Ribosome Assembly Research Articles

OsTHA8 encodes a pentatricopeptide repeat protein required for RNA editing and splicing during rice chloroplast development

In higher plants, the chloroplast is the most important organelle for photosynthesis and for numerous essential metabolic processes in the cell. Although many genes involved in chloroplast development have been identified, the mechanisms underlying such development are not fully understood. In this study, a rice (Oryza sativa) mutant exhibiting pale green color and seedling lethality was isolated from a mutant library. The mutated gene was identified as an ortholog of THA8 (thylakoid assembly 8) in Arabidopsis and maize. This gene is designated as OsTHA8 hereafter. OsTHA8 showed a typical pentatricopeptide repeat (PPR) characteristic of only four PPR motifs. Inactivation of OsTHA8 led to a deficiency in chloroplast development in the rice seedling stage. OsTHA8 was expressed mainly in young leaves and leaf sheaths. The OsTHA8 protein was localized to the chloroplast. Loss of function of OsTHA8 weakened the editing efficiency of ndhB-611/737 and rps8-182 transcripts under normal conditions. Y2H and BiFC indicated that OsTHA8 facilitates RNA editing by forming an editosome with multiple organellar RNA editing factor (OsMORF8) and thioredoxin z (OsTRXz), which function in RNA editing in rice chloroplasts. Defective OsTHA8 impaired chloroplast ribosome assembly and resulted in reduced expression of PEP-dependent genes and photosynthesis-related genes. Abnormal splicing of the chloroplast gene ycf3 was detected in ostha8. These findings reveal a synergistic regulatory mechanism of chloroplast biogenesis mediated by RNA, broaden the function of the PPR family, and shed light on the RNA editing complex in rice.

Molecular functions of RNA helicases during ribosomal subunit assembly.

During their biogenesis, the ribosomal subunits undergo numerous structural and compositional changes to achieve their final architecture. RNA helicases are a key driving force of such remodelling events but deciphering their particular functions has long been challenging due to lack of knowledge of their molecular functions and RNA substrates. Advances in the biochemical characterisation ofRNA helicase activities together with new insights into RNA helicase binding sites on pre-ribosomes and structural snapshots of pre-ribosomal complexes containing RNA helicases now open the door to a deeper understanding of precisely how different RNA helicases contribute to ribosomal subunit maturation.

A novel Modulator of Ring Stage Translation (MRST) gene alters artemisinin sensitivity in Plasmodium falciparum.

The implementation of artemisinin (ART) combination therapies (ACTs) has greatly decreased deaths caused by Plasmodium falciparum malaria, but increasing ACT resistance in Southeast Asia and Africa could reverse this progress. Parasite population genetic studies have identified numerous genes, single-nucleotide polymorphisms (SNPs), and transcriptional signatures associated with altered artemisinin activity with SNPs in the Kelch13 (K13) gene being the most well-characterized artemisinin resistance marker. However, there is an increasing evidence that resistance to artemisinin in P. falciparum is not related only to K13 SNPs, prompting the need to characterize other novel genes that can alter ART responses in P. falciparum. In our previous analyses of P. falciparum piggyBac mutants, several genes of unknown function exhibited increased sensitivity to artemisinin that was similar to a mutant of K13. Further analysis of these genes and their gene co-expression networks indicated that the ART sensitivity cluster was functionally linked to DNA replication and repair, stress responses, and maintenance of homeostatic nuclear activity. In this study, we have characterized PF3D7_1136600, another member of the ART sensitivity cluster. Previously annotated as a conserved Plasmodium gene of unknown function, we now provide putative annotation of this gene as a Modulator of Ring Stage Translation (MRST). Our findings reveal that the mutagenesis of MRST affects gene expression of multiple translation-associated pathways during the early ring stage of asexual development via putative ribosome assembly and maturation activity, suggesting an essential role of MRST in protein biosynthesis and another novel mechanism of altering the parasite's ART drug response.IMPORTANCEPlasmodium falciparum malaria killed more than 600,000 people in 2021, though ACTs have been critical in reducing malaria mortality as a first-line treatment for infection. However, ACT resistance in Southeast Asia and emerging resistance in Africa are detrimental to this progress. Mutations to Kelch13 (K13) have been identified to confer increased artemisinin tolerance in field isolates, however, genes other than K13 are implicated in altering how the parasite responds to artemisinin prompts additional analysis. Therefore, in this study we have characterized a P. falciparum mutant clone with altered sensitivity to artemisinin and identified a novel gene (PF3D7_1136600) that is associated with alterations to parasite translational metabolism during critical timepoints for artemisinin drug response. Many genes of the P. falciparum genome remain unannotated, posing a challenge for drug-gene characterizations in the parasite. Therefore, through this study, we have putatively annotated PF3D7_1136600 as a novel MRST gene and have identified a potential link between MRST and parasite stress response mechanisms.

Emerging Quantitative Biochemical, Structural, and Biophysical Methods for Studying Ribosome and Protein-RNA Complex Assembly.

Ribosome assembly is one of the most fundamental processes of gene expression and has served as a playground for investigating the molecular mechanisms of how protein-RNA complexes (RNPs) assemble. A bacterial ribosome is composed of around 50 ribosomal proteins, several of which are co-transcriptionally assembled on a ~4500-nucleotide-long pre-rRNA transcript that is further processed and modified during transcription, the entire process taking around 2 min in vivo and being assisted by dozens of assembly factors. How this complex molecular process works so efficiently to produce an active ribosome has been investigated over decades, resulting in the development of a plethora of novel approaches that can also be used to study the assembly of other RNPs in prokaryotes and eukaryotes. Here, we review biochemical, structural, and biophysical methods that have been developed and integrated to provide a detailed and quantitative understanding of the complex and intricate molecular process of bacterial ribosome assembly. We also discuss emerging, cutting-edge approaches that could be used in the future to study how transcription, rRNA processing, cellular factors, and the native cellular environment shape ribosome assembly and RNP assembly at large.

Ribosomal Protein uS5 and Friends: Protein-Protein Interactions Involved in Ribosome Assembly and Beyond.

Ribosomal proteins are fundamental components of the ribosomes in all living cells. The ribosomal protein uS5 (Rps2) is a stable component of the small ribosomal subunit within all three domains of life. In addition to its interactions with proximal ribosomal proteins and rRNA inside the ribosome, uS5 has a surprisingly complex network of evolutionarily conserved non-ribosome-associated proteins. In this review, we focus on a set of four conserved uS5-associated proteins: the protein arginine methyltransferase 3 (PRMT3), the programmed cell death 2 (PDCD2) and its PDCD2-like (PDCD2L) paralog, and the zinc finger protein, ZNF277. We discuss recent work that presents PDCD2 and homologs as a dedicated uS5 chaperone and PDCD2L as a potential adaptor protein for the nuclear export of pre-40S subunits. Although the functional significance of the PRMT3-uS5 and ZNF277-uS5 interactions remain elusive, we reflect on the potential roles of uS5 arginine methylation by PRMT3 and on data indicating that ZNF277 and PRMT3 compete for uS5 binding. Together, these discussions highlight the complex and conserved regulatory network responsible for monitoring the availability and the folding of uS5 for the formation of 40S ribosomal subunits and/or the role of uS5 in potential extra-ribosomal functions.

A novel protein encoded by circRsrc1 regulates mitochondrial ribosome assembly and translation during spermatogenesis

BackgroundCircular RNAs (circRNAs) are a large class of mammalian RNAs. Several protein products translated by circRNAs have been reported to be involved in the development of various tissues and systems; however, their physiological functions in male reproduction have yet not been explored.ResultsHere, we report an endogenous circRNA (circRsrc1) that encodes a novel 161-amino-acid protein which we named Rsrc1-161aa through circRNA sequencing coupled with mass spectrometry analysis on mouse testicular tissues. Deletion of Rsrc1-161aa in mice impaired male fertility with a significant decrease in sperm count and motility due to dysfunctions of mitochondrial energy metabolism. A series of in vitro rescue experiments revealed that circRsrc1 regulates mitochondrial functions via its encoded protein Rsrc1-161aa. Mechanistically, Rsrc1-161aa directly interacts with mitochondrial protein C1qbp and enhances its binding activity to mitochondrial mRNAs, thereby regulating the assembly of mitochondrial ribosomes and affecting the translation of oxidative phosphorylation (OXPHOS) proteins and mitochondrial energy metabolism.ConclusionsOur studies reveal that Rsrc1-161aa protein encoded by circRsrc1 regulates mitochondrial ribosome assembly and translation during spermatogenesis, thereby affecting male fertility.

A Photoactivated Ru (II) Polypyridine Complex Induced Oncotic Necrosis of A549 Cells by Activating Oxidative Phosphorylation and Inhibiting DNA Synthesis as Revealed by Quantitative Proteomics.

The ruthenium polypyridine complex [Ru(dppa)2(pytp)] (PF6)2 (termed as ZQX-1), where dppa = 4,7-diphenyl-1,10-phenanthroline and pytp = 4'-pyrene-2,2':6',2''-terpyridine, has been shown a high and selective cytotoxicity to hypoxic and cisplatin-resistant cancer cells either under irradiation with blue light or upon two-photon excitation. The IC50 values of ZQX-1 towards A549 cancer cells and HEK293 health cells are 0.16 ± 0.09 µM and >100 µM under irradiation at 420 nm, respectively. However, the mechanism of action of ZQX-1 remains unclear. In this work, using the quantitative proteomics method we identified 84 differentially expressed proteins (DEPs) with |fold-change| ≥ 1.2 in A549 cancer cells exposed to ZQX-1 under irradiation at 420 nm. Bioinformatics analysis of the DEPs revealed that photoactivated ZQX-1 generated reactive oxygen species (ROS) to activate oxidative phosphorylation signaling to overproduce ATP; it also released ROS and pyrene derivative to damage DNA and arrest A549 cells at S-phase, which synergistically led to oncotic necrosis and apoptosis of A549 cells to deplete excess ATP, evidenced by the elevated level of PRAP1 and cleaved capase-3. Moreover, the DNA damage inhibited the expression of DNA repair-related proteins, such as RBX1 and GPS1, enhancing photocytotoxicity of ZQX-1, which was reflected in the inhibition of integrin signaling and disruption of ribosome assembly. Importantly, the photoactivated ZQX-1 was shown to activate hypoxia-inducible factor 1A (HIF1A) survival signaling, implying that combining use of ZQX-1 with HIF1A signaling inhibitors may further promote the photocytotoxicity of the prodrug.

Novel rRNA transcriptional activity of NhaR revealed by its growth recovery for the bipA-deleted Escherichia coli at low temperature.

The BipA protein is a universally conserved GTPase in bacterial species and is structurally similar to translational GTPases. Despite its wide distribution, BipA is dispensable for growth under optimal growth conditions but is required under stress conditions. In particular, bipA-deleted cells (ESC19) have been shown to display a variety of phenotypic changes in ribosome assembly, capsule production, lipopolysaccharide (LPS) synthesis, biofilm formation, and motility at low temperature, suggesting its global regulatory roles in cold adaptation. Here, through genomic library screening, we found a suppressor clone containing nhaR, which encodes a Na+-responsive LysR-type transcriptional regulator and whose gene product partially restored the growth of strain ESC19 at 20°C. The suppressed cells showed slightly reduced capsule production and improved biofilm-forming ability at 20°C, whereas the defects in the LPS core and swimming motility were not restored but aggravated by overexpression of nhaR. Notably, the overexpression partially alleviated the defects in 50S ribosomal subunit assembly and rRNA processing of ESC19 cells by enhancing the overall transcription of rRNA. Electrophoretic mobility shift assay revealed the association of NhaR with the promoter of seven rrn operons, suggesting that NhaR directly regulates rRNA transcription in ESC19 at 20°C. The suppressive effects of NhaR on ribosomes, capsules, and LPS were dependent on its DNA-binding activity, implying that NhaR might be a transcriptional factor involved in regulating these genes at 20°C. Furthermore, we found that BipA may be involved in adaptation to salt stress, designating BipA as a global stress-responsive regulator, as the deletion of bipA led to growth defects at 37°C and high Na+ concentrations without ribosomal defects.

A co-transcriptional ribosome assembly checkpoint controls nascent large ribosomal subunit maturation

During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large ribosomal subunits. While the formation of an early large subunit assembly checkpoint precedes the separation of small and large subunit maturation, its mechanism of action and function remain unknown. Here, we report the cryo-electron microscopy structure of the yeast co-transcriptional large ribosomal subunit assembly intermediate that serves as a checkpoint. The structure provides the mechanistic basis for how quality-control pathways are established through co-transcriptional ribosome assembly factors, that structurally interrogate, remodel and, together with ribosomal proteins, cooperatively stabilize correctly folded pre-ribosomal RNA. Our findings thus provide a molecular explanation for quality control during eukaryotic ribosome assembly in the nucleolus.

Abstract 3500: A noncatalytic role for mettl1 in oncogenic translation in soft tissue sarcoma

Abstract Amplification of chromosomal material derived from 12q is pathognomonic in a subset of soft tissue sarcomas (STS), including well-differentiated and dedifferentiated liposarcoma. However, it remains unclear which of the amplified genes are oncogenic drivers, obstacles to rational therapeutic development. We defined a minimal 12q amplicon in human liposarcoma, and tested the oncogenic activity of each recurrently amplified gene to accelerate the onset of AKT-induced STS in a genetically engineered zebrafish model of the disease. This revealed several genes whose overexpression accelerated the onset of AKT-induced STS, including METTL1, best-known for its ability to catalyze methylation of tRNAs. Surprisingly, catalytically defective mutants of METTL1 exhibited oncogenic activity that was at least as potent as the wild-type gene in zebrafish and human STS, indicating enzyme-independent oncogenic functions. We found that METTL1 promotes ribosome assembly and oncogenic translation independent of its enzymatic activity. In human liposarcoma cells and mouse embryonic fibroblasts, METTL1 overexpression was sufficient to stimulate polysome formation, and this effect was phenocopied by catalytically dead mutants of METTL1. METTL1 overexpression also robustly stimulated the rate of protein synthesis in cells (P < 0.0001), and in a cell-free biochemical assay for translation of a reporter mRNA (P < 0.001). The ability of METTL1 to stimulate ribosomal function triggered a therapeutically targetable dependency, because small molecule inhibitors of ribosome biogenesis had potent therapeutic activity against METTL1-amplified (P < 0.0001), but not METTL1 wild-type, sarcoma cells. Thus, METTL1 is a soft tissue sarcoma oncogene that stimulates oncogenic translation independent of its enzymatic activity, an oncogenic function that can be targeted therapeutically. Citation Format: Raja Hussain Ali, Brianna Silverman, Esteban A. Orellana, Alyssa Kennedy, Caitlin Bowers, Ashley Gutierrez, David Papke, Christopher D. Fletcher, Antonio P. Atayde, Akiko Shimamura, Richard I. Gregory, Alejandro Gutierrez. A noncatalytic role for mettl1 in oncogenic translation in soft tissue sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3500.

Abstract 3906: The mRNA translational plasticity of small cell lung carcinoma is associated with its phenotypic transdifferentiation

Abstract Small cell lung carcinoma(SCLC), classified as a recalcitrant cancer type, accounts for 13-15% of all lung cancers with a median overall survival less than 10 months in patients with extensive stage SCLC. SCLC was once considered as molecularly homogeneous due to nearly universal loss of TP53 and RB1, whereas subsets of MYC-driven, non-neuroendocrine (non-NE) SCLC variants, possess chemoresistance, suggesting its high plasticity and intertumoral heterogeneity related to rapid refractory to first-line chemotherapies. Current pathognomonic classification stratifies SCLC into two NE subtypes (ASCL1+, NEUROD1+) and two non-NE subtypes (POU2F3+ and triple negative), associated with different expression patterns of MYC family proteins. MYC is a master regulator of mRNA translation, whether translational regulation actively tunes SCLC plasticity and the associated chemosensitivity remains an enigma. Here, we delineate the translation landscape of NE and non-NE SCLC cells by using paired human NE SCLC cell line and constitutive or induced non-NE SCLC cells. As expected, non-NE SCLC cells are more resistant to the clinically relevant first-line chemotherapy than NE SCLC cells. With polysome profiling in combination with polysome RNA-seq, we showed differential expression at both transcription- and translation-level. However, transcription-level changes minimally overlapped with the translational changes between NE and non-NE SCLC cells, with a global reduction of translation activity observed in non-NE SCLC cells. Through analyzing the polysome profiles, we observed a striking accumulation of 60S monosomes and diminishment of 80S ribosomes derived from non-NE SCLC cells compared to those from NE SCLC cells. This unique translational pattern in non-NE SCLC cells was correlated with an increased expression of eIF6, a ‘sitting on the bench’ member of eukaryotic initiation factor (eIF) family. The eIF6 couples 60S monosome maturation and recycling, enrolling a quintessential role in functional 80S ribosome formation. Positive correlation between eIF6 expression and non-NE SCLC tumors was observed in 81 tumor samples derived from patients with limited stage SCLC and CCLE cell consortium, in which c-MYC expression was also positively correlated with eIF6 level. By using both ultracentrifugation and fraction analysis from polysome profiling, we found that less association of eIF6 with 60S may mitigate 80S ribosome assembly in non-NE SCLC cells. In addition, in vivo xenograft models showed that eIF6 expression was higher in residual tumors upon chemotherapy. Taken together, these data revealed an eIF6-dependent translational regulation in non-NE transdifferentiation of SCLC subtypes, suggesting an underappreciated function of translational regulation of SCLC plasticity and the potential therapeutic value of eIF6 for overcoming SCLC resistance to chemotherapy. Citation Format: Haoning Peng, Mengyao Wang, Lu Li, Lunxu Liu, Shensi Shen. The mRNA translational plasticity of small cell lung carcinoma is associated with its phenotypic transdifferentiation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3906.

Abstract 3814: In CLL the U1 snRNA driver mutation alters splicing in multiple genes and pathways

Abstract 5-10% of patients with the IGHV wild type form of chronic lymphocytic leukemia (CLL) carry a g.3A>C driver mutation in the U1 small nuclear RNA (snRNA). We investigatedthe patterns of mis-splicing and their pathway consequences in three U1-mutant CLL cellline models using long read sequencing in order to better understand the mechanisms ofoncogenicity in tumors carrying this mutation. CLL cell lines (HG3, JVM3 and MEC1) expressing the U1 mutation and their wild-type counterparts were submitted to Oxford Nanopore sequencing to generate full-length RNA transcripts. This transcriptomic datawas evaluated, together with previously obtained short-read data. The g.3A>C mutation occurs at a specific location of the U1 snRNA, a core component of the eukaryotic spliceosome, and acts by altering the 5' splice site (5'SS) recognition sequence to cause consistent patterns of mis-splicing. Long read analysis identified multiple instances of intron retention and suppression of exon skipping, and in silico translation of these mis-splicing events predicted stop-gain and other loss of function mutations in several expressed genes, as well as widespread changes in gene expression levels. We performed a pathway overrepresentation analysis of the altered gene expression patterns in the mutant cells using the Reactome knowledgebase and identified an enrichment in the processes of translation, non-sense mediated decay (NMD) and immune signaling, including interferon signaling. In particular, there was a marked down-regulation of genes related to ribosomal assembly and the translational machinery. A more comprehensiveanalysis of the U1 mutation phenotype in CLL may accelerate the development ofbetter therapeutic and diagnostic approaches in patients. Citation Format: Andrea Senff-Ribeiro, Fatemeh Almodaresi, Quang Trinh, Shimin Shuai, David Spaner, Xose S. Puente, Elias Campo, Lincoln D. Stein. In CLL the U1 snRNA driver mutation alters splicing in multiple genes and pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3814.

Arabidopsis HOT3/eIF5B1 constrains rRNA RNAi by facilitating 18S rRNA maturation

Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S ribosomal RNA (rRNA) 3' end maturation during late-stage 40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the genome-wide effects of eIF5B have not been studied at the single-nucleotide resolution in any organism, and 18S rRNA 3' end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat stress acclimation by translational regulation, but its molecular function remained unknown. Here, we show that HOT3 is a late-stage ribosome biogenesis factor that facilitates 18S rRNA 3' end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3' end maturation or metabolism. We quantitatively defined processing hotspots and identified adenylation as the prevalent nontemplated RNA addition at the 3' ends of pre-18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNA interference to generate RDR1- and DCL2/4-dependent risiRNAs mainly from a 3' portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome-free fractions and were not responsible for the 18S rRNA maturation or translation initiation defects in hot3. Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late 40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, messenger RNA (mRNA) translation initiation, and siRNA biogenesis in plants.

Motif conservation, stability, and host gene expression are the main drivers of snoRNA expression across vertebrates

Small nucleolar RNAs (snoRNAs) are structured noncoding RNAs present in multiple copies within eukaryotic genomes. snoRNAs guide chemical modifications on their target RNA and regulate processes like ribosome assembly and splicing. Most human snoRNAs are embedded within host gene introns, the remainder being independently expressed from intergenic regions. We recently characterized the abundance of snoRNAs and their host gene across several healthy human tissues and found that the level of most snoRNAs does not correlate with that of their host gene, with the observation that snoRNAs embedded within the same host gene often differ drastically in abundance. To better understand the determinants of snoRNA expression, we trained machine learning models to predict whether snoRNAs are expressed or not in human tissues based on more than 30 collected features related to snoRNAs and their genomic context. By interpreting the models' predictions, we find that snoRNAs rely on conserved motifs, a stable global structure and terminal stem, and a transcribed locus to be expressed. We observe that these features explain well the varying abundance of snoRNAs embedded within the same host gene. By predicting the expression status of snoRNAs across several vertebrates, we notice that only one-third of all annotated snoRNAs are expressed per genome, as in humans. Our results suggest that ancestral snoRNAs disseminated within vertebrate genomes, sometimes leading to the development of new functions and a probable gain in fitness and thereby conserving features favorable to the expression of these few snoRNAs, the large remainder often degenerating into pseudogenes.

Noncatalytic regulation of 18S rRNA methyltransferase DIMT1 in acute myeloid leukemia.

Several rRNA-modifying enzymes install rRNA modifications while participating in ribosome assembly. Here, we show that 18S rRNA methyltransferase DIMT1 is essential for acute myeloid leukemia (AML) proliferation through a noncatalytic function. We reveal that targeting a positively charged cleft of DIMT1, remote from the catalytic site, weakens the binding of DIMT1 to rRNA and mislocalizes DIMT1 to the nucleoplasm, in contrast to the primarily nucleolar localization of wild-type DIMT1. Mechanistically, rRNA binding is required for DIMT1 to undergo liquid-liquid phase separation, which explains the distinct nucleoplasm localization of the rRNA binding-deficient DIMT1. Re-expression of wild-type or a catalytically inactive mutant E85A, but not the rRNA binding-deficient DIMT1, supports AML cell proliferation. This study provides a new strategy to target DIMT1-regulated AML proliferation via targeting this essential noncatalytic region.

TELOMERE BIOLOGY AND RIBOSOME BIOGENESIS: STRUCTURAL AND FUNCTIONAL INTERCONNECTIONS.

Telomeres are the nucleoprotein structures which play a pivotal role in the protection and maintenance of eukaryotic chromosomes. Telomeres and the enzyme telomerase which replenishes telomeric DNA lost during replication are important factors necessary to ensure continued cell proliferation. Cell proliferation is also dependent on proper and efficient protein synthesis, which is carried out by ribosomes. Mutations in genes involved in either ribosome biogenesis or telomere biology result in cellular abnormalities and can cause human genetic diseases, defined as ribosomopathies and telomeropathies, respectively. Interestingly, recent discoveries indicate that many of the ribosome assembly and rRNA maturation factors have additional non-canonical functions in telomere biology. Similarly, several key proteins and enzymes involved in telomere biology, including telomerase, have unexpected roles in rRNA transcription and maturation. These observations point to an intriguing crosstalk mechanism potentially explaining the multiple pleiotropic symptoms of mutations in many causal genes identified in various telomeropathy and ribosomopathy diseases. In this review, we provide a brief summary of eukaryotic telomere and rDNA loci structures, highlight several universal features of rRNA and telomerase biogenesis, evaluate intriguing interconnections between telomere biology and ribosome assembly, and conclude with an assessment of overlapping features of human diseases of telomeropathies and ribosomopathies.

Cryo-EM reconstruction of the human 40S ribosomal subunit at 2.15 Å resolution.

The chemical modification of ribosomal RNA and proteins is critical for ribosome assembly, for protein synthesis and may drive ribosome specialisation in development and disease. However, the inability to accurately visualise these modifications has limited mechanistic understanding of the role of these modifications in ribosome function. Here we report the 2.15 Å resolution cryo-EM reconstruction of the human 40S ribosomal subunit. We directly visualise post-transcriptional modifications within the 18S rRNA and four post-translational modifications of ribosomal proteins. Additionally, we interpret the solvation shells in the core regions of the 40S ribosomal subunit and reveal how potassium and magnesium ions establish both universally conserved and eukaryote-specific coordination to promote the stabilisation and folding of key ribosomal elements. This work provides unprecedented structural details for the human 40S ribosomal subunit that will serve as an important reference for unravelling the functional role of ribosomal RNA modifications.

Concurrent remodeling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase.

Biogenesis intermediates of nucleolar ribosomal 60S precursor particles undergo a number of structural maturation steps before they transit to the nucleoplasm and are finally exported into the cytoplasm. The AAA+-ATPase Rea1 participates in the nucleolar exit by releasing the Ytm1-Erb1 heterodimer from the evolving pre-60S particle. Here, we show that the DEAD-box RNA helicase Spb4 with its interacting partner Rrp17 is further integrated into this maturation event. Spb4 binds to a specific class of late nucleolar pre-60S intermediates, whose cryo-EM structure revealed how its helicase activity facilitates melting and restructuring of 25S rRNA helices H62 and H63/H63a prior to Ytm1-Erb1 release. In vitro maturation of such Spb4-enriched pre-60S particles, incubated with purified Rea1 and its associated pentameric Rix1-complex in the presence of ATP, combined with cryo-EM analysis depicted the details of the Rea1-dependent large-scale pre-ribosomal remodelling. Our structural insights unveil how the Rea1 ATPase and Spb4 helicase remodel late nucleolar pre-60S particles by rRNA restructuring and dismantling of a network of several ribosomal assembly factors.

Pleiotropic effects of PipX, PipY, or RelQ overexpression on growth, cell size, photosynthesis, and polyphosphate accumulation in the cyanobacterium Synechococcus elongatus PCC7942.

The cyanobacterial protein PipY belongs to the Pyridoxal-phosphate (PLP)-binding proteins (PLPBP/COG0325) family of pyridoxal-phosphate-binding proteins, which are represented in all three domains of life. These proteins share a high degree of sequence conservation, appear to have purely regulatory functions, and are involved in the homeostasis of vitamin B6 vitamers and amino/keto acids. Intriguingly, the genomic context of the pipY gene in cyanobacteria connects PipY with PipX, a protein involved in signaling the intracellular energy status and carbon-to-nitrogen balance. PipX regulates its cellular targets via protein-protein interactions. These targets include the PII signaling protein, the ribosome assembly GTPase EngA, and the transcriptional regulators NtcA and PlmA. PipX is thus involved in the transmission of multiple signals that are relevant for metabolic homeostasis and stress responses in cyanobacteria, but the exact function of PipY is still elusive. Preliminary data indicated that PipY might also be involved in signaling pathways related to the stringent stress response, a pathway that can be induced in the unicellular cyanobacterium Synechococcus elongatus PCC7942 by overexpression of the (p)ppGpp synthase, RelQ. To get insights into the cellular functions of PipY, we performed a comparative study of PipX, PipY, or RelQ overexpression in S. elongatus PCC7942. Overexpression of PipY or RelQ caused similar phenotypic responses, such as growth arrest, loss of photosynthetic activity and viability, increased cell size, and accumulation of large polyphosphate granules. In contrast, PipX overexpression decreased cell length, indicating that PipX and PipY play antagonistic roles on cell elongation or cell division. Since ppGpp levels were not induced by overexpression of PipY or PipX, it is apparent that the production of polyphosphate in cyanobacteria does not require induction of the stringent response.

Nucleolar URB1 ensures 3' ETS rRNA removal to prevent exosome surveillance.

The nucleolus is the most prominent membraneless condensate in the nucleus. It comprises hundreds of proteins with distinct roles in the rapid transcription of ribosomal RNA (rRNA) and efficient processing within units comprising a fibrillar centre and a dense fibrillar component and ribosome assembly in a granular component1. The precise localization of most nucleolar proteins and whether their specific localization contributes to the radial flux of pre-rRNA processing have remained unknown owing to insufficient resolution in imaging studies2-5. Therefore, how these nucleolar proteins are functionally coordinated with stepwise pre-rRNA processing requires further investigation. Here we screened 200 candidate nucleolar proteins using high-resolution live-cell microscopy and identified 12 proteins that are enriched towards the periphery of the dense fibrillar component (PDFC). Among these proteins, unhealthy ribosome biogenesis 1 (URB1) is a static, nucleolar protein that ensures 3' end pre-rRNA anchoring and folding for U8 small nucleolar RNA recognition and the subsequent removal of the 3' external transcribed spacer (ETS) at the dense fibrillar component-PDFC boundary. URB1 depletion leads to a disrupted PDFC, uncontrolled pre-rRNA movement, altered pre-rRNA conformation and retention of the 3' ETS. These aberrant 3' ETS-attached pre-rRNA intermediates activate exosome-dependent nucleolar surveillance, resulting in decreased 28S rRNA production, head malformations in zebrafish and delayed embryonic development in mice. This study provides insight into functional sub-nucleolar organization and identifies a physiologically essential step in rRNA maturation that requires the static protein URB1 in the phase-separated nucleolus.