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Karbstein, K.

Publications and source records attributed to Karbstein, K..

17 recordsLinked to original sources

Data Fusion for Integrative Species Identification Using Deep Learning

AO_SCPLOWBSTRACTC_SCPLOWDNA analyses have revolutionized species identification and taxonomic work. Yet, persistent challenges arise from little differentiation among species and considerable variation within species, particularly among closely related groups. While images are commonly used as an alternative modality for automated identification tasks, their usability is limited by the same concerns. An integrative strategy, fusing molecular and image data through machine learning, holds significant promise for fine-grained species identification. However, a systematic overview and rigorous statistical testing concerning molecular and image preprocessing and fusion techniques, including practical advice for biologists, are missing so far. We introduce a machine learning scheme that integrates both molecular and image data for species identification. Initially, we systematically assess and compare three different DNA arrangements (aligned, unaligned, SNP-reduced) and two encoding methods (fractional, ordinal). Later, artificial neural networks are used to extract visual and molecular features, and we propose strategies for fusing this information. Specifically, we investigate three strategies: I) fusing directly after feature extraction, II) fusing features that passed through a fully connected layer after feature extraction, and III) fusing the output scores of both unimodal models. We systematically and statistically evaluate these strategies for four eukaryotic datasets, including two plant (Asteraceae, Poaceae) and two animal families (Lycaenidae, Coccinellidae) using Leave-One-Out Cross-Validation (LOOCV). In addition, we developed an approach to understand molecular-and image-specific identification failure. Aligned sequences with nucleotides encoded as decimal number vectors achieved the highest identification accuracy among DNA data preprocessing techniques in all four datasets. Fusing molecular and visual features directly after feature extraction yielded the best results for three out of four datasets (52-99%). Overall, combining DNA with image data significantly increased accuracy in three out of four datasets, with plant datasets showing the most substantial improvement (Asteraceae: +19%, Poaceae: +13.6%). Even for Lycaenidae with high identification accuracy based on molecular data (>96%), a statistically significant improvement (+2.1%) was observed. Detailed analysis of confusion rates between and within genera shows that DNA alone tends to identify the genus correctly, but often fails to recognize the species. The failure to resolve species is alleviated by including image data in the training. This increase in resolution hints at. a hierarchical role of modalities in which molecular data coarsely groups the specimens to then be guided towards a more fine-grained identification by the connected image. We systematically showed and explained, for the first time, that optimizing the preprocessing and integration of molecular and image data otters significant benefits, particularly for genetically similar and morphologically indistinguishable species, enhancing species identification by reducing modality-specific failure rates and information gaps. Our results can inform integration efforts for various organism groups, improving automated identification across a wide range of eukaryotic species.

bioinformatics↗

Phylogenetic and taxonomic insights into Betula: low-coverage whole genome sequencing and plastome analysis with focus on the rare Ukrainian endemic species Betula klokovii Zaverucha

Betula klokovii Zaverucha is a rare endemic species of Ukraine that is still not well taxonomically studied. In the current pilot study, we performed low-coverage whole genome sequencing for B. klokovii, related species (B. pendula Roth and B. pubescens Ehrh.) and the assumed hybrid B. klokovii x B. pendula, assessed the genomic structure of taxa with different mapping settings using UMAP non-linear dimension reduction algorithm, extracted and assembled whole plastomes. Single Nucleotide Polymorphism (SNP) analysis based on low-coverage whole genome sequencing (LC-WGS) followed by UMAP visualization reveals the separation of B. klokovii from the other analysed taxa. The best taxonomic resolution was achieved with reads filtered for contamination. In contrast, the best result in obtaining complete plastome assemblies was achieved via NOVOPlasty pipeline and raw reads. The size for the eight newly assembled plastid genomes ranges between 160,535 and 160,625bp, GC content is 36,1%. We annotated 130 genes (113 unique) for all eight assemblies. In addition, we investigated B. klokoviis relationships with 20 other birch species and two intraspecific taxa by reconstructing plastome-based Bayesian inference and maximum likelihood phylogenies. Overall, plastome phylogeny provides a better resolution in comparison to phylogenies based on a few plastid or nuclear molecular markers. However, it could be affected by chloroplast capture, and some other factors like quality of sequencing and assembly, and is not suitable to detect hybrids when used alone. In particular, we found that B. klokovii is likely a separate taxon that is closely related to B. pubescens but morphologically and genetically distinct. The study shows that genome-wide SNP data and plastome phylogenies have a certain potential for addressing issues with specific taxa within the genus Betula L. However, to fully leverage the potential of this approach, we suggest collecting a much larger number of birch plastome sequences to be sequenced and assembled. For a better understanding of birch phylogeny there is a need for reference-grade chromosome scale genome assemblies for polyploid species.

plant biology↗

The ubiquitin-proteasome system regulates the formation of specialized ribosomes during high salt stress in yeast

Rps26-deficient ribosomes are a physiologically relevant ribosome population which arises during osmotic stress to support the translation of mRNAs involved in the response to high salt in yeast. They are formed by binding of the chaperone Tsr2 to fully assembled ribosomes to release Rps26 when intracellular Na+ concentrations rise. Tsr2-mediated Rps26 release is reversible, enabling a rapid response that conserves ribosomes. However, because the concentration of Tsr2 relative to ribosomes is low, how the released Rps26*Tsr2 complex is managed to allow for accumulation of Rps26-deficient ribosomes to nearly 50% of all ribosomes remains unclear. Here we show that released Rps26 is degraded via the Pro/N-degron pathway, enabling the accumulation of Rps26-deficient ribosomes. Substitution of the N-terminal proline of Rps26 to serine increases the stability of free Rps26, limits the accumulation of Rps26-deficient ribosomes and renders yeast sensitive to high salt. The GID-complex, an E3 ubiquitin ligase, and its adaptor Gid4, mediate polyubiquitination of Rps26 at Lys66 and Lys70. Moreover, this ubiquitination event is required for Rps26 degradation, the accumulation of Rps26-deficient ribosomes and the high salt stress resistance. Together, the data show that targeted degradation of released Rps26 from the Rps26*Tsr2 complex allows Tsr2 to be recycled, thus facilitating multiple rounds of Rps26 release.

biochemistry↗

Unstructured Domains in Assembly Factors Promote the Hierarchy of Ribosome Maturation via Their Structural Plasticity.

Ribosomes are assembled with the help of a large machinery of assembly factors (AFs), whose roles remain incompletely characterized. Recent structural studies of assembly intermediates have provided tremendous context for such studies and helped reveal novel roles, including as RNA chaperones. These structures have also revealed that a subset of the AFs have entirely non-globular structures, or large non-globular extensions, which extend across the assembling ribosomal subdomains, contacting many rRNA regions, ribosomal proteins (RPs) and other AFs. How these unusual structures could help promote assembly has remained unclear although it has been suggested that their potential for making multiple interactions helps constrain early assembly steps. By studying the roles of the AF Ltv1, an entirely non-globular protein, during late maturation steps of the 40S subunits head, we show here how the structural plasticity that derives from the non-globular structure is used to communicate maturation steps across the nascent subunit, thereby establishing the previously described hierarchy in head assembly. Our data indicate that while this structural plasticity enables integration of distinct folding and assembly steps, it also creates the potential for mutations that allow for bypass of these QC steps. These mutations are pathogenic in humans, further demonstrating the importance of proper 40S subunit assembly for protein homeostasis.

biochemistry↗

Efficient hybrid strategies for assembling the plastome, mitochondriome, and large nuclear genome of diploid Ranunculus cassubicifolius (Ranunculaceae)

Currently, it is still a challenge - in terms of laboratory effort and cost, as well as assembly quality - to unravel the sequence of large and complex genomes from non-model plants. This often hampers the study of evolutionarily intricate species groups. The species-rich genus Ranunculus (Ranunculaceae) is an angiosperm model system for the study of polyploidy, apomixis, reticulate evolution, and biogeography. However, neither mitochondrial, nor high-quality nuclear genome sequences are available. This limits phylogenomic, functional, and taxonomic analyses thus far. Here, we tested Illumina short-read, Oxford Nanopore Technology (ONT) or PacBio/HiFi long-read, and hybrid-read assembly strategies. We used the diploid progenitor species R. cassubicifolius (R. auricomus complex), and selected the best assemblies in terms of completeness, contiguity, and quality scores. We first assembled the plastome (156 kbp, 85 genes) and mitogenome (1.18 Mbp, 40 genes) sequences using Illumina and Illumina-PacBio-hybrid strategies, respectively. We also present an updated plastome and the first mitogenome phylogeny of Ranunculaceae, including studies of gene loss (e.g., infA, ycf15, or rps) with evolutionary implications. For the nuclear genome, we favored a PacBio-based assembly three-times polished with filtered reads and subsequently scaffolded into 8 pseudochromosomes by chromatin conformation data (Hi-C) as the representative sequence. We obtained a haploid genome sequence with 2.69 Gbp, 94.5% complete BUSCO embryophyta_odb10 genes found, and 31,322 annotated genes. The genomic information presented here will improve phylogenomic analyses in this species complex, and will enable advanced functional, evolutionary, and biogeographic analyses for the genus and beyond Ranunculaceae in the future. Significance StatementThe genus Ranunculus is a model system in flowering plants for polyploidy, apomixis, evolution, and biogeography research. We present the first nuclear and mitochondrial genome sequence, and the plastid evolution of Ranunculaceae. Using Illumina, ONT, and PacBio data, we developed an efficient assembly strategy that can be applied to other non-model plants. Results presented here are useful for improving population genomic and phylogenomic analyses, and enable better functional analyses at species, genus and family level.

genomics↗

A disease associated mutant reveals how Ltv1 orchestrates RP assembly and rRNA folding of the small ribosomal subunit head

Ribosomes are complex macromolecules assembled from 4 rRNAs and 79 ribosomal proteins (RPs). Their assembly is organized in a highly hierarchical manner, which is thought to avoid dead-end pathways, thereby enabling efficient assembly of ribosomes in the large quantities needed for healthy cellular growth. Moreover, hierarchical assembly also can help ensure that each RP is included in the mature ribosome. Nonetheless, how this hierarchy is achieved remains unknown, beyond the examples that depend on direct RP-RP interactions, which account for only a fraction of the observed dependencies. Using assembly of the small subunit head and a disease-associated mutation in the assembly factor Ltv1 as a model system, we dissect here how the hierarchy in RP binding is constructed. Our data demonstrate that the LIPHAK-disease-associated Ltv1 mutation leads to global defects in head assembly, which are explained by direct binding of Ltv1 to 5 out of 15 RPs, and indirect effects that affect 4 additional RPs. These indirect effects are mediated by conformational transitions in the nascent subunit that are regulated by Ltv1. Mechanistically, Ltv1 aids the recruitment of some RPs via direct protein-protein interactions, but surprisingly also delays the recruitment of other RPs. Delayed binding of key RPs also delays the acquisition of RNA structure that is stabilized by these proteins. Finally, our data also indicate direct roles for Ltv1 in chaperoning the folding of a key rRNA structural element, the three-helix junction j34-35-38. Thus, Ltv1 plays critical roles in organizing the order of both RP binding to rRNA and rRNA folding, thereby enabling efficient 40S subunit assembly.

molecular biology↗

The kinase Rio1 and a ribosome collision-dependent decay pathway survey the integrity of 18S rRNA cleavage

The 18S rRNA sequence is highly conserved, particularly at its 3-end. In contrast, the sequence around the 3-end is degenerate with similar sites nearby. How RNA is correctly processed by the endonuclease Nob1 is not known, especially because in vitro experiments have shown it to be error-prone. Here we used yeast genetics, biochemistry, and next generation sequencing to investigate a role for Rio1 in monitoring the 3-end of 18S rRNA. We demonstrate that Nob1 can miscleave its rRNA substrate and that miscleaved rRNA accumulates upon bypassing the Rio1-mediated quality control step, but not in healthy cells with intact quality control mechanisms. Mechanistically, we show that Rio1 binding to miscleaved rRNA is weakened. Accordingly, excess Pno1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA enter the polysomes and produce dominant negative growth defects, suggesting that they cause defects during translation. Our data strongly suggest that ribosome collisions identify these miscleaved 18S rRNA-containing ribosomes as partially functional and target them for degradation. Altogether, the data support a model in which Rio1 inspects the 3-end of the nascent 18S rRNA, only removing Nob1 and Pno1 from the ribosomes with precisely cleaved 18S rRNA to prevent miscleaved 18S rRNA-containing ribosomes from erroneously engaging in translation, where they induce ribosome collisions. The data also demonstrate how ribosome collisions "purify" the cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.

molecular biology↗

Chaperone-Directed Ribosome Repair after Oxidative Damage

Reactive oxygen species are ubiquitous in cells, where they damage RNA and protein. While relief mechanisms, including effects on translation, have been described, whether ribosomes are functionally compromised by oxidation, and how this damage is mitigated, remains unknown. Here we show that cysteines in ribosomal proteins, including Rps26, are readily oxidized and rendered non-functional, which is exacerbated when yeast are exposed to H2O2. Oxidized Rps26 is released from ribosomes by its chaperone Tsr2, which allows for repair of the damaged ribosomes with newly made Rps26. Ribosomes containing damaged Rpl10 or Rpl23 are similarly repaired by their chaperones, Sqt1 and Bcp1. Ablation of this pathway impairs growth, which is exacerbated under oxidative stress. These findings reveal a novel mechanism for chaperone-mediated ribosome repair with implications for aging and health. One-Sentence SummaryChaperones repair thiol-oxidized ribosomes by release of damaged components and incorporation of newly made ribosomal proteins.

molecular biology↗

The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate "handover" from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

biochemistry↗

Blocking a dead-end assembly pathway creates a point of regulation for the DEAD-box ATPase Has1 and prevents platform misassembly

Assembly of ribosomal subunits occurs via parallel pathways, which accelerate the process and render it more robust. Nonetheless, in vitro analyses have also demonstrated that some assembly pathways are dead-ends, presumably due to rRNA misfolding. If and how these non-productive pathways are avoided during assembly in vivo remains unknown. Here we use a combination of biochemical, genetic, proteomic and structural analyses to demonstrate a role for assembly factors in biasing the folding landscape away from non-productive intermediates. By binding Rrp36, Rrp5 is prevented from forming a premature interaction with the platform, which leads to a dead-end intermediate, and a misassembled platform that is functionally defective. The DEAD-box ATPase Has1 separates Rrp5 and Rrp36, allowing Rrp5 to reposition to the platform, thereby promoting ribosome assembly and enabling rRNA processing. Thus, Rrp36 establishes an ATP-dependent regulatory point that ensures correct platform assembly by opening a new folding channel that avoids funnels to misfolding.

biochemistry↗

Unraveling phylogenetic relationships, reticulate evolution, and genome composition of polyploid plant complexes by RAD-Seq and Hyb-Seq

Complex genome evolution of young polyploid complexes is poorly understood. Besides challenges caused by hybridization, polyploidization, and incomplete lineage sorting, bioinformatic analyses are often exacerbated by missing information on progenitors, ploidy, and reproduction modes. By using a comprehensive, self-developed bioinformatic pipeline integrating phylogenetic, structure, network, and SNP-origin analyses, we for the first time unraveled polyploid phylogenetic relationships and genome evolution within the large Eurasian Ranunculus auricomus species complex comprising more than 840 taxa. Our results rely on 97,312 genomic RAD-Seq loci, target enrichment of 576 nuclear genes (48 phased), and 71 plastid regions (Hyb-Seq; OMICS-data) derived from the 75 most widespread polyploid apomictic taxa and four di- and one tetraploid potential sexual progenitor species. Phylogenetic tree and structure analyses consistently showed 3-5 supported polyploid groups, each containing sexual progenitor species. In total, analyses revealed four diploid sexual progenitors and a one unknown, probably extinct progenitor, contributing to the genome composition of R. auricomus polyploids. Phylogenetic network, structure, and SNP-origin analyses based on RAD-Seq loci and phased nuclear genes completed by plastid data demonstrated predominantly allopolyploid origins, each involving 2-3 different diploid sexual subgenomes. Allotetraploid genomes were characterized by subgenome dominance and large proportions of interspecific, non-hybrid SNPs, indicating an enormous degree of post-origin evolution (i.e., Mendelian segregation of the diploid hybrid generations, back-crossings, and gene flow due to facultative sexuality of apomicts), but only low proportions of lineage-specific SNPs. The R. auricomus model system is the first large European polyploid species complex studied with reduced representation OMICS data. Our bioinformatic pipeline underlines the importance of combining different approaches and datasets to successfully unveil how reticulate evolution and post-origin processes shape the diversity of polyploid plant complexes.

evolutionary biology↗

The chaperone Tsr2 regulates Rps26 release and reincorporation from mature ribosomes to enable a reversible, ribosome-mediated response to stress

Although ribosome assembly is quality controlled to maintain protein homeostasis, different ribosome populations have been described. How these form, especially under stress conditions that impact energy levels and stop the energy-intensive production of ribosomes, remains unknown. Here we demonstrate how a physiologically relevant ribosome population arises during high Na+ and pH stress via dissociation of Rps26 from fully assembled ribosomes to enable a translational response to these stresses. The chaperone Tsr2 releases Rps26 in the presence of high Na or pH in vitro and is required for Rps26 release in vivo. Moreover, Tsr2 stores free Rps26 and promotes re-incorporation of the protein, thereby repairing the subunit after the stress subsides. Our data implicate a residue in Rps26 involved in Diamond Blackfan Anemia in mediating the effects of Na+. These data demonstrate how different ribosome populations can arise rapidly, without major energy input, and without bypass of quality control mechanisms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/438496v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@151e234org.highwire.dtl.DTLVardef@d9d3fcorg.highwire.dtl.DTLVardef@ec628corg.highwire.dtl.DTLVardef@13a0917_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LITsr2 releases Rps26 from mature ribosomes to remodel ribosome populations C_LIO_LITsr2 stores released Rps26 and allows for ribosome repair after stress C_LIO_LIRps26 exploits a Mg binding site linked to Diamond Blackfan Anemia (DBA) C_LIO_LIChaperone-mediated ribosome remodeling might be common for chaperoned RPs C_LI

molecular biology↗

Ribosome assembly factors modulate the RNA folding landscape to prevent misfolding

While RNAs are known to misfold, the underlying molecular causes remain unclear, and focused on alternative secondary structures. Conversely, how RNA chaperones function in a biological context to promote folding beyond duplex annealing, remains unknown. Here we show in a combination of DMS-MaPseq, structural analyses, biochemical experiments, and yeast genetics that three-way junctions are prone to misfolding during assembly of the small ribosomal subunit in vivo. We identify ubiquitous roles for ribosome assembly factors in chaperoning their folding by preventing the formation of tertiary interactions. In the absence of these assembly factors, tertiary interactions kinetically trap misfolded three-way junctions, thereby blocking further progress in the assembly cascade. While these protein chaperones act indirectly by binding the interaction partners, our analyses also suggest direct roles for snoRNAs in binding and chaperoning three-way junctions during transcription. This work furthermore shows that the dissociation of assembly factors renders reversible folding steps irreversible, thereby setting up a timer that regulates not just the flux of assembly but also dictates the propensity of misfolded intermediates to escape quality control. Finally, the data demonstrate how RNA chaperones act locally to unfold specific tertiary interactions, in contrast to protein chaperones, which globally unfold misfolded proteins.

biochemistry↗

Phylogenomics supported by geometric morphometrics reveals delimitation of sexual species within the polyploid apomictic Ranunculus auricomus complex (Ranunculaceae)

Species are the basic units of biodiversity and evolution. Nowadays, they are widely considered as ancestor-descendant lineages. Their definition remains a persistent challenge for taxonomists due to lineage evolutionary role and circumscription, i.e., persistence in time and space, ecological niche or a shared phenotype of a lineage. Recognizing and delimiting species is particularly methodically challenging in fast-evolving, evolutionary young species complexes often characterized by low genetic divergence, hybrid origin, introgression and incomplete lineage sorting (ILS). Ranunculus auricomus is a large Eurasian apomictic polyploid complex that probably has arisen from the hybridization of a few sexual progenitor species. However, even delimitation and relationships of diploid sexual progenitors have been unclearly ranging from two to twelve species. Here, we present an innovative workflow combining phylogenomic methods based on 86,782 parameter-optimized RADseq loci and target enrichment of 663 nuclear genes together with geometric morphometrics to delimit sexual species in this evolutionary young complex (< 1 Mya). For the first time, we revealed a fully resolved and well-supported maximum likelihood (ML) tree phylogeny congruent to neighbor-net network and STRUCTURE results based on RADseq data. In a few clades, we found evidence of discordant patterns indicated by quartet sampling (QS) and reticulation events in the neighbor-net network probably caused by introgression and ILS. Together with coalescent-based species delimitation approaches based on target enrichment data, we found five main genetic lineages, with an allopatric distribution in Central and Southern Europe. A concatenated geometric morphometric data set including basal and stem leaves, as well as receptacles, revealed the same five main clusters. We accept those five morphologically differentiated, geographically isolated, genetic main lineages as species: R. cassubicifolius s.l. (incl. R. carpaticola), R. flabellifolius, R. envalirensis s.l. (incl. R. cebennensis), R. marsicus and R. notabilis s.l. (incl. R. austroslovenicus, R. calapius, R. mediocompositus, R. peracris and R. subcarniolicus). Our comprehensive workflow combing phylogenomic methods supported by geometric morphometrics proved to be successful in delimiting closely related sexual taxa and applying an evolutionary species concept, which is also transferable to other evolutionarily young species complexes.

evolutionary biology↗

Phylogenomics unravels speciation patterns in temperate-montane plant species: a case study on the recently radiating Ranunculus auricomus species complex

The time frame and geographical patterns of diversification processes in European temperate-montane herbs are still not well understood. We used the sexual species of the Ranunculus auricomus complex as a model system to understand how vicariance vs. dispersal processes in the context of Pleistocene climatic fluctuations have triggered speciation in temperate-montane plant species. We employed Target Enrichment sequence data from about 600 nuclear genes and coalescent-based species tree inference methods to resolve phylogenetic relationships among the sexual taxa of the complex. We estimated absolute divergence times and, using ancestral range reconstruction, we tested if speciation was rather enhanced by vicariance or dispersal processes. Phylogenetic relationships among taxa were fully resolved. Incongruence among species trees mainly concerned the intraspecific relationships in R. notabilis s.l., R. cassubicifolius s.l., and the position of the tetraploid R. marsicus. Speciation events took place in a very short time at the end of the Mid-Pleistocene Transition (830-580 ka). A second wave of intraspecific geographical differentiation within and around the European mountain systems happened between 200-100 ka. Ancestral range reconstruction supports the existence of a widespread European ancestor of the R. auricomus complex. Vicariance processes have triggered allopatric speciation in temperate-montane plant species during the climatic deterioration occurred in the last phase of the Mid-Pleistocene Transition. Vegetation restructuring from forest into tundra could have confined these forest species into isolated glacial refugia. During subsequent warming periods, range expansions of these locally distributed species could have been hampered by congeneric competitors in the same habitat.

evolutionary biology↗

A kinase-dependent checkpoint prevents escape of immature ribosomes into the translating pool

Premature release of nascent ribosomes into the translating pool must be prevented, as these do not support viability and may be prone to mistakes. Here we show that the kinase Rio1, the nuclease Nob1, and its binding partner Pno1 cooperate to establish a checkpoint that prevents the escape of immature ribosomes into polysomes. Nob1 blocks mRNA recruitment, and rRNA cleavage is required for its dissociation from nascent 40S subunits, thereby setting up a checkpoint for maturation. Rio1 releases Nob1 and Pno1 from pre-40S ribosomes to discharge nascent 40S into the translating pool. Weakly binding Nob1 and Pno1 mutants can bypass the requirement for Rio1, and Pno1 mutants rescue cell viability. In these strains, immature ribosomes escape into the translating pool, where they cause fidelity defects and perturb protein homeostasis. Thus, the Rio1-Nob1-Pno1 network establishes a checkpoint that safeguards against the release of immature ribosomes into the translating pool.

molecular biology↗

Subunit joining exposes nascent pre-40S rRNA for processing and quality control

During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control remains unknown. To address these questions, we determined the structure of an 80S-like ribosome assembly intermediate to an overall resolution of 3.4 [A]. The structure, validated by biochemical data, resolves a large body of previously paradoxical data and illustrates how assembly and translation factors cooperate to promote the formation of an interface that lacks many mature subunit contacts but is stabilized by the universally conserved Dim1. We also show how Tsr1 enables this interface by blocking the canonical binding of eIF5B to 40S subunits, while maintaining its binding to 60S. The structure also shows how this interface leads to unfolding of the platform, which allows for temporal regulation of the ATPase Fap7, thus linking 40S maturation to quality-control during ribosome assembly.

biochemistry↗