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.

Evolutionarily divergent mTOR remodels translatome for tissue regeneration.

An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot1. Here, we demonstrate that rapid activation of protein synthesis is a unique feature of the injury response critical for limb regeneration in the axolotl (Ambystoma mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components that are selectively activated at the level of translation from pre-existing messenger RNAs in response to injury. By contrast, protein synthesis is not activated in response to non-regenerative digit amputation in the mouse. We identify the mTORC1 pathway as a key upstream signal that mediates tissue regeneration and translational control in the axolotl. We discover unique expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR (axmTOR) in human cells, we show that these changes create a hypersensitive kinase that allows axolotls to maintain this pathway in a highly labile state primed for rapid activation. This change renders axolotl mTOR more sensitive to nutrient sensing, and inhibition of amino acid transport is sufficient to inhibit tissue regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in a highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.

Subfunctionalized expression drives evolutionary retention of ribosomal protein paralogs Rps27 and Rps27l 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 ribosomal protein paralogs Rps27 (eS27) and Rps27l (eS27L) as a case study. Evolutionary analysis suggests that Rps27 and Rps27l likely arose during whole-genome duplication(s) in a common vertebrate ancestor. We show that Rps27 and Rps27l have inversely correlated mRNA abundance across mouse cell types, with the highest Rps27 in lymphocytes and the highest Rps27l in mammary alveolar cells and hepatocytes. By endogenously tagging the Rps27 and Rps27l proteins, we demonstrate that Rps27- and Rps27l-ribosomes associate preferentially with different transcripts. Furthermore, murine Rps27 and Rps27l loss-of-function alleles are homozygous lethal at different developmental stages. However, strikingly, expressing Rps27 protein from the endogenous Rps27l locus or vice versa completely rescues loss-of-function lethality and yields mice with no detectable deficits. Together, these findings suggest that Rps27 and Rps27l 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.

Diversity of ribosomes at the level of rRNA variation associated with human health and disease.

With hundreds of copies of ribosomal DNA (rDNA) it is unknown whether they possess sequence variations that ultimately form different types of ribosomes. Here, we developed an algorithm for variant-calling between paralog genes (termed RGA) and compared rDNA variations with rRNA variations from long-read sequencing of translating ribosomes (RIBO-RT). Our analyses identified dozens of highly abundant rRNA variants, largely indels, that are incorporated into translationally active ribosomes and assemble into distinct ribosome subtypes encoded on different chromosomes. We developed an in-situ rRNA sequencing method (SWITCH-seq) revealing that variants are co-expressed within individual cells and found that they possess different structures. Lastly, we observed tissue-specific rRNA-subtype expression and linked specific rRNA variants to cancer. This study therefore reveals the variation landscape of translating ribosomes within human cells.

Animal models of Diamond-Blackfan anemia: updates and challenges.

Diamond-Blackfan anemia (DBA) is a ribosomopathy that is characterized by macrocytic anemia, congenital malformations, and early onset during childhood. Genetic studies have demonstrated that most patients carry mutations in one of the 20 related genes, most of which encode ribosomal proteins (RP). Treatment of DBA includes corticosteroid therapy, chronic red blood cell transfusion, and other forms of immunosuppression. Currently, hematopoietic stem cell transplantation is the only cure for DBA. Interestingly, spontaneous remissions occur in 10-20% of transfusion-dependent DBA patients. However, there is no consistent association between specific mutations and clinical manifestations. In the past decades, researchers have made significant progress in understanding the pathogenesis of DBA, but it remains unclear how the ubiquitous RP haploinsufficiency causes the erythroid-specific defect in hematopoiesis in DBA patients, and why there is a difference in penetrance and spontaneous remission among individuals who carry identical mutations. In this paper, we provide a comprehensive review of the development of DBA animal models and discuss the future research directions for these important experimental systems.

Regulation of eIF4E guides a unique translational program to control erythroid maturation.

Translation control is essential in balancing hematopoietic precursors and differentiation; however, the mechanisms underlying this program are poorly understood. We found that the activity of the major cap-binding protein eIF4E is unexpectedly regulated in a dynamic manner throughout erythropoiesis that is uncoupled from global protein synthesis rates. Moreover, eIF4E activity directs erythroid maturation, and increased eIF4E expression maintains cells in an early erythroid state associated with a translation program driving the expression of PTPN6 and Igf2bp1. A cytosine-enriched motif in the 5' untranslated region is important for eIF4E-mediated translation specificity. Therefore, selective translation of key target genes necessary for the maintenance of early erythroid states by eIF4E highlights a unique mechanism used by hematopoietic precursors to rapidly elicit erythropoietic maturation upon need.

A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production.

Recent findings suggest that the ribosome itself modulates gene expression. However, whether ribosomes change composition across cell types or control cell fate remains unknown. Here, employing quantitative mass spectrometry during human embryonic stem cell differentiation, we identify dozens of ribosome composition changes underlying cell fate specification. We observe upregulation of RPL10A/uL1-containing ribosomes in the primitive streak followed by progressive decreases during mesoderm differentiation. An Rpl10a loss-of-function allele in mice causes striking early mesodermal phenotypes, including posterior trunk truncations, and inhibits paraxial mesoderm production in culture. Ribosome profiling in Rpl10a loss-of-function mice reveals decreased translation of mesoderm regulators, including Wnt pathway mRNAs, which are also enriched on RPL10A/uL1-containing ribosomes. We further show that RPL10A/uL1 regulates canonical and non-canonical Wnt signaling during stem cell differentiation and in the developing embryo. These findings reveal unexpected ribosome composition modularity that controls differentiation and development through the specialized translation of key signaling networks.

The promises and pitfalls of specialized ribosomes.

The concept of specialized ribosomes has garnered equal amounts of interest and skepticism since it was first introduced. We ask researchers in the field to provide their perspective on the topic and weigh in on the evidence (or lack thereof) and what the future may bring.

The Mettl3 epitranscriptomic writer amplifies p53 stress responses.

The p53 transcription factor drives anti-proliferative gene expression programs in response to diverse stressors, including DNA damage and oncogenic signaling. Here, we seek to uncover new mechanisms through which p53 regulates gene expression using tandem affinity purification/mass spectrometry to identify p53-interacting proteins. This approach identified METTL3, an m6A RNA-methyltransferase complex (MTC) constituent, as a p53 interactor. We find that METTL3 promotes p53 protein stabilization and target gene expression in response to DNA damage and oncogenic signals, by both catalytic activity-dependent and independent mechanisms. METTL3 also enhances p53 tumor suppressor activity in in vivo mouse cancer models and human cancer cells. Notably, METTL3 only promotes tumor suppression in the context of intact p53. Analysis of human cancer genome data further supports the notion that the MTC reinforces p53 function in human cancer. Together, these studies reveal a fundamental role for METTL3 in amplifying p53 signaling in response to cellular stress.

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 an 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 highly structured "superfolder" mRNAs can be designed to improve both stability and expression with further enhancement 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.

Controlling tissue patterning by translational regulation of signaling transcripts through the core translation factor eIF3c.

Although gene expression is tightly regulated during embryonic development, the impact of translational control has received less experimental attention. Here, we find that eukaryotic translation initiation factor-3 (eIF3) is required for Shh-mediated tissue patterning. Analysis of loss-of-function eIF3 subunit c (Eif3c) mice reveal a unique sensitivity to the Shh receptor patched 1 (Ptch1) dosage. Genome-wide in vivo enhanced cross-linking immunoprecipitation sequence (eCLIP-seq) shows unexpected specificity for eIF3 binding to a pyrimidine-rich motif present in subsets of 5'-UTRs and a corresponding change in the translation of these transcripts by ribosome profiling in Eif3c loss-of-function embryos. We further find a transcript specific effect in Eif3c loss-of-function embryos whereby translation of Ptch1 through this pyrimidine-rich motif is specifically sensitive to eIF3 amount. Altogether, this work uncovers hidden specificity of housekeeping translation initiation machinery for the translation of key developmental signaling transcripts.

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. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and in vivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how in vivo tissue-specific phenotypes emerge in ribosomopathies.

Functional and structural basis of extreme conservation in vertebrate 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. Here, we reveal an unexpected role of extremely conserved 5' untranslated regions (UTRs) in noncanonical translational regulation that is linked to the emergence of essential developmental features in vertebrate species. Endogenous deletion of conserved elements within these 5' UTRs decreased gene expression, and extremely conserved 5' UTRs possess cis-regulatory elements that promote cell-type-specific regulation of translation. We further developed in-cell mutate-and-map (icM2), a new methodology that maps RNA structure inside cells. Using icM2, we determined that an extremely conserved 5' UTR encodes multiple alternative structures and that each single nucleotide within the 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.

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.

Therapy and prevention of obesity: nutrition, physical activity and medical treatment / Az elhízás kezelése és megelozése: táplálkozás, testmozgás, orvosi lehetoségek.

Összefoglaló. Az elhízás és következményes megbetegedései fontos népegészségügyi problémát jelentenek hazánkban is. Kezelése komoly szakmai kihívás, ugyanakkor prevenciója eredményesebb lehet. Az elhízott betegekkel leggyakrabban találkozó háziorvosok, más szakorvosok és egészségügyi szakemberek részérol nagy igény van egy viszonylag rövid, áttekintheto, naprakész gyakorlatias útmutatóra. A különbözo orvosszakmai társaságokban tevékenykedo, évtizedes szakmai tapasztalatokkal rendelkezo szerzok összefoglalják tudományosan megalapozott, bizonyítékokon alapuló ismereteiket. Az elhízás kezelését lépcsozetesen célszeru megkezdeni, elotte felmérve a beteg motivációját, általános állapotát, lehetoségeit. A szerzok leírják az energiaszükséglet meghatározásával, az étrenddel és a fizikai aktivitás megtervezésével kapcsolatos alapveto szempontokat. Felsorolják a hazánkban elérheto gyógyszereket és metabolikus sebészeti beavatkozásokat, az életmódi támogatás igényét. Az elhízás megelozésében az élet elso 1000 napjának táplálkozása, a késobbiekben a szüloi minta a meghatározó. Sok kihasználatlan lehetosége van a háziorvosok, a lakóközösségek, az állami szervek koordinált együttmuködésének, helyi kezdeményezéseknek. Az elhízás betegségként való meghatározása egyaránt igényel egészségpolitikai és kormányzati támogatást, az elhízottak ellátására szakosodott multidiszciplináris centrumok számának és kompetenciájának növelését. Orv Hetil. 2021; 162(9): 323-335. Summary. Obesity and related morbidities have a high public health impact in Hungary. The treatment is a challenge, but prevention seems more effective. General practitioners, other specialists and health care professionals who are treating obese persons require short, summarized, updated and practical guideline. Hungarian medical professionals of different scientific societies, having decennial practices, are summarizing their evidence-based knowledge. Obesity management requires step by step approach, evaluating previously the general health condition, motivation and options of the patients. The measurement of energy requirement, planning of diet and physical activities, available surgical methods and medications are described in detail with life style and mental support needed. The most important period in the prevention of obesity is the first 1000 days from conception. Other significant factors are the life style habits of the parents. Proper obesity prevention requires better coordination of primary health care, community and governmental activities. Obesity should be defined as morbidity, therefore stronger governmental support and more health-policy initiatives are needed, beside increasing number and developing of multidisciplinary centres. Orv Hetil. 2021; 162(9): 323-335.

Optogenetic manipulation of cellular communication using engineered myosin motors.

Cells achieve highly efficient and accurate communication through cellular projections such as neurites and filopodia, yet there is a lack of genetically encoded tools that can selectively manipulate their composition and dynamics. Here, we present a versatile optogenetic toolbox of artificial multi-headed myosin motors that can move bidirectionally within long cellular extensions and allow for the selective transport of GFP-tagged cargo with light. Utilizing these engineered motors, we could transport bulky transmembrane receptors and organelles as well as actin remodellers to control the dynamics of both filopodia and neurites. Using an optimized in vivo imaging scheme, we further demonstrate that, upon limb amputation in axolotls, a complex array of filopodial extensions is formed. We selectively modulated these filopodial extensions and showed that they re-establish a Sonic Hedgehog signalling gradient during regeneration. Considering the ubiquitous existence of actin-based extensions, this toolbox shows the potential to manipulate cellular communication with unprecedented accuracy.

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

Roles for ribosomal RNA (rRNA) in gene regulation remain largely unexplored. With hundreds of rDNA units positioned across multiple loci, it is not possible to genetically modify rRNA in mammalian cells, hindering understanding of ribosome function. It remains elusive whether expansion segments (ESs), tentacle-like rRNA extensions that vary in sequence and size across eukaryotic evolution, may have functional roles in translation control. Here, we develop variable expansion segment-ligand chimeric ribosome immunoprecipitation RNA sequencing (VELCRO-IP RNA-seq), a versatile methodology to generate species-adapted ESs and to map specific mRNA regions across the transcriptome that preferentially associate with ESs. Application of VELCRO-IP RNA-seq to a mammalian ES, ES9S, identified a large array of transcripts that are selectively recruited to ribosomes via an ES. We further characterize a set of 5' UTRs that facilitate cap-independent translation through ES9S-mediated ribosome binding. Thus, we present a technology for studying the enigmatic ESs of the ribosome, revealing their function in gene-specific translation.