, or whether they exist to maintain appropriate total expression of equivalent proteins. Here, we systematically tested evolutionary models of paralog function using the mammalian ribosomal protein paralogs eS27 (Rps27) and eS27L (Rps27l) as a case study. We first showed that eS27 and eS27L have inversely correlated mRNA abundance across cell types, with the highest eS27 in lymphocytes and the highest eS27L in mammary alveolar cells and hepatocytes. By endogenously tagging the eS27 and eS27L proteins, we demonstrated that eS27- and eS27L- ribosomes associate preferentially with different transcripts. Furthermore, we generated murine eS27 and eS27L loss-of-function alleles that are homozygous lethal at different developmental stages. However, strikingly, we found that expressing eS27 protein from the endogenous eS27L locus, or vice versa, completely rescues loss-of-function lethality and yields mice with no detectable deficits. Together, these findings suggest that eS27 and eS27L are evolutionarily retained because their subfunctionalized expression patterns render both genes necessary to achieve the requisite total expression of two equivalent proteins across cell types. Our work represents the most in-depth characterization of a mammalian ribosomal protein paralog to date and highlights the importance of considering both protein function and expression when investigating paralogs.

genetics↗

bioRxiv2021

Evolutionarily divergent mTOR remodels the translatome to drive rapid wound closure and regeneration

An outstanding mystery in biology is why some species, such as the axolotl, can scarlessly heal and regenerate tissues while most mammals cannot. Here, we demonstrate that rapid activation of protein synthesis is a unique, and previously uncharacterized, feature of the injury response critical for limb regeneration in the axolotl (A. mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components, which do not change in their overall mRNA abundance but are selectively activated at the level of translation from pre-existing mRNAs in response to injury. In contrast, we show that protein synthesis is not activated in response to digit amputation in the non-regenerative mouse. We further identify the mTORC1 pathway as a key upstream signal that mediates this regenerative translation response in the axolotl. Inhibition of this pathway is sufficient to suppress translation and axolotl regeneration. Surprisingly, although mTOR is highly evolutionarily conserved, we discover unappreciated expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR in human cells, we demonstrate that this change creates a hypersensitive kinase that may allow axolotls to maintain this pathway in a highly labile state primed for rapid activation. This may underlie metabolic differences and nutrient sensing between regenerative and non-regenerative species that are key to regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.

developmental biology↗

bioRxiv2021

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop a new RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that "superfolder" mRNAs can be designed to improve both stability and expression that are further enhanced through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

molecular biology↗

bioRxiv2020

VELCRO-IP RNA-seq explores ribosome expansion segment function in translation genome-wide

SUMMARYRoles for ribosomal RNA (rRNA) in gene regulation remain largely unexplored. With hundreds of rDNA units scattered across multiple chromosomal loci, it is not possible to genetically modify rRNA in mammalian cells, hindering understanding of ribosome function. Emerging evidence suggests that expansion segments (ESs), tentacle-like rRNA extensions that vary in sequence and size across eukaryotic evolution, may provide platforms for the binding of proteins and mRNAs. Here, we develop VELCRO-IP RNA-seq: a versatile methodology to generate species-adapted ESs and map specific mRNA regions across the transcriptome that preferentially associate with ESs. By applying VELCRO-IP RNA-seq to a mammalian ES, ES9S, we identified a large array of mRNAs that are selectively recruited to ribosomes via an ES. We further characterize a set of specific 5’ UTRs that facilitate cap-independent translation through ES9S-mediated ribosome recruitment. These data provide a novel technology for studying the enigmatic ESs of the ribosome in gene-specific translation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Functional and structural basis of extreme non-coding conservation in vertebrate mRNA 5' untranslated regions

The lack of knowledge about extreme conservation in genomes remains a major gap in our understanding of the evolution of gene regulation. While previous findings have mainly focused on the role of extreme conservation at the level of DNA in transcriptional regulation, its implications for RNA biology remains largely unexplored. Here, we reveal an unexpected role of extremely conserved 5UTRs in translational regulation that is linked to the emergence of essential developmental features in vertebrate species. Endogenous deletion of conserved elements within these 5UTRs decreased gene expression at the post-transcriptional level. A large-scale reporter library of extremely conserved 5UTRs revealed the widespread presence of cis-regulatory elements that promote cell-type specific regulation of translation. As these elements function as RNA molecules, further understanding of their potential structures was essential. We therefore developed in-cell mutate-and-map (icM2), a novel methodology that maps RNA structure using high-throughput mutational analysis, previously impossible to perform inside cells. Using icM2, we determined that an extremely conserved 5UTR encodes multiple alternative structures whose relative proportions are actively maintained by ATP-dependent RNA helicases. We further show that each single nucleotide within the extremely conserved element maintains the balance of alternative structures important to control the dynamic range of protein expression. These results explain how extreme sequence conservation can lead to RNA-level biological functions encoded in the untranslated regions of vertebrate genomes.

genetics↗

bioRxiv2020

A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes, such as limb and craniofacial defects as well as bone marrow failure. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining comprehensive mouse genetics and in vivo ribosome profiling, we observe limb patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos and uncover corresponding selective translational changes of transcripts controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are reconciled by the unexpected identification that p53 functions as a master regulator of protein synthesis through transcriptional activation of 4E-BP1. 4E-BP1, a key regulator of translation, in turn, facilitates selective changes in the translatome downstream of p53 and thereby explains, at least in part, how RP haploinsufficiency elicits specificity to gene expression. These results provide an integrative model to explain how in vivo tissue-specific phenotypes emerge from a mutation in a ribosome component.

developmental biology↗