Small Nucleolar Ribonucleoprotein Research Articles

The deubiquitinase USP36 promotes snoRNP group SUMOylation and is essential for ribosome biogenesis.

SUMOylation plays a crucial role in regulating diverse cellular processes including ribosome biogenesis. Proteomic analyses and experimental evidence showed that a number of nucleolar proteins involved in ribosome biogenesis are modified by SUMO. However, how these proteins are SUMOylated in cells is less understood. Here, we report that USP36, a nucleolar deubiquitinating enzyme (DUB), promotes nucleolar SUMOylation. Overexpression of USP36 enhances nucleolar SUMOylation, whereas its knockdown or genetic deletion reduces the levels of SUMOylation. USP36 interacts with SUMO2 and Ubc9 and directly mediates SUMOylation in cells and in vitro. We show that USP36 promotes the SUMOylation of the small nucleolar ribonucleoprotein (snoRNP) components Nop58 and Nhp2 in cells and in vitro and their binding to snoRNAs. It also promotes the SUMOylation of snoRNP components Nop56 and DKC1. Functionally, we show that knockdown of USP36 markedly impairs rRNA processing and translation. Thus, USP36 promotes snoRNP group SUMOylation and is critical for ribosome biogenesis and protein translation.

A PRC2-independent function for EZH2 in regulating rRNA 2'-O methylation and IRES-dependent translation.

Dysregulated translation is a common feature of cancer. Uncovering its governing factors and underlying mechanism are important for cancer therapy. Here, we report that enhancer of zeste homologue 2 (EZH2), previously known as a transcription repressor and lysine methyltransferase, can directly interact with fibrillarin (FBL) to exert its role in translational regulation. We demonstrate that EZH2 enhances rRNA 2'-O methylation via its direct interaction with FBL. Mechanistically, EZH2 strengthens the FBL-NOP56 interaction and facilitates the assembly of box C/D small nucleolar ribonucleoprotein. Strikingly, EZH2 deficiency impairs the translation process globally and reduces internal ribosome entry site (IRES)-dependent translation initiation in cancer cells. Our findings reveal a previously unrecognized role of EZH2 in cancer-related translational regulation.

Tah1, A Key Component of R2TP Complex that Regulates Assembly of snoRNP, is Involved in De Novo Generation and Maintenance of Yeast Prion [URE3

The cellular chaperone machinery plays key role in the de novo formation and propagation of yeast prions (infectious protein). Though the role of Hsp70s in the prion maintenance is well studied, how Hsp90 chaperone machinery affects yeast prions remains unclear. In the current study, we examined the role of Hsp90 and its co-chaperones on yeast prions [PSI+] and [URE3]. We show that the overproduction of Hsp90 co-chaperone Tah1, cures [URE3] which is a prion form of native protein Ure2 in yeast. The Hsp90 co-chaperone Tah1 is involved in the assembly of small nucleolar ribonucleoproteins (snoRNP) and chromatin remodelling complexes. We found that Tah1 deletion improves the frequency of de novo appearance of [URE3]. The Tah1 was found to interact with Hsp70. The lack of Tah1 not only represses antagonizing effect of Ssa1 Hsp70 on [URE3] but also improves the prion strength suggesting role of Tah1 in both fibril growth and replication. We show that the N-terminal tetratricopeptide repeat domain of Tah1 is indispensable for [URE3] curing. Tah1 interacts with Ure2, improves its solubility in [URE3] strains, and affects the kinetics of Ure2 fibrillation in vitro. Its inhibitory role on Ure2 fibrillation is proposed to influence [URE3] propagation. The present study shows a novel role of Tah1 in yeast prion propagation, and that Hsp90 not only promotes its role in ribosomal RNA processing but also in the prion maintenance. SummaryPrions are self-perpetuating infectious proteins. What initiates the misfolding of a protein into its prion form is still not clear. The understanding of cellular factors that facilitate or antagonize prions is crucial to gain insight into the mechanism of prion formation and propagation. In the current study, we reveal that Tah1 is a novel modulator of yeast prion [URE3]. The Hsp90 co-chaperone Tah1, is required for the formation of small nucleolar ribonucleoprotein complex. We show that the absence of Tah1 improves the induction of [URE3] prion. The overexpressed Tah1 cures [URE3], and this function is promoted by Hsp90 chaperones. The current study thus provides a novel cellular factor and the underlying mechanism, involved in the prion formation and propagation

The box C/D snoRNP assembly factor Bcd1 interacts with the histone chaperone Rtt106 and controls its transcription dependent activity

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins initiates co-transcriptionally and requires the action of the assembly machinery including the Hsp90/R2TP complex, the Rsa1p:Hit1p heterodimer and the Bcd1 protein. We present genetic interactions between the Rsa1p-encoding gene and genes involved in chromatin organization including RTT106 that codes for the H3-H4 histone chaperone Rtt106p controlling H3K56ac deposition. We show that Bcd1p binds Rtt106p and controls its transcription-dependent recruitment by reducing its association with RNA polymerase II, modulating H3K56ac levels at gene body. We reveal the 3D structures of the free and Rtt106p-bound forms of Bcd1p using nuclear magnetic resonance and X-ray crystallography. The interaction is also studied by a combination of biophysical and proteomic techniques. Bcd1p interacts with a region that is distinct from the interaction interface between the histone chaperone and histone H3. Our results are evidence for a protein interaction interface for Rtt106p that controls its transcription-associated activity.

RNA Metabolism Guided by RNA Modifications: The Role of SMUG1 in rRNA Quality Control.

RNA modifications are essential for proper RNA processing, quality control, and maturation steps. In the last decade, some eukaryotic DNA repair enzymes have been shown to have an ability to recognize and process modified RNA substrates and thereby contribute to RNA surveillance. Single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) is a base excision repair enzyme that not only recognizes and removes uracil and oxidized pyrimidines from DNA but is also able to process modified RNA substrates. SMUG1 interacts with the pseudouridine synthase dyskerin (DKC1), an enzyme essential for the correct assembly of small nucleolar ribonucleoproteins (snRNPs) and ribosomal RNA (rRNA) processing. Here, we review rRNA modifications and RNA quality control mechanisms in general and discuss the specific function of SMUG1 in rRNA metabolism. Cells lacking SMUG1 have elevated levels of immature rRNA molecules and accumulation of 5-hydroxymethyluridine (5hmU) in mature rRNA. SMUG1 may be required for post-transcriptional regulation and quality control of rRNAs, partly by regulating rRNA and stability.

ROLE OF R2TP COMPLEX IN LYMPHOMA AND ITS THERAPEUTIC POTENTIAL

The R2TP complex which comprises of RUVBL1, RUVBL2, PIH1D1 and RPAP3 in humans is known to be a specialized Co-chaperone of Hsp-90 protein. This multimeric-protein complex is involved in the assembly and maturation of several multi-subunit complexes including RNA polymerase II, small nucleolar ribonucleoproteins, and complexes containing phosphatidylinositol-3-kinase-like kinases. Since their discovery as a co-chaperone of Hsp90, the R2TP complex is involved in multitude of cellular processes including, chromatin remodelling, transcription regulation, ribonucleoprotein complex biogenesis, mitotic assembly, telomerase complex assembly, and apoptosis. Lymphoma arises from the abnormal proliferation of B-cells and the R2TP complex have been reported to play an important role in the activation of p53 and RB. Therefore, the inactivation in any of the tumor suppressor pathways can drive cells to malignancy.However, there are multiple factors which may contribute towards malignancy but the folding defects in these tumor suppressor pathways could be one of the reasons. R2TP is tightly linked with oncogenesis and its inhibition can decrease the proliferation activity of cancer cells. So, the multisubunit chaperone complex as well as its components could be promising candidates for cancer chemotherapy.

Jouvence, a new human snoRNA involved in the control of cell proliferation

BackgroundSmall nucleolar RNAs (snoRNAs) are non-coding RNAs that are conserved from archaebacteria to mammals. They are associated in the nucleolus, with proteins to form small nucleolar ribonucleoprotein (snoRNPs). They modify ribosomal RNAs, for example, the H/ACA box that converts uridine to pseudouridine. In humans, various pathologies have been associated with snoRNAs, and several snoRNAs have been reported to participate in many cancer processes. Recently, a new H/ACA box snoRNA named jouvence has been identified in Drosophila and has been shown to be involved in lifespan determination in relation to gut homeostasis. Because snoRNAs are conserved through evolution, both structurally and functionally, a jouvence orthologue has been identified in humans. RT-PCR has revealed that jouvence is expressed, suggesting that it might be functional. These results suggest the hypothesis that jouvence may display similar functions, including increasing the healthy lifespan in humans.ResultsHere, we report the characterization of the human snoRNA jouvence, which has not yet been annotated in the genome. We show that its overexpression significantly stimulates cell proliferation, both in various stable cancerous cell lines as well as in primary cells. By contrast, its knockdown by siRNA leads to the opposite phenotype, a rapid decrease in cell proliferation. Transcriptomic analysis (RNA-Seq) revealed that the overexpression of jouvence leads to a dedifferentiation signature of the cells. Conversely, the knockdown of jouvence led to a striking decrease in the expression levels of genes involved in ribosome biogenesis and the spliceosome.ConclusionThe overexpression of a single and short non-coding RNA of 159 nucleotides, the snoRNA-jouvence, seems to be sufficient to reorient cells toward stemness, while its depletion blocks cell proliferation. In this context, we speculate that the overexpression of jouvence, which appears to be a non-canonical H/ACA snoRNA, could represent a new tool to fight against the deleterious effects of aging, while inversely, its knockdown by siRNA could represent a new approach in cancer therapy.

SnR30/U17 Small Nucleolar Ribonucleoprotein: A Critical Player during Ribosome Biogenesis.

The small nucleolar RNA snR30 (U17 in humans) plays a unique role during ribosome synthesis. Unlike most members of the H/ACA class of guide RNAs, the small nucleolar ribonucleoprotein (snoRNP) complex assembled on snR30 does not direct pseudouridylation of ribosomal RNA (rRNA), but instead snR30 is critical for 18S rRNA processing during formation of the small subunit (SSU) of the ribosome. Specifically, snR30 is essential for three pre-rRNA cleavages at the A0/01, A1/1, and A2/2a sites in yeast and humans, respectively. Accordingly, snR30 is the only essential H/ACA guide RNA in yeast. Here, we summarize our current knowledge about the interactions and functions of snR30, discuss what remains to be elucidated, and present two non-exclusive hypotheses on the possible molecular function of snR30 during ribosome biogenesis. First, snR30 might be responsible for recruiting other proteins including endonucleases to the SSU processome. Second, snR30 may contribute to the refolding of pre-rRNA into a required conformation that serves as a checkpoint during ribosome biogenesis facilitating pre-rRNA cleavage. In both scenarios, the snR30 snoRNP may have scaffolding and RNA chaperoning activity. In conclusion, the snR30 snoRNP is a crucial player with an unknown molecular mechanism during ribosome synthesis, posing many interesting future research questions.

H/ACA snoRNA levels are regulated during stem cell differentiation

H/ACA small nucleolar RNAs (snoRNAs) guide pseudouridylation as part of a small nucleolar ribonucleoprotein complex (snoRNP). Disruption of H/ACA snoRNA levels in stem cells impairs pluripotency, yet it remains unclear how H/ACA snoRNAs contribute to differentiation. To determine if H/ACA snoRNA levels are dynamic during differentiation, we comprehensively profiled H/ACA snoRNA abundance in multiple murine cell types and during differentiation in three cellular models, including mouse embryonic stem cells and mouse myoblasts. We determined that the profiles of H/ACA snoRNA abundance are cell-type specific, and we identified a subset of snoRNAs that are specifically regulated during differentiation. Additionally, we demonstrated that a decrease in Snora27 abundance upon differentiation corresponds to a decrease in pseudouridylation of its target site within the E-site transfer RNA (tRNA) binding region of the 28S ribosomal RNA (rRNA) in the large ribosomal subunit. Together, these data point toward a potential model in which H/ACA snoRNAs are specifically regulated during differentiation to alter pseudouridylation and fine tune ribosome function.

SnoRNPs: Functions in Ribosome Biogenesis.

Ribosomes are perhaps the most critical macromolecular machine as they are tasked with carrying out protein synthesis in cells. They are incredibly complex structures composed of protein components and heavily chemically modified RNAs. The task of assembling mature ribosomes from their component parts consumes a massive amount of energy and requires greater than 200 assembly factors. Among the most critical of these are small nucleolar ribonucleoproteins (snoRNPs). These are small RNAs complexed with diverse sets of proteins. As suggested by their name, they localize to the nucleolus, the site of ribosome biogenesis. There, they facilitate multiple roles in ribosomes biogenesis, such as pseudouridylation and 2′-O-methylation of ribosomal (r)RNA, guiding pre-rRNA processing, and acting as molecular chaperones. Here, we reviewed their activity in promoting the assembly of ribosomes in eukaryotes with regards to chemical modification and pre-rRNA processing.

BMAL1 Associates with NOP58 in the Nucleolus and Contributes to Pre-rRNA Processing.

SummaryThe transcription factor BMAL1 is a core element of the circadian clock that contributes to cyclic control of genes transcribed by RNA polymerase II. By using biochemical cellular fractionation and immunofluorescence analyses we reveal a previously uncharacterized nucleolar localization for BMAL1. We used an unbiased approach to determine the BMAL1 interactome by mass spectrometry and identified NOP58 as a prominent nucleolar interactor. NOP58, a core component of the box C/D small nucleolar ribonucleoprotein complex, associates with Snord118 to control specific pre-ribosomal RNA (pre-rRNA) processing steps. These results suggest a non-canonical role of BMAL1 in ribosomal RNA regulation. Indeed, we show that BMAL1 controls NOP58-associated Snord118 nucleolar levels and cleavage of unique pre-rRNA intermediates. Our findings identify an unsuspected function of BMAL1 in the nucleolus that appears distinct from its canonical role in the circadian clock system.

MicroRNAs and long non-coding RNAs as novel regulators of ribosome biogenesis.

Ribosome biogenesis is the fine-tuned, essential process that generates mature ribosomal subunits and ultimately enables all protein synthesis within a cell. Novel regulators of ribosome biogenesis continue to be discovered in higher eukaryotes. While many known regulatory factors are proteins or small nucleolar ribonucleoproteins, microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) are emerging as a novel modulatory layer controlling ribosome production. Here, we summarize work uncovering non-coding RNAs (ncRNAs) as novel regulators of ribosome biogenesis and highlight their links to diseases of defective ribosome biogenesis. It is still unclear how many miRNAs or lncRNAs are involved in phenotypic or pathological disease outcomes caused by impaired ribosome production, as in the ribosomopathies, or by increased ribosome production, as in cancer. In time, we hypothesize that many more ncRNA regulators of ribosome biogenesis will be discovered, which will be followed by an effort to establish connections between disease pathologies and the molecular mechanisms of this additional layer of ribosome biogenesis control.

The PAQosome, a novel molecular chaperoning machine for assembly of human protein complexes and networks

The human proteome comprises the products of more than 20,000 genes, most being arranged in complexes and networks that underlie essential cell functions. The Particle for Arrangement of Quaternary structure (PAQosome), formerly known as the R2TP/Prefoldin‐like complex, is to our knowledge the only molecular chaperoning machine designed by the evolution to assemble large protein complexes and regulatory networks in humans. The human PAQosome is a 12‐subunit complex made of two multisubunit modules, namely R2TP (RUVBL1, RUVBL2, RPAP3 and PIH1D1) and Prefoldin‐like (URI1, UXT, PDRG1, PFDN2, PFDN6 and the newly discovered ASDURF), as well as two other accessory proteins (POLR2E and WDR92). Several PAQosome clients/substrates have previously been identified, including all three nuclear RNA polymerases, various small nucleolar ribonucleoproteins (snoRNPs) and small nuclear RNPs (snRNPs), all six members of the phosphatidylinositol 3‐kinase‐related kinase (PIKK) family and others. Client specificity appears to be regulated by a number of different mechanisms, including the use of alternative core PAQosome subunits that recognize different clients directly, and the action of specificity adaptors that connect the PAQosome core to specific clients. Here, we present data identifying additional PAQosome clients, some being recruited through novel mechanisms that will be discussed. For example, and similar to what has been reported for axonemal dynein complexes involved in cilium motility, we now identify cytoplasmic dynein complexes that are responsible for cargo transport along microtubules as new clients of the PAQosome. Our recent data also indicate that specific phosphorylation of PAQosome subunits is at play for recognition of an important newly‐identified client, the pre‐ribosome, which is recruited to the PAQosome along with multiple ribosome biogenesis factors. Detailed mechanisms of assembly of some client complexes as well as the effect of mutations causing genetic diseases on complex/network integrity and PAQosome function will be presented. Characterization of the PAQosome is important not only to elucidate a newly‐recognized basal mechanism of gene regulation in eukaryotes but also to explore new research avenues that can lead to development of novel therapeutic and diagnostic tools.

Box C/D snoRNPs: solid-state NMR fingerprint of an early-stage 50 kDa assembly intermediate.

Many cellular functions rely on stable protein-only or protein-RNA complexes. Deciphering their assembly mechanism is a key question in cell biology. We here focus on box C/D small nucleolar ribonucleoproteins involved in ribosome biogenesis. The mature particles contain four core proteins and a guide RNA. Despite their relatively simple composition, these particles don't self-assemble in eukaryote and the production of a native and functional particle requires a large number of transient other proteins, called assembly factors. We present here 13C and 15N solid-state NMR assignment of yeast 126-residue core protein Snu13 in the context of its 50 kDa pre-complex with assembly factors Rsa1p:Hit1p. In this sample, only one third of the protein is labelled, leading to a low sensitivity. We could nevertheless obtain assignment data for 91% of the residues. Secondary structure derived from our assignments shows that Snu13p overall structure is maintained in the context of the complex. Chemical shift perturbations are analysed to evaluate Snu13p conformational changes and interaction interface upon binding to its partner proteins. While indirect perturbations are observed in the hydrophobic core, we find other good candidate residues belonging to the interaction interface. We describe the role of some Snu13p N-terminal and C-terminal residues, not identified in previous structural studies. These preliminary results will serve as a basis for future interaction studies, especially by adding RNA, to decipher box C/D snoRNP particles assembly pathway.

Molecular determinants that govern scaRNA processing by Drosha/DGCR8.

ABSTRACTThe Cajal body (CB) is a subnuclear domain that participates in the biogenesis of many different types of ribonucleoproteins (RNPs), including small nuclear RNPs (snRNPs), small Cajal body-specific RNPs (scaRNPs) and telomerase. Most scaRNAs, the RNA component of scaRNPs, accumulate in CBs. However, there are three scaRNAs (scaRNA 2, 9, and 17) that are known to be processed into small, nucleolar-enriched fragments. Evidence suggests that these fragments are packaged into a new class of RNPs, called regulatory RNPs (regRNPs), and may modify small nucleolar RNP (snoRNP) activity, thus playing a role in rRNA modification. However, the mechanism by which these fragments are produced is unknown. Previous work has reported the involvement of Drosha and DGCR8 in the cleavage of primary-scaRNA9. Here, we expand on that knowledge by identifying sequence elements necessary for the efficient production of these RNA fragments and demonstrate that primary scaRNA 2 and 17 are also processed by the Drosha-DGCR8 complex. Collectively, our work establishes new factors in the scaRNP biogenesis pathway and adds to the ever-expanding list of noncanonical functions for the microprocessor complex.

R2TP/PAQosome as a promising chemotherapeutic target in cancer

R2TP/PAQosome (particle for arrangement of quaternary structure) is a novel multisubunit chaperone specialized in the assembly/maturation of protein complexes that are involved in essential cellular processes such as PIKKs (phosphatidylinositol 3-kinase-like kinases) signaling, snoRNP (small nucleolar ribonucleoprotein) biogenesis, and RNAP II (RNA polymerase II) complex formation. In this review article, we describe the current understanding of R2TP/PAQosome functions and characteristics as well as how the chaperone complex is involved in oncogenesis, highlighting DNA damage response, mTOR (mammalian target of rapamycin) pathway as well as snoRNP biogenesis. Also, we discuss its possible involvement in HNSCC (head and neck squamous cell carcinoma) including OSCC (oral squamous cell carcinoma). Finally, we provide an overview of current anti-cancer drug development efforts targeting R2TP/PAQosome.

Dyskerin Mutations Present in Dyskeratosis Congenita Patients Increase Oxidative Stress and DNA Damage Signalling in Dictyostelium Discoideum.

Dyskerin is a protein involved in the formation of small nucleolar and small Cajal body ribonucleoproteins. These complexes participate in RNA pseudouridylation and are also components of the telomerase complex required for telomere elongation. Dyskerin mutations cause a rare disease, X-linked dyskeratosis congenita, with no curative treatment. The social amoeba Dictyostelium discoideum contains a gene coding for a dyskerin homologous protein. In this article D. discoideum mutant strains that have mutations corresponding to mutations found in dyskeratosis congenita patients are described. The phenotype of the mutant strains has been studied and no alterations were observed in pseudouridylation activity and telomere structure. Mutant strains showed increased proliferation on liquid culture but reduced growth feeding on bacteria. The results obtained indicated the existence of increased DNA damage response and reactive oxygen species, as also reported in human Dyskeratosis congenita cells and some other disease models. These data, together with the haploid character of D. discoideum vegetative cells, that resemble the genomic structure of the human dyskerin gene, located in the X chromosome, support the conclusion that D. discoideum can be a good model system for the study of this disease.

Base-pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar ribonucleoproteins.

H/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They target many RNAs site-specifically through base-pairing interactions between H/ACA guide and substrate RNA. Besides ribosomal RNA (rRNA) and small nuclear RNA (snRNA), H/ACA snoRNPs are thought to also modify messenger RNA (mRNA) with potential impacts on gene expression. However, the base pairing between known target RNAs and H/ACA guide RNAs varies widely in nature, and therefore the rules governing substrate RNA selection are still not fully understood. To provide quantitative insight into substrate RNA recognition, we systematically altered the sequence of a substrate RNA target by the Saccharomyces cerevisiae H/ACA guide RNA snR34. Time courses measuring pseudouridine formation revealed a gradual decrease in the initial velocity of pseudouridylation upon reducing the number of base pairs between substrate and guide RNA. Changing or inserting nucleotides close to the target uridine severely impairs pseudouridine formation. Interestingly, filter binding experiments show that all substrate RNA variants bind to H/ACA snoRNPs with nanomolar affinity. Next, we showed that binding of inactive, near-cognate RNAs to H/ACA snoRNPs does not inhibit their activity for cognate RNAs, presumably because near-cognate RNAs dissociate rapidly. We discuss that the modulation of initial velocities by the base-pairing strength might affect the order and efficiency of pseudouridylation in rRNA during ribosome biogenesis. Moreover, the binding of H/ACA snoRNPs to near-cognate RNAs may be a mechanism to search for cognate target sites. Together, our data provide critical information to aid in the prediction of productive H/ACA guide—substrate RNA pairs.

PS987 RIBOMETHYLOME CHANGES DEPEND ON DISTINCT PATTERNS OF C/D BOX SNORNAS IN AML SUBTPYES

Background:Small nucleolar RNAs (snoRNAs) are a group of non‐coding RNAs. The most prominent role for snoRNA is to guide small nucleolar ribonucleoproteins (snoRNPs) to complementary regions of ribosomal (rRNA), where the snoRNP complexes catalyze ribosomal modifications. snoRNAs are divided into the two major classes: box C/D and box H/ACA snoRNAs. C/D box snoRNAs are involved in 2‐O‐methylation, whereas H/ACA snoRNAs guide pseudouridylation. Beyond the role in rRNA modification, functions in splicing and chromatin have been proposed. Recently, we demonstrated that snoRNAs play a critical role for AML1‐ETO induced leukemia (Zhou et al., Nat Cell Biol 2017).Aims:We aimed to elucidate snoRNA expression patterns and functions in acute myeloid leukemia and establish the first global map of 2’‐methylation of rRNA in AML patients.Methods:We analyzed snoRNA expression patterns in a cohort of 161 AML patients using small‐RNA sequencing. All patient samples were obtained at the time of diagnosis. Expression levels were calculated for known C/D‐box and H/ACA‐box snoRNA. Only regions with at least one reads per million (RPM) in at least one patient sample were included for further investigation. We applied ribomethseq to elucidate the entire ribosomal 2’‐0 methylation pattern in AML patients. We calculated the methylation scoreC from 5’‐read ends for all known sites from snOPY and an updated list from Krogh et al. 2016, Further, methylation patterns and snoRNA patterns were analysed with regard to AML mutations as analysed by panel sequencing.Results:Next generation sequencing data revealed expression o 354 known C/D box snoRNAs and 160 H/ACA box snoRNAs in primary AML patient samples. Expression patterns of snoRNAs varied according to specific genetic risk groups. High levels of snoRNA expression were observed in intermediate molecular risk group patients (215 C/D box and 68 H/ACA box snoRNAs, p≤0.05). Also, high levels of snoRNA were found in patients with a poor response to the first cycle of induction therapy (144 C/D box and 61 H/ACA box snoRNAs, p≤0.05). Of note, NPM1 mutations were associated with wide ranging changes in expression (almost all downregulation) in 232 C/D box and 61 H/ACA box snoRNA. In contrast, AMLs with ASXL1 or RUNX1 mutations predominantly expressed higher levels of snoRNA expression. We established the first map of ribosomal 2’‐0 methylation patterns in AML patients on published modification sites. This resulted in RiboMethylome data constituting 107 high quality ribosomal sites which were methylated in AML patients. In most instances, C/D box snoRNA expression was positively correlated with the respective levels of 2́‐O‐methylation in rRNA. Importantly, NPM1 mutation status strongly influenced the RiboMethylome in affected AML blasts.Summary/Conclusion:AML with different mutation patterns exhibit striking differences in snoRNA expression levels. Our data reveal widespread plasticity of epitranscriptomic modifications in the ribosomes of AML patients. These findings suggest that driver mutations in AML could induce specialized ribosomes which may contribute to leukemic transformation.

Bcd1p controls RNA loading of the core protein Nop58 during C/D box snoRNP biogenesis.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins (C/D snoRNPs) is guided by conserved trans-acting factors that act collectively to assemble the core proteins SNU13/Snu13, NOP58/Nop58, NOP56/Nop56, FBL/Nop1, and box C/D small nucleolar RNAs (C/D snoRNAs), in human and in yeast, respectively. This finely elaborated process involves the sequential interplay of snoRNP-related proteins and RNA through the formation of transient pre-RNP complexes. BCD1/Bcd1 protein is essential for yeast cell growth and for the specific accumulation of box C/D snoRNAs. In this work, chromatin, RNA, and protein immunoprecipitation assays revealed the ordered loading of several snoRNP-related proteins on immature and mature snoRNA species. Our results identify Bcd1p as an assembly factor of C/D snoRNP biogenesis that is likely recruited cotranscriptionally and that directs the loading of the core protein Nop58 on RNA.