40S Ribosomal Subunit Research Articles

Assessment of the cyst wall and surface microbiota in dormant embryos of the Antarctic calanoid copepod, Boeckella poppei.

Embryos of zooplankton from inland waters and estuaries can remain viable for years in an extreme state of metabolic suppression. How these embryos resist microbial attack with limited metabolic capacity for immune defence or repair is unknown. As a first step in evaluating resistance to microbial attack in dormant zooplankton, surface colonization of the Antarctic freshwater copepod, Boeckella poppei, was evaluated. Scanning electron micrographs demonstrate the outer two layers of a five-layered cyst wall in B. poppei fragment and create a complex environment for microbial colonization. By contrast, the third layer remains undamaged during years of embryo storage in native sediment. The absence of damage to the third layer indicates that it is resistant to degradation by microbial enzymes. Scanning electron microscopy and microbiome analysis using the 16S ribosomal subunit gene and internal transcribed spacer (ITS) region demonstrate the presence of a diverse microbial community on the embryo surface. Coverage of the embryos with microbial life varies from a sparse population with individual microbes to complete coverage by a thick biofilm. Extracellular polymeric substance binds debris and provides a structural element for the microbial community. Frequent observation of bacterial fission indicates that the biofilm is viable in stored sediments.

Proteomic profiling of the Macrobrachium rosenbergii nodavirus infection: A study of early to late-stage infection in vitro

The Macrobrachium rosenbergii nodavirus (MrNV), belonging to the Nodaviridae family, is responsible for a deadly infection in freshwater prawns, especially impacting the post-larvae of Macrobrachium rosenbergii with a mortality rate reaching 100 %. Recent research has shed light on the typical process of MrNV trafficking within the host, illustrating how the virus navigates through cells as the infection advances and the subsequent cellular alterations. Yet, the specific cellular pathways disrupted by MrNV, leading to these alterations, are underexplored. Furthermore, the precise effects of the MrNV capsid protein, known for its strong immune response, on the host cells are not well understood. This study seeks to clarify these impacts by analysing and comparing the protein expression profiles in healthy, MrNV virus-like particle (VLP) invaded, and MrNV-infected Sf9 cells over a 24-h period using a mass spectrometry based proteomics approach. Our findings show that the protein expression in MrNV VLP-invaded and MrNV-infected Sf9 cells during the mid-infection stages is similar, involving key signalling pathways like the eukaryotic translation system, cell cycle, actin cytoskeleton regulation, and the mTOR pathway. However, changes in protein expression for key proteins such as 40S and 60S ribosomal subunits, 14–3-3 protein epsilon, and tubulin beta chain persisted only within the MrNV-infection group whilst the protein expression in the VLP-invasion group reverted to baseline levels over time, underscoring the transient nature of VLP effects due to their inability to replicate. Additionally, a reduction in peroxiredoxin levels was observed in the later stages of MrNV infection, indicating a potential viral strategy to trigger apoptosis and release virions. Our results suggest that MrNV increases expression of 40S ribosome activity to boost viral protein synthesis while suppressing 60S ribosome expression, which impedes the synthesis of host proteins. MrNV also appears to extend the lifespan of host cells by interfering with their cell cycle and blocking apoptotic pathways, thus facilitating viral replication. This research enhances our comprehension of MrNV's infectious mechanism, delineates the pathways exploited by the virus, and identifies crucial molecular targets for potential therapeutic intervention.

RPS21 Enhances hepatocellular carcinoma development through GPX4 stabilization

The study highlights that RPS21, a gene encoding a component of the 40S ribosomal subunit, plays an oncogenic role in hepatocellular carcinoma (HCC) and may influence tumor aggressiveness by affecting antioxidant capacity. RPS21 was found to be upregulated in HCC through RNA-sequencing of clinical samples and analysis of the TCGA database. Kaplan-Meier survival analyses linked higher RPS21 expression to lower survival rates across multiple metrics (OS, PFS, RFS, DSS). Mutation analysis via the cBioPortal showed that primarily amplifications in RPS21 are associated with a poorer prognosis. Tissue microarrays confirmed higher RPS21 levels in tumor samples, which were associated with more advanced clinical stages and grades. Experimental interventions involving lentiviral knockdown or overexpression of RPS21 significantly altered HCC cell proliferation and migration. These findings were supported by mouse models, which showed impacts on tumor growth and metastasis. Further mechanistic studies indicated that RPS21 modulates the ubiquitination and stability of GPX4, a key player in ferroptosis and oxidative stress regulation in HCC cells. This comprehensive study, which merges bioinformatic analysis with laboratory research, positions RPS21 as a viable target for HCC therapy and opens new pathways for understanding and treating liver cancer.

Critical and differential roles of eIF4A1 and eIF4A2 in B-cell development and function.

Eukaryotic initiation factor 4 A (eIF4A) plays critical roles during translation initiation of cellular mRNAs by forming the cap-binding eIF4F complex, recruiting the 40S small ribosome subunit, and scanning the 5' untranslated region (5' UTR) for the start codon. eIF4A1 and eIF4A2, two isoforms of eIF4A, are highly conserved and exchange freely within eIF4F complexes. The understanding of their biological and molecular functions remains incomplete if not fragmentary. In this study, we showed that eIF4A1 and eIF4A2 exhibit different expression patterns during B-cell development and activation. Mouse genetic analyses showed that they play critical but differential roles during B-cell development and humoral immune responses. While eIF4A1 controls global protein synthesis, eIF4A2 regulates the biogenesis of 18S ribosomal RNA and the 40S ribosome subunit. This study demonstrates the distinct cellular and molecular functions of eIF4A1 and eIF4A2 and reveals a new role of eIF4A2 in controlling 40S ribosome biogenesis.

Crystal structure of the GDP-bound GTPase Era from Staphylococcus aureus

GTPase Era from Staphylococcus aureus belongs to the TRAFAC superfamily of the TrmE-Era-EngA-EngB-Septin-like GTPases class and plays a significant role in the vital activity of this pathogenic microorganism as a maturation factor of the 30S ribosome subunit. However, the functions of this protein are not fully understood, making it a promising object for further study. Here, the 2.76 Å resolution crystal structure of Staphylococcus aureus Era in complex with GDP is presented. Structural comparison with other GTP-bound and GDP-bound homologous proteins, GTPase domain and the KH domain revealed a mutual orientation in S. aureus which has not been described before. The GDP-bound Era structure presented here will facilitate efforts to elucidate its interactions with its regulators and lay the foundation for a structure-based search for specific inhibitors.

Resistance to linezolid in Staphylococcus aureus by mutation, modification, and acquisition of genes.

Linezolid binds to the 50S subunit of the bacterial ribosome, inhibiting bacterial protein synthesis by preventing the formation of the initiation complex. Oxazolidinone antimicrobial drugs represent the last line of defense in treating Staphylococcus aureus infections; thus, resistance to linezolid in S. aureus warrants high priority. This article examines the major mechanisms of resistance to linezolid in S. aureus, which include: mutations in the domain V of 23S rRNA (primarily G2576); chromosomal mutations in the rplC, rplD, and rplV genes (encoding the ribosomal uL3, uL4, and uL22 proteins, respectively); the exogenous acquisition of the methylase encoded by the chloramphenicol-florfenicol resistance (cfr) gene; the endogenous methylation or demethylation of 23S rRNA; the acquisition of optrA and poxtA resistance genes; and the existence of the LmrS multidrug efflux pump. In conclusion, these mechanisms mediate resistance through mutations or modifications to the bacterial target, thereby reducing the affinity of linezolid for the peptidyl transferase center (PTC) binding site or by preventing the binding of linezolid to the PTC through a ribosomal protective effect. The existence of additional, unexplained resistance mechanisms requires further investigation and verification.

Broad‐Spectrum Antiviral Agents against SARS‐CoV‐2 Variants Inhibit the Conserved Viral Protein Nsp1–RNA Interaction

AbstractThe ongoing global threats posed by COVID‐19 pandemic, catalyzed by SARS‐CoV‐2, underscores the pressing need for effective antiviral strategies. The viral non‐structural protein 1 (Nsp1) significantly influences pathogenicity by impeding host protein expression and enhancing viral RNA translation through its interaction with the stem‐loop 1 (SL1) in the 5′ untranslated region (UTR). We have developed a novel dual‐luciferase reporter assay, designed to investigate the critical Nsp1–SL1 interaction, and identified P23E02 as a potential inhibitor. Our investigation, combining molecular docking studies and alanine mutagenesis, has unveiled that P23E02 disrupts Nsp1–40S ribosomal subunit interaction, liberating translational inhibition and empowering host antiviral responses. P23E02 exhibits antiviral efficacy against various sarbecoviruses, making it a promising candidate for combatting COVID‐19 and related diseases. This study underscores the therapeutic potential of targeting the Nsp1/SL1 axis and lays the foundation for innovative antiviral interventions, ultimately fortifying global preparedness against future viral threats.

Ribosome-inactivation by a class of widely distributed C-tail anchored membrane proteins

Ribosome hibernation is a commonly used strategy that protects ribosomes under unfavorable conditions and regulates developmental processes. Multiple ribosome-hibernation factors have been identified in all domains of life, but due to their structural diversity and the lack of a common inactivation mechanism, it is currently unknown how many different hibernation factors exist. Here, we show that the YqjD/ElaB/YgaM paralogs, initially discovered as membrane-bound ribosome binding proteins in E.coli, constitute an abundant class of ribosome-hibernating proteins, which are conserved across all proteobacteria and some other bacterial phyla. Our data demonstrate that they inhibit invitro protein synthesis by interacting with the 50S ribosomal subunit. Invivo cross-linking combined with mass spectrometry revealed their specific interactions with proteins surrounding the ribosomal tunnel exit and even their penetration into the ribosomal tunnel. Thus, YqjD/ElaB/YgaM inhibit translation by blocking the ribosomal tunnel and thus mimic the activity of antimicrobial peptides and macrolide antibiotics.

The Drosophila ribonucleoprotein Clueless is required for ribosome biogenesis in vivo

As hubs of metabolism, mitochondria contribute critical processes to coordinate and optimize energy and intermediate metabolites. Drosophila Clueless (Clu) and vertebrate CLUH are ribonucleoproteins critical for supporting mitochondrial function yet do so in multiple ways. Clu/CLUH bind mRNAs and CLUH regulates mRNA localization and translation of mRNAs encoding proteins destined for mitochondrial import. In addition, Clu associates with ribosomal proteins and translation factors, yet whether it is required for fundamental ribosome function in vivo is not clear. In this study, we examine the Clu interactome and probe Clu’s requirement in ribosome biogenesis. We previously showed that Clu associates with ribosomal proteins. In this study, we extend these observations to show that clu null mutants display a significant decrease in overall protein synthesis. In addition, Clu associates with ribosomal proteins in an mRNA-independent manner, suggesting Clu’s core ribosomal function may be separate from its role in localizing and translating specific mRNAs. We find that Clu is present in the nucleus and associates with the ribosomal RNA (rRNA) processing protein fibrillarin but, surprisingly, that processed rRNA products are normal in the absence of Clu. Furthermore, Clu loss does not affect ribosomal protein levels, but does result in a decrease in 40S and 60S ribosomal subunits abundance. Together, these results demonstrate that Clu is present in the nucleus and required for 40S and 60S biogenesis and global translation in vivo. These results highlight the multifaceted role of Clu in supporting cell function through regulation of mRNA encoding mitochondrial proteins and ribosome biogenesis.

Chloramphenicol Interferes with 50S Ribosomal Subunit Maturation via Direct and Indirect Mechanisms.

Chloramphenicol (CAM), a well-known broad-spectrum antibiotic, inhibits peptide bond formation in bacterial ribosomes. It has been reported to affect ribosome assembly mainly through disrupting the balance of ribosomal proteins. The present study investigates the multifaceted effects of CAM on the maturation of the 50S ribosomal subunit in Escherichia coli (E. coli). Using label-free quantitative mass spectrometry (LFQ-MS), we observed that CAM treatment also leads to the upregulation of assembly factors. Further cryo-electron microscopy (cryo-EM) analysis of the ribosomal precursors characterized the CAM-treatment-accumulated pre-50S intermediates. Heterogeneous reconstruction identified 26 distinct pre-50S intermediates, which were categorized into nine main states based on their structural features. Our structural analysis highlighted that CAM severely impedes the formation of the central protuberance (CP), H89, and H58 during 50S ribosomal subunit maturation. The ELISA assay further demonstrated the direct binding of CAM to the ribosomal precursors, suggesting that the interference with 50S maturation occurs through a combination of direct and indirect mechanisms. These findings provide new insights into the mechanism of the action of CAM and provide a foundation for a better understanding of the assembly landscapes of the ribosome.

CLONING OF CDNA-GENE OF ARABIDOPSIS THALIANA RIBOSOMAL PROTEIN S6, ITS EXPRESSION IN ESCHERICHIA COLI AND PURIFICATION OF ATRPS6B1 RECOMBINANT PROTEIN

Living organisms must adapt their growth and development in response to external factors like stress and nutrient availability throughout their lifespan. Consequently, they have evolved various regulatory pathways to enhance environmental perception and facilitate necessary metabolic adjustments. These pathways involve key proteins that, under stress and nutrient limitation, initiate anabolic and catabolic cellular processes. One critical pathway present in all eukaryotes involves the protein kinase TOR (Target of rapamycin), a large kinase that governs numerous biological processes, including the activation of S6K kinase, which phosphorylates the S6 protein (RPS6). Crystal structures have revealed the precise location of RPS6 within the 40S ribosomal subunit, highlighting structural features such as the C-terminal helix containing multiple phosphorylation sites. RPS6 primarily regulates mechanisms controlling cell growth and division. In plants, eS6 is encoded by two conserved genes, and its activation and phosphorylation are closely linked to S6Ks. Recent studies emphasize the significance of S6 protein phosphorylation as a key event in signaling pathways related to cell growth and survival factors, crucially regulating translation initiation and protein synthesis. S6 protein can be phosphorylated on various serine and threonine residues by S6K kinase, and further research shows its interaction with other molecules and proteins, expanding its functional roles in cell signaling. Further exploration of ribosomal protein S6 could unveil new insights into its molecular interactions, roles in cellular physiology and pathophysiology, and potential applications in enhancing plant biomass and yield. This study involved cloning and site-directed mutagenesis of cDNA for the second isoform of AtRPS6 protein (AtRPS6B), followed by expression of natural and phosphomimetic forms of the protein in E. coli ArcticExpress (DE3) cells. The proteins were then purified using metal-chelate affinity chromatography (IMAC), and their presence and degree of purification were confirmed via Western blotting. These findings contribute to the broader research context surrounding ribosomal protein S6. RPS6, a key effector in the TOR signaling pathway, plays a crucial role in regulating ribosome biosynthesis by controlling transcription and translation processes. Phosphorylation of RPS6, indicative of S6K activity, is associated with actively dividing cells. However, the precise role of RPS6 in ribosome biosynthesis regulation remains a subject of ongoing investigation. The data obtained from the study on AtRPS6B expand our understanding of the molecular mechanisms underlying ribosomal biosynthesis regulation. These insights may significantly contribute to our knowledge of the interplay between signaling pathways, ribosomal proteins, cellular metabolism, and offer new perspectives for therapeutic interventions targeting cellular processes associated with ribosomes.

A PARP14/TARG1-Regulated RACK1 MARylation Cycle Drives Stress Granule Dynamics in Ovarian Cancer Cells.

Mono(ADP-ribosyl)ation (MARylation) is emerging as a critical regulator of ribosome function and translation. Herein, we demonstrate that RACK1, an integral component of the ribosome, is MARylated on three acidic residues by the mono(ADP-ribosyl) transferase (MART) PARP14 in ovarian cancer cells. MARylation of RACK1 is required for stress granule formation and promotes the colocalization of RACK1 in stress granules with G3BP1, eIF3η, and 40S ribosomal proteins. In parallel, we observed reduced translation of a subset of mRNAs, including those encoding key cancer regulators (e.g., AKT). Treatment with a PARP14 inhibitor or mutation of the sites of MARylation on RACK1 blocks these outcomes, as well as the growth of ovarian cancer cells in culture and in vivo. To re-set the system after prolonged stress and recovery, the ADP-ribosyl hydrolase TARG1 deMARylates RACK1, leading to the dissociation of the stress granules and the restoration of translation. Collectively, our results demonstrate a therapeutically targetable pathway that controls stress granule assembly and disassembly in ovarian cancer cells.

Structural analysis of noncanonical translation initiation complexes

Translation initiation is a highly regulated, multi-step process that is critical for efficient and accurate protein synthesis. In bacteria, initiation begins when mRNA, initiation factors, and a dedicated initiator fMet-tRNAfMet bind the small (30S) ribosomal subunit. Specific binding of fMet-tRNAfMet in the peptidyl (P) site is mediated by the inspection of the fMet moiety by initiation factor IF2 and of three conserved G-C base pairs in the tRNA anticodon stem by the 30S head domain. Tandem A-minor interactions form between 16S ribosomal RNA nucleotides A1339 and G1338 and tRNA base pairs G30-C40 and G29-C41, respectively. Swapping the G30-C40 pair of tRNAfMet with C-G (called tRNAfMet M1) reduces discrimination against the noncanonical start codon CUG invitro, suggesting crosstalk between the gripping of the anticodon stem and recognition of the start codon. Here, we solved electron cryomicroscopy structures of Escherichia coli 70S initiation complexes containing the fMet-tRNAfMet M1 variant paired to the noncanonical CUG start codon, in the presence or absence of IF2 and the non-hydrolyzable GTP analog GDPCP, alongside structures of 70S initiation complexes containing this tRNAfMet variant paired to the canonical bacterial start codons AUG, GUG, and UUG. We find that the M1 mutation weakens A-minor interactions between tRNAfMet and 16S nucleotides A1339 and G1338, with IF2 strengthening the interaction of G1338 with the tRNA minor groove. These structures suggest how even slight changes to the recognition of the fMet-tRNAfMet anticodon stem by the ribosome can impact the start codon selection.

Assigning roles in Chlamydomonas ribosome biogenesis: The conserved factor NIP7

Ribosome biogenesis (RB) is a highly conserved process across eukaryotes that results in the assembly of functional ribosomal subunits. Studies in Saccharomyces cerevisiae and Homo sapiens have identified numerous RB factors (RBFs), including the NIP7 protein, which is involved in late-stage pre-60S ribosomal maturation. NIP7 expression has also been observed in Chlamydomonas reinhardtii, highlighting its evolutionary significance. This study aimed to characterize the function of the NIP7 protein from C. reinhardtii (CrNip7) through protein complementation assays and a paromomycin resistance test, assessing its ability to complement the role of NIP7 in yeast. Protein interaction studies were conducted via yeast two-hybrid assay to identify potential protein partners of CrNip7. Additionally, rRNA modeling analysis was performed using the predicted structure of CrNip7 to investigate its interaction with rRNA. The study revealed that CrNip7 can complement the role of NIP7 in yeast, implicating CrNip7 in the biogenesis of the 60S ribosomal subunit. Furthermore, two possible partner proteins of CrNip7, UNC-p and G-patch, were identified through yeast two-hybrid assay. The potential of these proteins to interact with CrNip7 was explored through in silico analyses. Furthermore, nucleic acid interaction was also evaluated, indicating the involvement of the N- and C-terminal domains of CrNIP7 in interacting with rRNA. Collectively, our findings provide valuable insights into the RBFs CrNip7, offering novel information for comparative studies on RB among eukaryotic model organisms, shedding light on its evolutionary conservation and functional role across species.

SnoRNAs in making “snoman” ribosomes

In a recent study in Cell, Cheng and Wang etal.1 show that the small nucleolar RNA (snoRNA) SNORA13 has a non-canonical role in ribosome biogenesis and senescence by acting directly on RPL23 and regulating its assembly into the 60S ribosomal subunit.

RpL38 modulates germ cell differentiation by controlling Bam expression in Drosophila testis.

Switching from mitotic spermatogonia to meiotic spermatocytes is critical to producing haploid sperms during male germ cell differentiation. However, the underlying mechanisms of this switch remain largely unexplored. In Drosophila melanogaster, the gene RpL38 encodes the ribosomal protein L38, one component of the 60S subunit of ribosomes. We found that its depletion in spermatogonia severely diminished the production of mature sperms and thus led to the infertility of male flies. By examining the germ cell differentiation in testes, we found that RpL38-knockdown blocked the transition from spermatogonia to spermatocytes and accumulated spermatogonia in the testis. To understand the intrinsic reason for this blockage, we conducted proteomic analysis for these spermatogonia populations. Differing from the control spermatogonia, the accumulated spermatogonia in RpL38-knockdown testes already expressed many spermatocyte markers but lacked many meiosis-related proteins, suggesting that spermatogonia need to prepare some important proteins for meiosis to complete their switch into spermatocytes. Mechanistically, we found that the expression of bag of marbles (bam), a crucial determinant in the transition from spermatogonia to spermatocytes, was inhibited at both the mRNA and protein levels upon RpL38 depletion. We also confirmed that the bam loss phenocopied RpL38 RNAi in the testis phenotype and transcriptomic profiling. Strikingly, overexpressing bam was able to fully rescue the testis abnormality and infertility of RpL38-knockdown flies, indicating that bam is the key effector downstream of RpL38 to regulate spermatogonia differentiation. Overall, our data suggested that germ cells start to prepare meiosis-related proteins as early as the spermatogonial stage, and RpL38 in spermatogonia is required to regulate their transition toward spermatocytes in a bam-dependent manner, providing new knowledge for our understanding of the transition process from spermatogonia to spermatocytes in Drosophila spermatogenesis.

Source and variation of the amazing live Sea-Monkey microbiome.

An embryonic diapause in unfavourable conditions has allowed brine shrimp to thrive in hypersaline environments and, unexpectedly, mail-order sachets and small, novelty tanks. Marketed as Sea-Monkeys®, each kit involves a 3-step process to generate adult Artemia within a matter of weeks. Whether these kits also allow for the maintenance of a host-associated microbiome is unclear. Therefore, comparing five replicate tanks under the same culture conditions, we sequenced the 16S ribosomal small subunit (SSU) gene to analyse bacterial community compositions in adults, their surrounding tank water, and their feed. Adult Sea-Monkeys® harboured a bacterial microbiome that was clearly distinguishable from the tank water and food. Furthermore, individual tanks had a notable effect on fine-scale microbiome variation. Several Sea-Monkey bacterial variants appeared absent in environmental samples and included genera (Leucobacter and Microbacterium) known to confer desiccation resistance in other hosts. Although Sea-Monkeys® taxonomy is unclear, phylogenetic inference of the cytochrome c oxidase I (COXI) gene from the host animal suggests Sea-Monkeys® belong to the Artemia franciscana 'superspecies'. Overall, Sea-Monkeys® kits appear to be a convenient and scalable mesocosm for the study of host-microbiome interactions and could serve as a useful tool for future invertebrate microbiome research, outreach, and education.

Dominant-negative chaperonin mutation ptCPN60α1S57F uncovers redundancy in chloroplast rRNA processing.

The biogenesis of functional forms of chloroplast ribosomal RNAs (rRNAs) is crucial for the translation of chloroplast mRNAs into polypeptides. However, the molecular mechanisms underlying the proper processing and maturation of chloroplast rRNA species are poorly understood. Through a genetic approach, we isolated and characterized an Arabidopsis mutant, α1-4, harboring a missense mutation in the plastid chaperonin-60α1 gene. Using allelism tests and transgenic manipulation, we determined functional redundancy among ptCPN60 subunits. The ptCPN60α1S57F mutation caused specific defects in the formation of chloroplast rRNA species, including 23S, 5S, and 4.5S rRNAs, but not 16S rRNAs. Allelism tests suggested that the dysfunctional ptCPN60α1S57F competes with other members of the ptCPN60 family. Indeed, overexpression of the ptCPN60α1S57F protein in wild-type plants mimicked the phenotypes observed in the α1-4 mutant, while increasing the endogenous transcriptional levels of ptCPN60α2, β1, β2, and β3 in the α1-4 mutant partially mitigated the abnormal fragmentation processing of chloroplast 23S, 5S, and 4.5S rRNAs. Furthermore, we demonstrated functional redundancy between ptCPN60β1 and ptCPN60β2 in chloroplast rRNA processing through double-mutant analysis. Collectively, our data reveal a novel physiological role of ptCPN60 subunits in generating the functional rRNA species of the large 50S ribosomal subunit in Arabidopsis chloroplasts.

51 Epigenetic regulation of ribosomal RNA transcription and processing by the tumor suppressor SETD2

Abstract Background The methyltransferase SETD2 was originally identified as a tumor suppressor in clear cell renal cell carcinoma (ccRCC), and ccRCC with SETD2 mutations have increased metastatic potential and poor progression free survival. A known epigenetic regulator, SETD2 interacts with actively transcribing RNA polymerase II (RNAPII) and tri-methylates Histone 3 at Lysine 36 (H3K36me3), a modification that correlates with active gene transcription and is recognized by mRNA methylation and splicing factors. Despite these known roles in mRNA transcription, loss of SETD2 does not alter genic transcription, raising the question of whether or how SETD2 regulates RNA biology. Methods Given that SETD2 influences RNA processing, we sought to identify RNA-protein complexes that are regulated by SETD2. To do this in an unbiased and high-throughput manner, we used orthogonal organic phase separation (OOPS) coupled with mass spectrometry to identify changes in RNA binding proteins and pathways between control and SETD2-knockout cells. Results Corroborating previous findings, we identified that RNA binding proteins governing mRNA splicing and cellular metabolism were dysregulated in SETD2 mutant cells. However, the most robust changes in SETD2 knockout cells occurred in pathways regulated ribosome biogenesis (See Figure, orange highlights), resulting in a significant decrease in binding of ribosomal RNA (rRNA) processing factors to RNA. We confirm these alterations lead to defects in rRNA transcription, processing, and ribosome biogenesis. We identify that loss of SETD2 catalytic activity phenocopies these rRNA processing defects, suggesting H3K36me3 is essential for rRNA processing. Disruptions occur to both the large (60S) and small (40S) ribosome subunits, modifying protein translation. Lastly, we identify synthetic lethality between SETD2 deletion or inhibition and small molecules that interfere with rRNA transcription. Conclusions While the majority of research emphasizes the role SETD2 in the regulation of mRNA, our data identify that SETD2 and its catalytic activity play a critical role in the transcription and processing of rRNA. Importantly, rRNA comprises 80-90% of cellular RNA yet is typically overlooked and understudied. Next-generation RNA sequencing or transcriptional assays investigate mRNA or genic transcription while ignoring rRNA, demonstrating why SETD2-mediated rRNA processing has been unexplored. This may also have important implications for prospective clinical trials that utilized RNA-sequencing to identify optimal therapeutic regimens. Our work also identified that SETD2-mutated cells are more sensitive to compounds that inhibit rRNA transcription, potentially uncovering a new therapeutic vulnerability of ccRCC with mono- and bi-allelic loss of SETD2. Alterations in ribosome biogenesis leads to epithelial-to-mesenchymal transition and metastasis in many tumor types, and our future goal will be to investigate the role of ribosomes and rRNA transcription in transformation and metastasis in ccRCC as well as the molecular mechanisms connecting SETD2 to ribosome fidelity. DOD CDMRP Funding: yes

An atypical form of 60S ribosomal subunit in Diamond-Blackfan anemia linked to RPL17 variants.

Diamond-Blackfan anemia syndrome (DBA) is a ribosomopathy associated with loss-of-function variants in more than 20 ribosomal protein (RP) genes. Here, we report the genetic, functional, and biochemical dissection of 2 multigenerational pedigrees with variants in RPL17, a large ribosomal subunit protein-encoding gene. Affected individuals had clinical features and erythroid proliferation defects consistent with DBA. Further, RPL17/uL22 depletion resulted in anemia and micrognathia in zebrafish larvae, and in vivo complementation studies indicated that RPL17 variants were pathogenic. Lymphoblastoid cell lines (LCLs) derived from patients displayed a ribosomal RNA maturation defect reflecting haploinsufficiency of RPL17. The proteins encoded by RPL17 variants were not incorporated into ribosomes, but 10%-20% of 60S ribosomal subunits contained a short form of 5.8S rRNA (5.8SC), a species that is marginal in normal cells. These atypical 60S subunits were actively engaged in translation. Ribosome profiling showed changes of the translational profile, but those are similar to LCLs bearing RPS19 variants. These results link an additional RP gene to DBA. They show that ribosomes can be modified substantially by RPL17 haploinsufficiency but support the paradigm that translation alterations in DBA are primarily related to insufficient ribosome production rather than to changes in ribosome structure or composition.