RESUMEN
Small nucleolar RNAs (snoRNAs) are earning increasing attention from research communities due to their critical role in the post-transcriptional modification of various RNAs. These snoRNAs, along with their associated proteins, are crucial in regulating the expression of a vast array of genes in different human diseases. Primarily, snoRNAs facilitate modifications such as 2'-O-methylation, N-4-acetylation, and pseudouridylation, which impact not only ribosomal RNA (rRNA) and their synthesis but also different RNAs. Functionally, snoRNAs bind with core proteins to form small nucleolar ribonucleoproteins (snoRNPs). These snoRNAs then direct the protein complex to specific sites on target RNA molecules where modifications are necessary for either standard cellular operations or the regulation of pathological mechanisms. At these targeted sites, the proteins coupled with snoRNPs perform the modification processes that are vital for controlling cellular functions. The unique characteristics of snoRNAs and their involvement in various non-metabolic and metabolic diseases highlight their potential as therapeutic targets. Moreover, the precise targeting capability of snoRNAs might be harnessed as a molecular tool to therapeutically address various disease conditions. This review delves into the role of snoRNAs in health and disease and explores the broad potential of these snoRNAs as therapeutic agents in human pathologies.
Asunto(s)
ARN Nucleolar Pequeño , Ribonucleoproteínas Nucleolares Pequeñas , Humanos , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/genética , Animales , Procesamiento Postranscripcional del ARN , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
Embryo development in Arabidopsis (Arabidopsis thaliana) starts off with an asymmetric division of the zygote to generate the precursors of the embryo proper and the supporting extraembryonic suspensor. The suspensor degenerates as the development of the embryo proper proceeds beyond the heart stage. Until the globular stage, the suspensor maintains embryonic potential and can form embryos in the absence of the developing embryo proper. We report a mutant called meerling-1 (mrl-1), which shows a high penetrance of suspensor-derived polyembryony due to delayed development of the embryo proper. Eventually, embryos from both apical and suspensor lineages successfully develop into normal plants and complete their life cycle. We identified the causal mutation as a genomic rearrangement altering the promoter of the Arabidopsis U3 SMALL NUCLEOLAR RNA-ASSOCIATED PROTEIN 18 (UTP18) homolog that encodes a nucleolar-localized WD40-repeat protein involved in processing 18S preribosomal RNA. Accordingly, root-specific knockout of UTP18 caused growth arrest and accumulation of unprocessed 18S pre-rRNA. We generated the mrl-2 loss-of-function mutant and observed asynchronous megagametophyte development causing embryo sac abortion. Together, our results indicate that promoter rearrangement decreased UTP18 protein abundance during early stage embryo proper development, triggering suspensor-derived embryogenesis. Our data support the existence of noncell autonomous signaling from the embryo proper to prevent direct reprogramming of the suspensor toward embryonic fate.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Mutación , Ribonucleoproteínas Nucleolares Pequeñas , Semillas , Arabidopsis/genética , Arabidopsis/embriología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación/genética , ARN Ribosómico/genética , Semillas/genética , Semillas/crecimiento & desarrollo , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismoRESUMEN
Telomerase is a ribonucleoprotein (RNP) enzyme that extends telomeric repeats at eukaryotic chromosome ends to counterbalance telomere loss caused by incomplete genome replication. Human telomerase is comprised of two distinct functional lobes tethered by telomerase RNA (hTR): a catalytic core, responsible for DNA extension; and a Hinge and ACA (H/ACA) box RNP, responsible for telomerase biogenesis. H/ACA RNPs also have a general role in pseudouridylation of spliceosomal and ribosomal RNAs, which is critical for the biogenesis of the spliceosome and ribosome. Much of our structural understanding of eukaryotic H/ACA RNPs comes from structures of the human telomerase H/ACA RNP. Here we report a 2.7 Å cryo-electron microscopy structure of the telomerase H/ACA RNP. The significant improvement in resolution over previous 3.3 Å to 8.2 Å structures allows us to uncover new molecular interactions within the H/ACA RNP. Many disease mutations are mapped to these interaction sites. The structure also reveals unprecedented insights into a region critical for pseudouridylation in canonical H/ACA RNPs. Together, our work advances understanding of telomerase-related disease mutations and the mechanism of pseudouridylation by eukaryotic H/ACA RNPs.
Asunto(s)
Ribonucleoproteínas , Telomerasa , Humanos , Ribonucleoproteínas/genética , Telomerasa/genética , Microscopía por Crioelectrón , Ribonucleoproteínas Nucleolares Pequeñas/genética , ARN/genética , ARN RibosómicoRESUMEN
The DEAD-box helicase Dbp4 plays an essential role during the early assembly of the 40S ribosome, which is only poorly understood to date. By applying the yeast two-hybrid method and biochemical approaches, we discovered that Dbp4 interacts with the Efg1-Bud22 dimer. Both factors associate with early pre-90S particles and smaller complexes, each characterized by a high presence of the U14 snoRNA. A crosslink analysis of Bud22 revealed its contact to the U14 snoRNA and the 5' domain of the nascent 18S rRNA, close to its U14 snoRNA hybridization site. Moreover, depletion of Bud22 or Efg1 specifically affects U14 snoRNA association with pre-ribosomal complexes. Accordingly, we concluded that the role of the Efg1-Bud22 dimer is linked to the U14 snoRNA function on early 90S ribosome intermediates chaperoning the 5' domain of the nascent 18S rRNA. The successful rRNA folding of the 5' domain and the release of Efg1, Bud22, Dpb4, U14 snoRNA and associated snoRNP factors allows the subsequent recruitment of the Kre33-Bfr2-Enp2-Lcp5 module towards the 90S pre-ribosome.
Asunto(s)
Ribonucleoproteínas Nucleolares Pequeñas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribosomas/metabolismo , Precursores del ARN/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/química , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: Colorectal cancer (CRC) is a prevalent type of gastrointestinal cancer, and its poor prognosis is mainly attributed to the occurrence of invasion and metastasis. CYP1B1-AS1, as non-coding RNA, plays an important role in tumorigenesis and progression. However, the mechanism by which CYP1B1-AS1 acts in CRC is not yet understood. AIMS: The objective of this study was to investigate how CYP1B1-AS1 contributes to the development of CRC, and provide a base for CRC diagnosis and treatment. METHODS: RT-qPCR was used to detect the expression level of CYP1B1-AS1 in CRC and adjacent tissues. CCK-8, Edu, scratch healing, and transwell experiments were used to detect the changes of proliferation, migration, and invasion ability of CRC cells after overexpression or knockdown of CYP1B1-AS1 respectively. The RNA binding protein NOP58 combined with CYP1B1-AS1 was verified by RIP and RNA Pull-down experiments. Functional recovery experiments validated the interaction between CYP1B1-AS1 and NOP58 in CRC cells. The changes of EMT-related proteins were detected by Western blot, and the half-life of transcription factor SNAIL mRNA were detected by RT-qPCR after overexpression or knockdown of NOP58. RESULTS: CYP1B1-AS1 was found to be significantly downregulated in CRC compared to adjacent noncancerous tissues. Experiments conducted in vitro and in vivo confirmed that upregulation of CYP1B1-AS1 significantly inhibited the proliferation, migration, and invasion of CRC cells. In addition, CYP1B1-AS1 can directly bind to NOP58 and negatively regulate NOP58. The effect of overexpression CYP1B1-AS1 was reversed by NOP58 overexpression. NOP58 regulates the EMT process of CRC cells by affecting the stability of EMT-related transcription factor SNAIL mRNA, and then affects the progress of CRC. CONCLUSION: This research proves that CYP1B1-AS1 can inhibit the occurrence of EMT in CRC by binding with NOP58, thus delaying the progress of CRC. This finding indicates that CYP1B1-AS1 may be a novel biomarker to improve the diagnosis and treatment of CRC.
Asunto(s)
Neoplasias Colorrectales , MicroARNs , ARN Largo no Codificante , Humanos , Línea Celular Tumoral , MicroARNs/genética , Factores de Transcripción/genética , Neoplasias Colorrectales/patología , ARN Mensajero , Proliferación Celular/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Proteínas Nucleares/genética , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismoRESUMEN
The conserved H/ACA RNPs consist of one H/ACA RNA and 4 core proteins: dyskerin, NHP2, NOP10, and GAR1. Its assembly requires several assembly factors. A pre-particle containing the nascent RNAs, dyskerin, NOP10, NHP2 and NAF1 is assembled co-transcriptionally. NAF1 is later replaced by GAR1 to form mature RNPs. In this study, we explore the mechanism leading to the assembly of H/ACA RNPs. We performed the analysis of GAR1, NHP2, SHQ1 and NAF1 proteomes by quantitative SILAC proteomic, and analyzed purified complexes containing these proteins by sedimentation on glycerol gradient. We propose the formation of several distinct intermediate complexes during H/ACA RNP assembly, notably the formation of early protein-only complexes containing at least the core proteins dyskerin, NOP10, and NHP2, and the assembly factors SHQ1 and NAF1. We also identified new proteins associated with GAR1, NHP2, SHQ1 and NAF1, which can be important for box H/ACA assembly or function. Moreover, even though GAR1 is regulated by methylations, the nature, localization, and functions of these methylations are not well known. Our MS analysis of purified GAR1 revealed new sites of arginine methylations. Additionally, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs, even though with less efficiency than methylated ones.
Asunto(s)
Proteómica , Ribonucleoproteínas , Ribonucleoproteínas/genética , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Proteínas de Unión al ARN , ARN/genéticaRESUMEN
5-Methylcytosine (m5C) is a base modification broadly found on various RNAs in the human transcriptome. In eukaryotes, m5C is catalyzed by enzymes of the NSUN family composed of seven human members (NSUN1-7). NOP2/NSUN1 has been primarily characterized in budding yeast as an essential ribosome biogenesis factor required for the deposition of m5C on the 25S ribosomal RNA (rRNA). Although human NOP2/NSUN1 has been known to be an oncogene overexpressed in several types of cancer, its functions and substrates remain poorly characterized. Here, we used a miCLIP-seq approach to identify human NOP2/NSUN1 RNA substrates. Our analysis revealed that NOP2/NSUN1 catalyzes the deposition of m5C at position 4447 on the 28S rRNA. We also find that NOP2/NSUN1 binds to the 5'ETS region of the pre-rRNA transcript and regulates pre-rRNA processing through non-catalytic complex formation with box C/D snoRNAs. We provide evidence that NOP2/NSUN1 facilitates the recruitment of U3 and U8 snoRNAs to pre-90S ribosomal particles and their stable assembly into snoRNP complexes. Remarkably, expression of both WT and catalytically inactive NOP2/NSUN1 in knockdown background rescues the rRNA processing defects and the stable assembly of box C/D snoRNP complexes, suggesting that NOP2/NSUN1-mediated deposition of m5C on rRNA is not required for ribosome synthesis.
Asunto(s)
Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas , ARNt Metiltransferasas/metabolismo , 5-Metilcitosina/metabolismo , Humanos , Precursores del ARN/metabolismo , ARN Ribosómico/metabolismo , ARN Ribosómico 28S/metabolismo , ARN Nucleolar Pequeño/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Regulation of protein synthesis is critical for control of gene expression in all cells. Ribosomes are ribonucleoprotein machines responsible for translating cellular proteins. Defects in ribosome production, function, or regulation are detrimental to the cell and cause human diseases, such as progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PEHO) syndrome. PEHO syndrome is a devastating neurodevelopmental disorder caused by mutations in the ZNHIT3 gene, which encodes an evolutionarily conserved nuclear protein. The precise mechanisms by which ZNHIT3 mutations lead to PEHO syndrome are currently unclear. Studies of the human zinc finger HIT-type containing protein 3 homolog in budding yeast (Hit1) revealed that this protein is critical for formation of small nucleolar ribonucleoprotein complexes that are required for rRNA processing and 2'-O-methylation. Here, we use budding yeast as a model system to reveal the basis for the molecular pathogenesis of PEHO syndrome. We show that missense mutations modeling those found in PEHO syndrome patients cause a decrease in steady-state Hit1 protein levels, a significant reduction of box C/D snoRNA levels, and subsequent defects in rRNA processing and altered cellular translation. Using RiboMethSeq analysis of rRNAs isolated from actively translating ribosomes, we reveal site-specific changes in the rRNA modification pattern of PEHO syndrome mutant yeast cells. Our data suggest that PEHO syndrome is a ribosomopathy and reveal potential new aspects of the molecular basis of this disease in translation dysregulation.
Asunto(s)
Edema Encefálico , Enfermedades Neurodegenerativas , Proteínas Nucleares , Atrofia Óptica , Ribonucleoproteínas Nucleolares Pequeñas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Espasmos Infantiles , Factores de Transcripción , Edema Encefálico/genética , Humanos , Recién Nacido , Mutación , Enfermedades Neurodegenerativas/genética , Proteínas Nucleares/genética , Atrofia Óptica/genética , ARN Nucleolar Pequeño/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Espasmos Infantiles/genética , Factores de Transcripción/genéticaRESUMEN
Small nucleolar RNAs (snoRNAs) are commonly acknowledged as a class of homogeneous non-coding RNAs that guide ribosomal RNA modifications. However, snoRNAs referred to as orphans have largely unknown functions. Here, we systematically profile chromatin-associated snoRNAs (casnoRNAs) in mammalian cells and identify a subgroup of orphan casnoRNAs responding to DNA damage stress, among which SNORA73 shows the most marked reduction in chromatin enrichment. Downregulated SNORA73 maintains cancer genome stability and differentiation block in hematopoietic malignancy. Mechanistically, casnoRNA the 5' end non-canonical structure of SNORA73 is critical for its function and binding to poly (ADP-ribose) polymerase 1 (PARP1). SNORA73 inhibits PARP1 auto-PARylation to affect cancer genome stability by forming a small nucleolar ribonucleoprotein (snoRNP) with PARP1 and canonical H/ACA proteins DKC1/NHP2. Our findings reveal the role of an orphan snoRNA serving as casnoRNA and highlights a link between non-canonical structure of snoRNA and their functional diversity.
Asunto(s)
Cromatina , ARN Nucleolar Pequeño , Animales , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/genética , Cromatina/genética , Daño del ADN/genética , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/genéticaRESUMEN
Monitoring mitochondrial function is crucial for organismal survival. This task is performed by mitochondrial surveillance or quality control pathways, which are activated by signals originating from mitochondria and relayed to the nucleus (retrograde response) to start transcription of protective genes. In Caenorhabditis elegans, several systems are known to play this role, including the UPRmt, MAPKmt, and the ESRE pathways. These pathways are highly conserved and their loss compromises survival following mitochondrial stress. In this study, we found a novel interaction between the box C/D snoRNA core proteins (snoRNPs) and mitochondrial surveillance and innate immune pathways. We showed that box C/D, but not box H/ACA, snoRNPs are required for the full function of UPRmt and ESRE upon stress. The loss of box C/D snoRNPs reduced mitochondrial mass, mitochondrial membrane potential, and oxygen consumption rate, indicating overall degradation of mitochondrial function. Concomitantly, the loss of C/D snoRNPs increased immune response and reduced host intestinal colonization by infectious bacteria, improving host resistance to pathogenesis. Our data may indicate a model wherein box C/D snoRNP machinery regulates a "switch" of the cell's activity between mitochondrial surveillance and innate immune activation. Understanding this mechanism is likely to be important for understanding multifactorial processes, including responses to infection and aging.
Asunto(s)
Mitocondrias , Ribonucleoproteínas Nucleolares Pequeñas , Animales , Caenorhabditis elegans/microbiología , Inmunidad Innata/genética , Mitocondrias/genética , Mitocondrias/metabolismo , ARN Nucleolar Pequeño , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismoRESUMEN
Neddylation is a posttranslational modification that attaches ubiquitin-like protein Nedd8 to protein targets via Nedd8-specific E1-E2-E3 enzymes and modulates many important biological processes. Nedd8 attaches to a lysine residue of a substrate, not for degradation, but for modulation of substrate activity. We previously identified the HECT-type ubiquitin ligase Smurf1, which controls diverse cellular processes, is activated by Nedd8 through covalent neddylation. Smurf1 functions as a thioester bond-type Nedd8 ligase to catalyze its own neddylation. Numerous ubiquitination substrates of Smurf1 have been identified, but the neddylation substrates of Smurf1 remain unknown. Here, we show that Smurf1 interacts with RRP9, a core component of the U3 snoRNP complex, which is involved in pre-rRNA processing. Our in vivo and in vitro neddylation modification assays show that RRP9 is conjugated with Nedd8. RRP9 neddylation is catalyzed by Smurf1 and removed by the NEDP1 deneddylase. We identified Lys221 as a major neddylation site on RRP9. Deficiency of RRP9 neddylation inhibits pre-rRNA processing and leads to downregulation of ribosomal biogenesis. Consequently, functional studies suggest that ectopic expression of RRP9 promotes tumor cell proliferation, colony formation, and cell migration, whereas unneddylated RRP9, K221R mutant has no such effect. Furthermore, in human colorectal cancer, elevated expression of RRP9 and Smurf1 correlates with cancer progression. These results reveal that Smurf1 plays a multifaceted role in pre-rRNA processing by catalyzing RRP9 neddylation and shed new light on the oncogenic role of RRP9.
Asunto(s)
Carcinogénesis/metabolismo , Proteína NEDD8/metabolismo , Proteínas de Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Sustitución de Aminoácidos , Animales , Carcinogénesis/genética , Células HCT116 , Células HT29 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación Missense , Proteína NEDD8/genética , Proteínas de Neoplasias/genética , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: MEK1/ERK signaling pathway plays an important role in most tumor progression, including colorectal cancer (CRC), however, MEK1-targeting therapy has little effective in treating CRC patients, indicating there may be a complex mechanism to activate MEK1/ERK signaling pathway except RAS activated mechanism. METHODS: To investigate the clinical significance of IMP3, we analyzed its expression levels in publicly available dataset and samples from Fudan University Shanghai Cancer Center. The effects of IMP3 on proliferation, migration, and invasion were determined by in vitro and in vivo experiments. To investigate the role of IMP3 in colon carcinogenesis, conditional IMP3 knockout C57BL/6 mice was generated. The IMP3/MEKK1/MEK/ERK signaling axis in CRC was screened and validated by RNA-sequencing, RNA immunoprecipitation, luciferase reporter and western blot assays. RESULTS: We find RNA binding protein IMP3 directly bind to MEKK1 mRNA 3'-UTR, which regulates its stability, promote MEKK1 expression and sequentially activates MEK1/ERK signaling. Functionally, IMP3 promote the malignant biological process of CRC cells via MEKK1/MEK1/ERK signaling pathway both in vitro and in vivo, Moreover, IMP3-/- mice show decreased the expression of MEKK1 as well as colorectal tumors compared with wild-type mice after treatment with azoxymethane/dextran sodium sulfate. Clinically, the expression of IMP3 and MEKK1 are positive correlated, and concomitant IMP3 and MEKK1 protein levels negatively correlate with metastasis in CRC patients. In addition, MEK1 inhibitor in combination with shRNA-IMP3 have a synergistic effect both in vitro and in vivo. CONCLUSION: Our study demonstrates that IMP3 regulates MEKK1 in CRC, thus activating the MEK1/ERK signaling in the progression of colorectal cancer, Furthermore, these results provide new insights into potential applications for combining MEK1 inhibitors with other target therapy such as IMP3 in preclinical trials for CRC patients.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , Sistema de Señalización de MAP Quinasas , ARN Mensajero/metabolismo , Animales , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Humanos , Masculino , Ratones , ARN Mensajero/genética , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismoRESUMEN
Posttranscriptional modifications of RNA play an important role in promoting the maturation and functional diversity of many RNA species. Accordingly, understanding the enzymes and mechanisms that underlie RNA modifications is an important aspect in advancing our knowledge of the continually expanding RNA modification field. However, of the more than 160 currently identified RNA modifications, a large portion remains without quantitative detection assays for their biochemical characterization. Here, we describe the tritium release assay as a convenient tool allowing for the quantitative assessment of in vitro RNA pseudouridylation by stand-alone or box H/ACA RNA-guided pseudouridine synthases. This assay enables quantification of RNA pseudouridylation over a time course to effectively compare pseudouridylation activity between different substrates and/or different recombinant enzymes as well as to determine kinetic parameters. With the help of a quench-flow apparatus, the tritium release assay can be adapted for rapid kinetic measurements under single-turnover conditions to dissect reaction mechanisms. As examples, we show the formation of pseudouridines by a reconstituted Saccharomyces cerevisiae H/ACA small ribonucleoprotein (snoRNP) and an Escherichia coli stand-alone pseudouridine synthase.
Asunto(s)
Transferasas Intramoleculares/genética , Seudouridina/genética , ARN/genética , Ribonucleoproteínas Nucleolares Pequeñas/genética , Escherichia coli/genética , Cinética , Procesamiento Postranscripcional del ARN/genética , ARN Guía de Kinetoplastida/genética , Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: Lung cancer is a common tumor and a leading cause of death worldwide. DEAD/H box RNA helicases (DDX) include several family members which regulate mRNA translation in cancer cells. In this study, we demonstrated that DEAD/H box RNA helicase 10 (DDX10) was significantly upregulated in lung cancer tissues compared with adjacent nontumor tissues. METHODS: DDX10 expression was knocked down with shRNA in order to investigate the impact on A549 lung cancer cell growth and related molecular mechanisms in vitro and in vivo. DDX10 expression in lung cancer was assessed using online databases and patient samples. RESULTS: DDX10 knockdown significantly inhibited the proliferation of lung cancer cells in vitro and in vivo. Furthermore, the bioinformatic tool indicated the putative downstream protein U3 small nucleolar ribonucleoprotein 4 (IMP4). Our data showed a positive correlation between IMP4 and DDX10. We found that IMP4 overexpression could reverse the effect of DDX10 knockdown on the proliferation and apoptosis of A549 cells. CONCLUSIONS: The findings of this study suggest that DDX10/IMP4 might be a novel target for lung cancer diagnosis and treatment.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Animales , Proliferación Celular , ARN Helicasas DEAD-box/genética , Femenino , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Ribonucleoproteínas Nucleolares Pequeñas/genética , TransfecciónRESUMEN
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.
Asunto(s)
Ribonucleoproteínas Nucleolares Pequeñas , Sumoilación , Proteínas de Ciclo Celular/metabolismo , Enzimas Desubicuitinizantes/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteómica , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Ubiquitina Tiolesterasa/genéticaRESUMEN
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.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Genes de ARNr/genética , Humanos , Sitios Internos de Entrada al Ribosoma/genética , Neoplasias/genética , Neoplasias/terapia , Unión Proteica/genética , Biosíntesis de Proteínas/genética , Ribonucleoproteínas Nucleolares Pequeñas/genéticaRESUMEN
H/ACA Box ribonucleoprotein complexes (RNPs) play a major role in modification of rRNA and snRNA, catalyzing the sequence specific pseudouridylation in eukaryotes and archaea. This enzymatic reaction takes place on a substrate RNA recruited via base pairing to an internal loop of the snoRNA. Eukaryotic snoRNPs contain the four proteins Nop10, Cbf5, Gar1 and Nhp2, with Cbf5 as the catalytic subunit. In contrast to archaeal H/ACA RNPs, eukaryotic snoRNPs contain several conserved features in both the snoRNA as well as the protein components. Here, we reconstituted the eukaryotic H/ACA RNP containing snR81 as a guide RNA in vitro and report on the effects of these eukaryote specific features on complex assembly and enzymatic activity. We compare their contribution to pseudouridylation activity for stand-alone hairpins versus the bipartite RNP. Using single molecule FRET spectroscopy, we investigated the role of the different eukaryote-specific proteins and domains on RNA folding and complex assembly, and assessed binding of substrate RNA to the RNP. Interestingly, we found diverging effects for the two hairpins of snR81, suggesting hairpin-specific requirements for folding and RNP formation. Our results for the first time allow assessing interactions between the individual hairpin RNPs in the context of the full, bipartite snoRNP.
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Hidroliasas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , ARN Nucleolar Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Catálisis , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica , Hidroliasas/genética , Técnicas In Vitro , Secuencias Invertidas Repetidas , Proteínas Asociadas a Microtúbulos/genética , Modelos Moleculares , Proteínas Nucleares/genética , Dominios Proteicos , Pliegue del ARN , ARN Guía de Kinetoplastida , ARN Nucleolar Pequeño/genética , Proteínas de Unión al ARN/genética , Proteínas Recombinantes , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Chaperonas Moleculares/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Activación Transcripcional/fisiología , Proliferación Celular/fisiología , Cromatina/genética , Cristalografía por Rayos X , Histonas/metabolismo , Resonancia Magnética Nuclear Biomolecular , ARN Polimerasa II/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcripción Genética/genéticaRESUMEN
Reactivation of telomerase is a major hallmark observed in 90% of all cancers. Yet paradoxically, enhanced telomerase activity does not correlate with telomere length and cancers often possess short telomeres; suggestive of supplementary non-canonical roles that telomerase might play in the development of cancer. Moreover, studies have shown that aberrant expression of shelterin proteins coupled with their release from shortening telomeres can further promote cancer by mechanisms independent of their telomeric role. While targeting telomerase activity appears to be an attractive therapeutic option, this approach has failed in clinical trials due to undesirable cytotoxic effects on stem cells. To circumvent this concern, an alternative strategy could be to target the molecules involved in the non-canonical functions of telomeric proteins. In this review, we will focus on emerging evidence that has demonstrated the non-canonical roles of telomeric proteins and their impact on tumorigenesis. Furthermore, we aim to address current knowledge gaps in telomeric protein functions and propose future research approaches that can be undertaken to achieve this.
Asunto(s)
Neoplasias/patología , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Disqueratosis Congénita/genética , Disqueratosis Congénita/patología , Humanos , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Telómero/metabolismo , Proteínas de Unión a Telómeros/química , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding, and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2'-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive and partially conflicting structural information exists for the archaeal enzyme, while no structural data is available for the eukaryotic enzyme. The yeast Box C/D RNP consists of a guide RNA, the RNA-primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58, and the enzymatic module Nop1. Here we present the high-resolution structure of the eukaryotic Box C/D methyltransferase Nop1 from Saccharomyces cerevisiae bound to the amino-terminal domain of Nop56. We discuss similarities and differences between the interaction modes of the two proteins in archaea and eukaryotes and demonstrate that eukaryotic Nop56 recruits the methyltransferase to the Box C/D RNP through a protein-protein interface that differs substantially from the archaeal orthologs. This study represents a first achievement in understanding the evolution of the structure and function of these proteins from archaea to eukaryotes.