Affinage

NOL9

Polynucleotide 5'-hydroxyl-kinase NOL9 · UniProt Q5SY16

Round 2 corrected
Length
702 aa
Mass
79.3 kDa
Annotated
2026-04-29
43 papers in source corpus 10 papers cited in narrative 10 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

NOL9 is a nucleolar polynucleotide 5'-hydroxyl-kinase essential for ribosome biogenesis, specifically the processing of the 32S precursor into mature 5.8S and 28S rRNAs. NOL9 forms a tetrameric complex with the endoribonuclease Las1L in which Las1L reciprocally activates NOL9 kinase activity toward single-stranded RNA, coupling ITS2 endonucleolytic cleavage to 5'-phosphorylation of cleavage products required for subsequent exonucleolytic trimming (PMID:28652339, PMID:29440475). NOL9 encodes a nucleolar localization sequence that directs the assembled Las1L–NOL9 complex to the nucleolus for late pre-rRNA processing (PMID:31288032). Loss of NOL9 function in zebrafish causes defective 28S rRNA processing, p53-dependent hematopoietic failure, and visceral organ hypoplasia, and disruption of the LAS1–NOL9 interaction pharmacologically activates the NPM1–MDM2–p53 surveillance pathway (PMID:26624285, PMID:36796466).

Mechanistic history

Synthesis pass · year-by-year structured walk · 7 steps
  1. 2010 High

    Establishing NOL9/Grc3 as an RNA 5'-kinase linked to ribosome biogenesis resolved the enzymatic identity of the gene and showed its kinase activity is required for 32S pre-rRNA processing to generate 5.8S and 28S rRNAs.

    Evidence siRNA knockdown in HeLa cells, in vitro polynucleotide kinase assay, sucrose gradient sedimentation, Northern blotting (human); genetic depletion, ChIP, Pol I transcription run-on (yeast)

    PMID:20814424 PMID:21063389

    Open questions at the time
    • Identity of the direct RNA substrates phosphorylated by NOL9 in vivo was not determined
    • How NOL9 kinase activity feeds into exonucleolytic trimming remained unclear
    • Whether NOL9 acts alone or requires a partner was unknown
  2. 2012 High

    Identification of Las1 as the major Grc3/NOL9-interacting protein demonstrated that the kinase functions within a dedicated endonuclease–kinase module on pre-60S particles, explaining the epistatic overlap of their depletion phenotypes.

    Evidence Reciprocal co-immunoprecipitation, genetic depletion, AP-MS, Northern blotting in S. cerevisiae and S. pombe

    PMID:21385875 PMID:23175604

    Open questions at the time
    • Stoichiometry and architecture of the Las1–Grc3 complex were unresolved
    • Whether Las1 endonuclease activity depends on Grc3 was not tested biochemically
    • The Grc3 role in heterochromatic silencing reported in S. pombe was not confirmed in other organisms
  3. 2015 High

    A vertebrate loss-of-function model in zebrafish established that nol9 is essential for organismal development, linking defective 28S rRNA processing to p53-dependent hematopoietic failure and p53-independent visceral organ hypoplasia.

    Evidence Forward genetic screen, nol9 mutant zebrafish, Northern blot for rRNA, genetic epistasis with tp53

    PMID:26624285

    Open questions at the time
    • Whether the p53-independent pancreatic phenotype reflects a ribosome-independent NOL9 function was unclear
    • Mammalian in vivo consequences of NOL9 loss were not examined
  4. 2017 High

    Biochemical reconstitution revealed that Las1 and Grc3 cross-activate each other — Las1 stimulates Grc3 kinase activity toward ssRNA and Grc3 is required for Las1 C2 endonuclease cleavage — establishing a coupled cleavage-phosphorylation mechanism for ITS2 processing.

    Evidence In vitro reconstitution of endonuclease and kinase assays, active-site mutagenesis validated in vivo, native mass spectrometry for tetrameric stoichiometry in S. cerevisiae

    PMID:28652339 PMID:29440475

    Open questions at the time
    • Structural basis for reciprocal activation was not determined
    • Whether the human Las1L–NOL9 complex has identical stoichiometry and cross-activation was not directly shown
  5. 2019 High

    Mapping the human Las1L–NOL9 complex architecture showed that NOL9 carries a nucleolar localization sequence responsible for directing the assembled complex to the nucleolus, explaining how the ITS2 processing machinery reaches its substrate.

    Evidence Deletion mapping, co-immunoprecipitation, confocal and super-resolution microscopy of nucleolar sub-structures in human cells

    PMID:31288032

    Open questions at the time
    • High-resolution atomic structure of the human Las1L–NOL9 complex is still lacking
    • Whether additional factors regulate nucleolar import or retention of the complex is unknown
  6. 2023 Medium

    Pharmacological disruption of the LAS1–NOL9 interaction by a covalent LAS1 inhibitor confirmed the interaction is functionally essential for 28S rRNA maturation and demonstrated that its loss activates the NPM1–MDM2–p53 nucleolar stress pathway, with therapeutic selectivity in NPM1-mutant AML.

    Evidence Covalent small-molecule targeting of LAS1 C264, co-immunoprecipitation, Northern blot, subcellular fractionation, cell viability assays in AML lines

    PMID:36796466

    Open questions at the time
    • Pharmacological specificity of HEN-463 beyond LAS1 was not exhaustively profiled
    • Whether NOL9 protein stability or activity is directly affected by the compound is unclear
    • In vivo efficacy in AML models was not reported
  7. 2025 Medium

    NOL9 was linked to hepatocellular carcinoma cell proliferation via Wnt/β-catenin signaling, expanding its functional repertoire beyond ribosome biogenesis, though the mechanistic bridge between NOL9's kinase activity and Wnt pathway activation is undefined.

    Evidence siRNA knockdown and overexpression in HCC cell lines, in vivo xenograft, bisulfite sequencing, luciferase reporter for Wnt pathway

    PMID:39955289

    Open questions at the time
    • No mechanistic connection between NOL9 polynucleotide kinase activity and Wnt/β-catenin signaling was established
    • Whether the proliferative effect is secondary to ribosome biogenesis defects and p53 activation was not tested
    • Single-lab finding without independent replication

Open questions

Synthesis pass · forward-looking unresolved questions
  • An atomic-resolution structure of the human Las1L–NOL9 complex and identification of the in vivo RNA substrates phosphorylated by NOL9 remain key open questions for understanding how coupled cleavage-phosphorylation is orchestrated during ITS2 processing.
  • No high-resolution structure of the human Las1L–NOL9 complex exists
  • The precise in vivo RNA substrates of NOL9 kinase have not been mapped by crosslinking or related approaches
  • Whether NOL9 has physiologically relevant functions outside ribosome biogenesis remains unresolved

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016740 transferase activity 4 GO:0003723 RNA binding 3
Localization
GO:0005634 nucleus 2 GO:0005730 nucleolus 2
Pathway
R-HSA-8953854 Metabolism of RNA 4 R-HSA-1852241 Organelle biogenesis and maintenance 2
Partners
IPI1LAS1LNSA1RIX1
Complex memberships
Las1L–NOL9 ITS2 endonuclease-kinase complexpre-60S ribosomal particle

Evidence

Reading pass · 10 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2010 NOL9 (human) is a nucleolar polynucleotide 5'-kinase that sediments with pre-60S ribosomal particles in HeLa nuclear extracts. Knockdown of NOL9 severely impairs ribosome biogenesis; specifically, the kinase activity of NOL9 is required for efficient generation of 5.8S and 28S rRNAs from the 32S precursor, and depletion causes a maturation defect at the 5' end of the predominant 5.8S short-form rRNA (5.8S-S), likely by impairing 5'→3' exonucleolytic trimming. siRNA knockdown in HeLa cells, sucrose gradient sedimentation/fractionation, in vitro polynucleotide kinase assay, Northern blotting for rRNA intermediates The EMBO journal High 21063389
2010 Grc3 (the yeast ortholog of NOL9) encodes a polynucleotide kinase required for efficient transcription termination by RNA polymerase I in S. cerevisiae, acting by controlling the phosphorylation status of the downstream Rnt1 cleavage product to regulate its accessibility to the torpedo exonuclease Rat1. Genetic depletion of GRC3, in vitro polynucleotide kinase assay, ChIP and transcription run-on assays for Pol I termination EMBO reports High 20814424
2012 In S. cerevisiae, Las1 (ortholog of human Las1L, NOL9's binding partner) co-precipitates primarily with 27S rRNA and associates with an Nsa1/Rix1-containing pre-60S particle. Grc3 (NOL9 ortholog) is identified as a major Las1-interacting protein; Grc3 kinase activity is required for efficient pre-rRNA processing, and Grc3 depletion causes rRNA processing defects similar to Las1 depletion, indicating they function together. Co-immunoprecipitation, sucrose gradient sedimentation, genetic depletion, Northern blotting Nucleic acids research High 23175604
2011 In fission yeast S. pombe, Grc3 (NOL9 ortholog) is required for both 25S rRNA processing and heterochromatic gene silencing. Protein complex analysis identified Las1 and components of the IPI complex (Rix1, Ipi1, Crb3) as Grc3-interacting proteins, and Grc3 localizes dynamically between heterochromatic regions and the perinucleolar region in a manner dependent on Swi6/Clr4. Affinity purification/mass spectrometry, genetic depletion, cytological localization, Northern blotting, silencing reporter assays The Journal of biological chemistry Medium 21385875
2017 The Las1L endoribonuclease (human) requires its binding partner Grc3 (NOL9 ortholog in yeast) for specific C2 cleavage of pre-rRNA both in vitro and in vivo. Grc3 and Las1 assemble into a tetrameric complex; Las1 reciprocally activates Grc3 kinase activity exclusively toward single-stranded RNA substrates. This cross-activation establishes Grc3/Las1 as a unique member of the RNaseL/Ire1 RNA splicing family. In vitro reconstitution of endoribonuclease cleavage assay, in vitro polynucleotide kinase assay, site-directed mutagenesis, in vivo pre-rRNA processing assays in S. cerevisiae, native mass spectrometry for complex stoichiometry Proceedings of the National Academy of Sciences of the United States of America High 28652339
2018 Grc3 (NOL9 ortholog) has distinct substrate preference for RNA over DNA substrates in vitro. Disruption of conserved residues at the Grc3 kinase active site abolishes Grc3-directed Las1-mediated pre-rRNA cleavage both in vitro and in vivo, establishing that Grc3 kinase activity is directly coupled to Las1 endonuclease activity during pre-rRNA processing. In vitro polynucleotide kinase assay with RNA/DNA substrates, active-site mutagenesis, in vitro cleavage assay, in vivo pre-rRNA processing assays in S. cerevisiae RNA (New York, N.Y.) High 29440475
2019 Human NOL9 forms a higher-order complex with Las1L to constitute the ITS2 pre-rRNA endonuclease-kinase machinery. NOL9 contains a nucleolar localization sequence (NoLS) that is responsible for nucleolar transport of the assembled Las1L–NOL9 complex. Structural analysis by high-resolution imaging defines the spatial organization of Las1L–NOL9 within nucleolar sub-structures linked to late pre-rRNA processing. Specific regions of both proteins orchestrate the intricate higher-order architecture of the complex. Co-immunoprecipitation, deletion mapping, confocal and super-resolution fluorescence microscopy (nucleolar sub-structure imaging), subcellular fractionation, domain mutagenesis Journal of molecular biology High 31288032
2015 In zebrafish, loss-of-function mutation of nol9 causes defective 28S rRNA processing, hypoplastic pancreas, liver, and intestine, and reduced hematopoietic stem/progenitor cells, definitive erythrocytes, and lymphocytes due to impaired proliferation of progenitor cells. The hematopoietic (but not pancreatic) defects are rescued by genetic loss of Tp53, indicating that the hematopoietic phenotype is p53-dependent. mTOR pathway activation by L-Leucine does not rescue either defect. Forward genetic screen, loss-of-function zebrafish mutant, Northern blotting for rRNA, genetic epistasis with tp53 mutant, L-Leucine mTOR activation, flow cytometry, ultrastructural analysis PLoS genetics High 26624285
2023 The LAS1–NOL9 protein–protein interaction is required for 28S rRNA maturation; covalent inhibition of LAS1 at C264 by the compound HEN-463 disrupts LAS1–NOL9 interaction, causes cytoplasmic translocation of LAS1, inhibits 28S rRNA maturation, activates the NPM1–MDM2–p53 pathway leading to p53 stabilization, and selectively kills NPM1-mutant AML cells. Covalent small-molecule targeting, co-immunoprecipitation (LAS1–NOL9 interaction), subcellular fractionation/immunofluorescence (LAS1 translocation), Northern/qRT-PCR (rRNA processing), Western blotting (p53 pathway), cell viability and apoptosis assays Pharmacological research Medium 36796466
2025 NOL9 expression in hepatocellular carcinoma (HCC) is regulated by DNA methylation at specific CpG sites (involving DNMT1) and by the transcription factor ZNF384. NOL9 knockdown inhibits HCC cell proliferation and promotes apoptosis in vitro and reduces tumor growth in vivo; overexpression has the opposite effect. Mechanistically, NOL9-mediated cell proliferation in HCC is dependent on activation of the Wnt/β-catenin signaling pathway. siRNA knockdown and overexpression in HCC cell lines, in vivo xenograft tumor growth assay, bisulfite sequencing/methylation analysis, luciferase reporter and Western blotting for Wnt/β-catenin pathway, flow cytometry (apoptosis/cell cycle) Cell death & disease Medium 39955289

Source papers

Stage 0 corpus · 43 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2002 Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proceedings of the National Academy of Sciences of the United States of America 1479 12477932
2015 The BioPlex Network: A Systematic Exploration of the Human Interactome. Cell 1118 26186194
2017 Architecture of the human interactome defines protein communities and disease networks. Nature 1085 28514442
2015 A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell 1015 26496610
2014 A proteome-scale map of the human interactome network. Cell 977 25416956
2002 Directed proteomic analysis of the human nucleolus. Current biology : CB 780 11790298
2003 Complete sequencing and characterization of 21,243 full-length human cDNAs. Nature genetics 754 14702039
2021 Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell 705 33961781
2011 Phylogenetic-based propagation of functional annotations within the Gene Ontology consortium. Briefings in bioinformatics 656 21873635
2018 High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies. Molecular cell 580 29395067
2004 The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome research 438 15489334
2022 OpenCell: Endogenous tagging for the cartography of human cellular organization. Science (New York, N.Y.) 432 35271311
2015 Panorama of ancient metazoan macromolecular complexes. Nature 407 26344197
1996 Normalization and subtraction: two approaches to facilitate gene discovery. Genome research 401 8889548
2002 Functional proteomic analysis of human nucleolus. Molecular biology of the cell 391 12429849
2021 A proximity-dependent biotinylation map of a human cell. Nature 339 34079125
2018 Mapping the Genetic Landscape of Human Cells. Cell 225 30033366
2016 A High-Density Map for Navigating the Human Polycomb Complexome. Cell reports 216 27705803
2018 An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations. Nature communications 201 29568061
2015 A deep proteomics perspective on CRM1-mediated nuclear export and nucleocytoplasmic partitioning. eLife 198 26673895
2014 Global mapping of herpesvirus-host protein complexes reveals a transcription strategy for late genes. Molecular cell 173 25544563
2020 UFMylation maintains tumour suppressor p53 stability by antagonizing its ubiquitination. Nature cell biology 168 32807901
2019 H4K20me0 recognition by BRCA1-BARD1 directs homologous recombination to sister chromatids. Nature cell biology 162 30804502
2020 AMPK, a Regulator of Metabolism and Autophagy, Is Activated by Lysosomal Damage via a Novel Galectin-Directed Ubiquitin Signal Transduction System. Molecular cell 152 31995728
2018 MYC Protein Interactome Profiling Reveals Functionally Distinct Regions that Cooperate to Drive Tumorigenesis. Molecular cell 152 30415952
2020 A High-Density Human Mitochondrial Proximity Interaction Network. Cell metabolism 148 32877691
2012 Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription. Molecular & cellular proteomics : MCP 145 22586326
2006 The DNA sequence and biological annotation of human chromosome 1. Nature 144 16710414
2009 Direct binding of CoREST1 to SUMO-2/3 contributes to gene-specific repression by the LSD1/CoREST1/HDAC complex. Molecular cell 140 19394292
2018 Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains. Molecular cell 136 30554943
2012 Las1 interacts with Grc3 polynucleotide kinase and is required for ribosome synthesis in Saccharomyces cerevisiae. Nucleic acids research 40 23175604
2007 Expression and function of transmembrane-4 superfamily (tetraspanin) proteins in osteoclasts: reciprocal roles of Tspan-5 and NET-6 during osteoclastogenesis. Allergology international : official journal of the Japanese Society of Allergology 35 17965585
2017 Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage. Proceedings of the National Academy of Sciences of the United States of America 33 28652339
2010 Nol9 is a novel polynucleotide 5'-kinase involved in ribosomal RNA processing. The EMBO journal 33 21063389
2010 Role of the RNA/DNA kinase Grc3 in transcription termination by RNA polymerase I. EMBO reports 31 20814424
2007 The tetraspanin superfamily member NET-6 is a new tumor suppressor gene. Journal of cancer research and clinical oncology 25 17486367
2015 The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish. PLoS genetics 22 26624285
2011 Roles of fission yeast Grc3 protein in ribosomal RNA processing and heterochromatic gene silencing. The Journal of biological chemistry 21 21385875
2019 Expression of tetraspanins NET-6 and CD151 in breast cancer as a potential tumor biomarker. Clinical and experimental medicine 14 31004251
2019 Nol9 Is a Spatial Regulator for the Human ITS2 Pre-rRNA Endonuclease-Kinase Complex. Journal of molecular biology 13 31288032
2018 Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex. RNA (New York, N.Y.) 13 29440475
2025 Nucleolar NOL9 regulated by DNA methylation promotes hepatocellular carcinoma growth through activation of Wnt/β-catenin signaling pathway. Cell death & disease 5 39955289
2023 Covalent targeting the LAS1-NOL9 axis for selective treatment in NPM1 mutant acute myeloid leukemia. Pharmacological research 5 36796466