Intracellular localization of CK2α as a prognostic factor in invasive breast carcinomas.

Overexpression of the ubiquitous protein kinase, CK2α, has been reported in various human cancers. Here, we demonstrate that nuclear and nucleolar CK2α localization in invasive ductal carcinomas of the breast is a reliable predictor of poor prognosis. Cellular localization of CK2α in nuclei and nucleoli was analyzed immunohistochemically using surgical tissue blocks from 112 patients, who had undergone surgery without neoadjuvant chemotherapy. Clinical data collection and median follow-up period were for more than 5 y. In total, 93.8% of patients demonstrated elevated CK2α expression in nuclei and 36.6% of them displayed elevated expression predominantly in nucleoli. Clinicopathological malignancy was strongly correlated with elevated nuclear and nucleolar CK2α expression. Recurrence-free survival was significantly worse (P = .0002) in patients with positive nucleolar CK2α staining. The 5-y survival rate decreased to a roughly 50% in nucleolar CK2α-positive patients of triple-negative (P = .0069) and p Stage 3 (P = .0073) groups. In contrast, no patients relapsed or died in the triple-negative group who exhibited a lack of nucleolar CK2α staining. Evaluation of nucleolar CK2α staining showed a high secondary index with a hazard ratio of 6.629 (P = .001), following lymph node metastasis with a hazard ratio of 14.30 (P = .0008). Multivariate analysis demonstrated that nucleolar CK2α is an independent factor for recurrence-free survival. Therefore, we propose that histochemical evaluation of nucleolar CK2α-positive staining may be a new and robust prognostic indicator for patients who need further treatment. Functional consequences of nucleolar CK2 dysfunction may be a starting point to facilitate development of novel treatments for invasive breast carcinoma.

Combined expression levels of KDM2A and KDM2B correlate with nucleolar size and prognosis in primary breast carcinomas.

Ribosome biogenesis is a fine-tuned cellular process and its deregulation is linked to cancer progression: tumors characterized by an intense ribosome biogenesis often display a more aggressive behavior. Ribosomal RNA (rRNA) synthesis is controlled at several levels, the higher one being the epigenetic regulation of the condensation of chromatin portions containing rRNA genes. KDM2A and KDM2B (Lysine (K)-specific demethylase 2A / B) are histone demethylases modulating the accessibility of ribosomal genes, thereby regulating their transcription. Both enzymes are able to demethylate lysins at relevant sites (e.g. K4, K36) on histone H3. We previously demonstrated that KDM2B is one of the factors regulating ribosome biogenesis in human breast cancer. In this study we aimed to define the combined contribution of KDM2A and KDM2B to breast cancer outcome. KDM2A and KDM2B mRNA levels, nucleolar area as a marker of ribosome biogenesis, and patients' prognosis were retrospectively assessed in a series of primary breast carcinomas. We observed that tumors characterized by reduced levels of both KDM2A and KDM2B displayed a particularly aggressive clinical behavior and increased nucleolar size. Our results suggest that KDM2A and KDM2B may cooperate in regulating ribosome biogenesis thus influencing the biological behavior and clinical outcome of human breast cancers.

Nucleolar RNA polymerase II drives ribosome biogenesis.

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.

Proliferation, cancer, and aging-novel functions of the nucleolar methyltransferase fibrillarin?

Fibrillarin is an essential nucleolar protein that catalyzes the 2'-O-methylation of ribosomal RNAs. Recently, experimental data have begun to accumulate that suggest that fibrillarin can influence various cellular processes, development of pathological processes, and even aging. The exact mechanism by which fibrillarin can influence these processes has not been found, but some experimental data indicate that up- or downregulation of fibrillarin can modify the ribosome structure and, thus, causе an alteration in relative efficiency with which various mRNAs are translated. Here, we discuss recent studies on the potential roles of fibrillarin in the regulation of cell proliferation, cancer progression, and aging.

DDX49 is an RNA helicase that affects translation by regulating mRNA export and the levels of pre-ribosomal RNA.

Among the proteins predicted to be a part of the DExD box RNA helicase family, the functions of DDX49 are unknown. Here, we characterize the enzymatic activities and functions of DDX49 by comparing its properties with the well-studied RNA helicase, DDX39B. We find that DDX49 exhibits a robust ATPase and RNA helicase activity, significantly higher than that of DDX39B. DDX49 is required for the efficient export of poly (A)+ RNA from nucleus in a splicing-independent manner. Furthermore, DDX49 is a resident protein of nucleolus and regulates the steady state levels of pre-ribosomal RNA by regulating its transcription and stability. These dual functions of regulating mRNA export and pre-ribosomal RNA levels enable DDX49 to modulate global translation. Phenotypically, DDX49 promotes proliferation and colony forming potential of cells. Strikingly, DDX49 is significantly elevated in diverse cancer types suggesting that the increased abundance of DDX49 has a role in oncogenic transformation of cells. Taken together, this study shows the physiological role of DDX49 in regulating distinct steps of mRNA and pre-ribosomal RNA metabolism and hence translation and potential pathological role of its dysregulation, especially in cancers.

NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus.

The PIDDosome (PIDD-RAIDD-caspase-2 complex) is considered to be the primary signaling platform for caspase-2 activation in response to genotoxic stress. Yet studies of PIDD-deficient mice show that caspase-2 activation can proceed in the absence of PIDD. Here we show that DNA damage induces the assembly of at least two distinct activation platforms for caspase-2: a cytoplasmic platform that is RAIDD dependent but PIDD independent, and a nucleolar platform that requires both PIDD and RAIDD. Furthermore, the nucleolar phosphoprotein nucleophosmin (NPM1) acts as a scaffold for PIDD and is essential for PIDDosome assembly in the nucleolus after DNA damage. Inhibition of NPM1 impairs caspase-2 processing, apoptosis, and caspase-2-dependent inhibition of cell growth, demonstrating that the NPM1-dependent nucleolar PIDDosome is a key initiator of the caspase-2 activation cascade. Thus we have identified the nucleolus as a novel site for caspase-2 activation and function.

S6 Kinase is essential for MYC-dependent rDNA transcription in Drosophila.

Increased rates of ribosome biogenesis and biomass accumulation are fundamental properties of rapidly growing and dividing malignant cells. The MYC oncoprotein drives growth predominantly via its ability to upregulate the ribosome biogenesis program, in particular stimulating the activity of the RNA Polymerase I (Pol I) machinery to increase ribosomal RNA (rRNA) transcription. Although MYC function is known to be highly dependent on the cellular signalling context, the pathways interacting with MYC to regulate transcription of ribosomal genes (rDNA) in vivo in response to growth factor status, nutrient availability and cellular stress are only beginning to be understood. To determine factors critical to MYC-dependent stimulation of rDNA transcription in vivo, we performed a transient expression screen for known oncogenic signalling pathways in Drosophila. Strikingly, from the broad range of pathways tested, we found that ribosomal protein S6 Kinase (S6K) activity, downstream of the TOR pathway, was the only factor rate-limiting for the rapid induction of rDNA transcription due to transiently increased MYC. Further, we demonstrated that one of the mechanism(s) by which MYC and S6K cooperate is through coordinate activation of the essential Pol I transcription initiation factor TIF-1A (RRN 3). As Pol I targeted therapy is now in phase 1 clinical trials in patients with haematological malignancies, including those driven by MYC, these data suggest that therapies dually targeting Pol I transcription and S6K activity may be effective in treating MYC-driven tumours.

Catalytically active telomerase holoenzyme is assembled in the dense fibrillar component of the nucleolus during S phase.

The maintenance of human telomeres requires the ribonucleoprotein enzyme telomerase, which is composed of telomerase reverse transcriptase (TERT), telomerase RNA component, and several additional proteins for assembly and activity. Telomere elongation by telomerase in human cancer cells involves multiple steps including telomerase RNA biogenesis, holoenzyme assembly, intranuclear trafficking, and telomerase recruitment to telomeres. Although telomerase has been shown to accumulate in Cajal bodies for association with telomeric chromatin, it is unclear where and how the assembly and trafficking of catalytically active telomerase is regulated in the context of nuclear architecture. Here, we show that the catalytically active holoenzyme is initially assembled in the dense fibrillar component of the nucleolus during S phase. The telomerase RNP is retained in nucleoli through the interaction of hTERT with nucleolin, a major nucleolar phosphoprotein. Upon association with TCAB1 in S phase, the telomerase RNP is transported from nucleoli to Cajal bodies, suggesting that TCAB1 acts as an S-phase-specific holoenzyme component. Furthermore, depletion of TCAB1 caused an increase in the amount of telomerase RNP associated with nucleolin. These results suggest that the TCAB1-dependent trafficking of telomerase to Cajal bodies occurs in a step separate from the holoenzyme assembly in nucleoli. Thus, we propose that the dense fibrillar component is the provider of active telomerase RNP for supporting the continued proliferation of cancer and stem cells.

Emerging roles of the nucleolus in regulating the DNA damage response: the noncanonical DNA repair enzyme APE1/Ref-1 as a paradigmatical example.

SIGNIFICANCE: An emerging concept in DNA repair mechanisms is the evidence that some key enzymes, besides their role in the maintenance of genome stability, display also unexpected noncanonical functions associated with RNA metabolism in specific subcellular districts (e.g., nucleoli). During the evolution of these key enzymes, the acquisition of unfolded domains significantly amplified the possibility to interact with different partners and substrates, possibly explaining their phylogenetic gain of functions. RECENT ADVANCES: After nucleolar stress or DNA damage, many DNA repair proteins can freely relocalize from nucleoli to the nucleoplasm. This process may represent a surveillance mechanism to monitor the synthesis and correct assembly of ribosomal units affecting cell cycle progression or inducing p53-mediated apoptosis or senescence. CRITICAL ISSUES: A paradigm for this kind of regulation is represented by some enzymes of the DNA base excision repair (BER) pathway, such as apurinic/apyrimidinic endonuclease 1 (APE1). In this review, the role of the nucleolus and the noncanonical functions of the APE1 protein are discussed in light of their possible implications in human pathologies. FUTURE DIRECTIONS: A productive cross-talk between DNA repair enzymes and proteins involved in RNA metabolism seems reasonable as the nucleolus is emerging as a dynamic functional hub that coordinates cell growth arrest and DNA repair mechanisms. These findings will drive further analyses on other BER proteins and might imply that nucleic acid processing enzymes are more versatile than originally thought having evolved DNA-targeted functions after a previous life in the early RNA world.

A nucleolar AAA-NTPase is required for parasite division.

Apicomplexa division involves several distinct phases shared with other eukaryote cell cycles including a gap period (G1) prior to chromosome synthesis, although how progression through the parasite cell cycle is controlled is not understood. Here we describe a cell cycle mutant that reversibly arrests in the G1 phase. The defect in this mutant was mapped by genetic complementation to a gene encoding a novel AAA-ATPase/CDC48 family member called TgNoAP1. TgNoAP1 is tightly regulated and expressed in the nucleolus during the G1/S phases. A tyrosine to a cysteine change upstream of the second AAA+ domain in the temperature sensitive TgNoAP1 allele leads to conditional protein instability, which is responsible for rapid cell cycle arrest and a primary defect in 28S rRNA processing as confirmed by knock-in of the mutation back into the parent genome. The interaction of TgNoAP1 with factors of the snoRNP and R2TP complexes indicates this protein has a role in pre-rRNA processing. This is a novel role for a cdc48-related chaperone protein and indicates that TgNoAP1 may be part of a dynamic mechanism that senses the health of the parasite protein machinery at the initial steps of ribosome biogenesis and conveys that information to the parasite cell cycle checkpoint controls.

Cyclin-dependent kinase 9 links RNA polymerase II transcription to processing of ribosomal RNA.

Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3' extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3' processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3' rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.

[Influence of ionizing radiation on enzymatic activity and state of nucleus-nucleolar apparatus in rat hepatocytes].

The effects of a single exposure of rats to the whole-body roentgen irradiation at the doses of 3.5 Gy and 4.5 Gy on the activity of creatine kinase, purine nucleoside phosphorylase, alanine aminotransferase, aspartate aminotransferase, as well as on the state of the nuclear-nucleolar apparatus in rat hepatocytes on the 6th and 13th days after radiation exposure have been studied. Irradiation at the above doses induced changes in the levels of enzymatic activity of different values and different directions within the same time periods, as well as oscillating changes in this type of enzymatic activity over time. This demonstrates various radiosensitivity and adaptation abilities of these enzymatic activities. The changes in the enzymatic activity significantly correspond to the changes in the morphometric indices of nuclear-nucleolar apparatus of hepatocytes, as well as the distribution of hepatocytes within the ploidy classes: in particular, stabilization of the enzymatic activity on the 13th day after irradiation correlates with the increased transcriptional activity, which is detectable through the increased number of nucleoli per nucleus and the expanded space of a hepatocyte nucleus. The compensation mechanisms are likely to be targeted at the changes in the functional activity of surviving hepatocytes, rather than at the replacement of the damaged cells by the new ones.

Structure-function relationships during transgenic telomerase expression in Arabidopsis.

Although telomerase (EC 2.7.7.49) is important for genome stability and totipotency of plant cells, the principles of its regulation are not well understood. Therefore, we studied subcellular localization and function of the full-length and truncated variants of the catalytic subunit of Arabidopsis thaliana telomerase, AtTERT, in planta. Our results show that multiple sites in AtTERT may serve as nuclear localization signals, as all the studied individual domains of the AtTERT were targeted to the nucleus and/or the nucleolus. Although the introduced genomic or cDNA AtTERT transgenes display expression at transcript and protein levels, they are not able to fully complement the lack of telomerase functions in tert -/- mutants. The failure to reconstitute telomerase function in planta suggests a more complex telomerase regulation in plant cells than would be expected based on results of similar experiments in mammalian model systems.

Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription.

Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription.

A non-catalytic role for inositol 1,3,4,5,6-pentakisphosphate 2-kinase in the synthesis of ribosomal RNA.

Fundamental to the life and destiny of every cell is the regulation of protein synthesis through ribosome biogenesis, which begins in the nucleolus with the production of ribosomal RNA (rRNA). Nucleolar organization is a highly dynamic and tightly regulated process; the structural factors that direct nucleolar assembly and disassembly are just as important in controlling rRNA synthesis as are the catalytic activities that synthesize rRNA. Here, we report that a signaling enzyme, inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5K) is also a structural component in the nucleolus. We demonstrate that IP5K has functionally significant interactions with three proteins that regulate rRNA synthesis: protein kinase CK2, TCOF1 and upstream-binding-factor (UBF). Through molecular modeling and mutagenic studies, we identified an Arg-Lys-Lys tripeptide located on the surface of IP5K that mediates its association with UBF. Nucleolar IP5K spatial dynamics were sensitive to experimental procedures (serum starvation or addition of actinomycin D) that inhibited rRNA production. We show that IP5K makes stoichiometrically sensitive contributions to the architecture of the nucleoli in intact cells, thereby influencing the degree of rRNA synthesis. Our study adds significantly to the biological significance of IP5K; previously, it was the kinase activity of this protein that had attracted attention. Our demonstration that IP5K 'moonlights' as a molecular scaffold offers an unexpected new example of how the biological sophistication of higher organisms can arise from gene products acquiring multiple functions, rather than by an increase in gene number.

The human homolog of Escherichia coli endonuclease V is a nucleolar protein with affinity for branched DNA structures.

Loss of amino groups from adenines in DNA results in the formation of hypoxanthine (Hx) bases with miscoding properties. The primary enzyme in Escherichia coli for DNA repair initiation at deaminated adenine is endonuclease V (endoV), encoded by the nfi gene, which cleaves the second phosphodiester bond 3' of an Hx lesion. Endonuclease V orthologs are widespread in nature and belong to a family of highly conserved proteins. Whereas prokaryotic endoV enzymes are well characterized, the function of the eukaryotic homologs remains obscure. Here we describe the human endoV ortholog and show with bioinformatics and experimental analysis that a large number of transcript variants exist for the human endonuclease V gene (ENDOV), many of which are unlikely to be translated into functional protein. Full-length ENDOV is encoded by 8 evolutionary conserved exons covering the core region of the enzyme, in addition to one or more 3'-exons encoding an unstructured and poorly conserved C-terminus. In contrast to the E. coli enzyme, we find recombinant ENDOV neither to incise nor bind Hx-containing DNA. While both enzymes have strong affinity for several branched DNA substrates, cleavage is observed only with E. coli endoV. We find that ENDOV is localized in the cytoplasm and nucleoli of human cells. As nucleoli harbor the rRNA genes, this may suggest a role for the protein in rRNA gene transactions such as DNA replication or RNA transcription.

Poly(ADP-Ribose) polymerase 1 (PARP-1) regulates ribosomal biogenesis in Drosophila nucleoli.

Poly(ADP-ribose) polymerase 1 (PARP1), a nuclear protein, utilizes NAD to synthesize poly(AD-Pribose) (pADPr), resulting in both automodification and the modification of acceptor proteins. Substantial amounts of PARP1 and pADPr (up to 50%) are localized to the nucleolus, a subnuclear organelle known as a region for ribosome biogenesis and maturation. At present, the functional significance of PARP1 protein inside the nucleolus remains unclear. Using PARP1 mutants, we investigated the function of PARP1, pADPr, and PARP1-interacting proteins in the maintenance of nucleolus structure and functions. Our analysis shows that disruption of PARP1 enzymatic activity caused nucleolar disintegration and aberrant localization of nucleolar-specific proteins. Additionally, PARP1 mutants have increased accumulation of rRNA intermediates and a decrease in ribosome levels. Together, our data suggests that PARP1 enzymatic activity is required for targeting nucleolar proteins to the proximity of precursor rRNA; hence, PARP1 controls precursor rRNA processing, post-transcriptional modification, and pre-ribosome assembly. Based on these findings, we propose a model that explains how PARP1 activity impacts nucleolar functions and, consequently, ribosomal biogenesis.

The AAA-ATPase NVL2 is a telomerase component essential for holoenzyme assembly.

Continued cell proliferation requires telomerase to maintain functional telomeres that are essential for chromosome integrity. Although the core enzyme includes a telomerase reverse transcriptase (TERT) and a telomerase RNA component (TERC), a number of auxiliary proteins have been identified to regulate telomerase assembly, localization, and enzymatic activity. Here we describe the characterization of the AAA-ATPase NVL2 as a novel hTERT-interacting protein. NVL2 interacts and co-localizes with hTERT in the nucleolus. NLV2 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT. Depletion of endogenous NVL2 by small interfering RNA led to a decrease in hTERT without affecting the steady-state levels of hTERT mRNA, thereby reducing telomerase activity, suggesting that NVL2 is an essential component of the telomerase holoenzyme. We also found that ATP-binding activity of NVL2 is required for hTERT binding as well as telomerase assembly. Our findings suggest that NVL2, in addition to its role in ribosome biosynthesis, is essential for telomerase biogenesis and provides an alternative approach for inhibiting telomerase activity in cancer.

Signals and pathways regulating nucleolar retention of novel putative nucleolar GTPase NGP-1(GNL-2).

NGP-1(GNL-2) is a putative GTPase, overexpressed in breast carcinoma and localized in the nucleolus. NGP-1 belongs to the MMR1-HSR1 family of large GTPases that are emerging as crucial coordinators of signaling cascades in different cellular compartments. The members of this family share very closely related G-domains, but the signals and pathways regulating their subcellular localization and their functional relevance remain unknown. To improve our understanding of the nuclear transport mechanism of NGP-1, we have identified two nucleolar localization signals (NoLS) that are independently shown to translocate NGP-1 as well the heterologous protein to the nucleolus. Site-specific mutagenesis and immunofluorescence studies suggest that the tandem repeats of positively charged amino acids are critical for NGP-1 NoLS function. Interestingly, amino-terminal (NGP-1(1-100)) and carboxyl-terminal (NGP-1(661-731)) signals independently interact with receptors importin-ß and importin-α, respectively. This investigation, for the first time, provides evidence that the interaction of importin-α with C-terminal NoLS (NGP-1(661-731)) was able to target the heterologous protein to the nucleolar compartment. Structural modeling analysis and alanine scanning mutagenesis of conserved G-domains suggest that G4 and G5 motifs are critical for GTP binding of NGP-1 and further show that the nucleolar localization of NGP-1 is regulated by a GTP gating-mediated mechanism. In addition, our data suggest that an ongoing transcription is essential for efficient localization of NGP-1 to the nucleolus. We have observed a high level of NGP-1 expression in the mitogen-activated primary human peripheral blood mononuclear cells (hPBMC) as well as in human fetal brain-derived neural precursor cells (hNPCs) in comparison to cells undergoing differentiation. Overall, the results suggest that multiple mechanisms are involved in the localization of NGP-1 to the nucleolus for the regulation of nucleolar function in cell growth and proliferation.

The Bowen-Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA.

The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen-Conradi syndrome (BCS) is caused by a specific Nep1(D86G) mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific (14)C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1(ts) mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome.