Affinage

CRNKL1

Crooked neck-like protein 1 · UniProt Q9BZJ0

Length
848 aa
Mass
100.5 kDa
Annotated
2026-06-09
52 papers in source corpus 14 papers cited in narrative 15 extracted findings
Cross-family judge vs UniProt: tie faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

CRNKL1 (yeast CLF1/SYF3) is an essential TPR-repeat scaffolding protein of the spliceosome required for the prespliceosome-to-spliceosome transition during pre-mRNA splicing (PMID:10445879). In yeast, depletion of the protein arrests spliceosome assembly after U2 snRNP addition but before productive U4/U6.U5 tri-snRNP association, blocking the first step of splicing (PMID:10445879), and the protein associates transiently with U6 and U5 snRNAs (PMID:11105756). It operates as a hub within the NTC/Prp19-associated network, interacting with Syf1p, Isy1p, Cef1p, Prp22p, and Ntc20p and functioning in the same genetic pathway as Prp45 and the second-step helicase Prp22 (PMID:11102353, PMID:19016306); structurally, its N-terminal crooked-neck TPR (crn-TPR1) motif is directly contacted by the first FF domain of Prp40, and it additionally interacts with the U2AF65 homolog Mud2p, consistent with a bridging role between snRNP and NTC components during assembly (PMID:10445879, PMID:16253993). Beyond splicing, yeast Clf1p has a direct role in DNA replication initiation, interacting with Orc2p and associating with replication origins in an Orc2p-dependent manner, and its loss causes G2/M cell cycle arrest (PMID:11105756, PMID:11973290). In human cells CRNKL1 acts as a nuclear retention factor that suppresses cytoplasmic accumulation of intron-retaining RNAs, including unspliced HIV-1 transcripts, with depletion enhancing cytoplasmic levels of unspliced RNA primarily by affecting nuclear export rather than splicing itself (PMID:33468685, PMID:39902983). Recurrent de novo missense variants at Arg267 cause severe microcephaly with pontocerebellar hypoplasia and seizures, and mutant Crnkl1 disrupts brain development, cell proliferation, and cell cycle gene expression in zebrafish, establishing CRNKL1 as essential for human brain development (PMID:40857589).

Mechanistic history

Synthesis pass · year-by-year structured walk · 8 steps
  1. 1999 High

    Established the core function of the protein by showing it is essential for the first catalytic step of splicing and acts at a defined point in spliceosome assembly, answering what stage of splicing requires this factor.

    Evidence In vitro splicing with Clf1p-depleted yeast extracts and spliceosome assembly analysis, plus two-hybrid/binding assays mapping interactions with Prp40p and Mud2p

    PMID:10445879

    Open questions at the time
    • Whether the Prp40p/Mud2p interactions are direct in a reconstituted system was not resolved
    • No structural model of Clf1p within the assembling spliceosome
  2. 2000 High

    Linked the splicing factor to cell cycle control and placed it within a defined protein complex, showing the protein has a dual role and operates in a network of NTC-associated factors.

    Evidence snRNA co-precipitation and in vivo depletion with cell cycle readouts; synthetic lethality screen with prp17/cdc40Δ plus two-hybrid and Co-IP mapping interactions with Isy1p, Cef1p, Prp22p, Ntc20p

    PMID:11102353 PMID:11105756

    Open questions at the time
    • Whether G2/M arrest is a direct consequence of splicing defects or a separate function was not separated
    • Stoichiometry and architecture of the complex not defined
  3. 2002 High

    Demonstrated a splicing-independent role in DNA replication initiation, answering whether the cell cycle phenotype reflects a direct function at replication origins.

    Evidence Temperature-sensitive clf1 mutants, SIC1-deletion epistasis, two-hybrid and Co-IP with Orc2p, and origin chromatin association assays in S. cerevisiae

    PMID:11973290

    Open questions at the time
    • Mechanism by which a TPR splicing scaffold acts at origins is unknown
    • Not tested whether the human ortholog retains a replication role
  4. 2005 High

    Provided structural definition of the Clf1p–Prp40 interaction, showing the crn-TPR1 motif is recognized by a distinct FF-domain binding site, clarifying interaction specificity.

    Evidence NMR solution structure of Prp40 FF1 and chemical shift perturbation mapping of the crn-TPR1 binding interface

    PMID:16253993

    Open questions at the time
    • No structure of full-length Clf1p or the complete TPR array
    • Functional consequence of disrupting this interface in vivo not tested
  5. 2009 Medium

    Extended the functional network by genetically connecting the protein to Prp45 and the second-step helicase Prp22, indicating involvement beyond initial assembly.

    Evidence Synthetic lethality screen with prp45(1-169) and Cwc2-pulldown stoichiometry analysis of spliceosomal complexes

    PMID:19016306

    Open questions at the time
    • Direct physical contact with Prp45 not demonstrated
    • Mechanistic basis of altered Prp22 stoichiometry unclear
  6. 2021 High

    Identified a conserved human function as a nuclear retention factor for intron-retaining RNAs, answering what the mammalian ortholog does beyond canonical splicing.

    Evidence Genome-wide CRISPR screen, targeted depletion, nuclear/cytoplasmic RNA fractionation and RNA-seq, with later validation via a dual-fluorescent HIV-1 reporter

    PMID:33468685 PMID:39902983

    Open questions at the time
    • Molecular mechanism by which CRNKL1 enforces nuclear retention/export control not defined
    • Whether retention is a direct activity or a consequence of altered spliceosome states unresolved
  7. 2021 Medium

    Refined the cotranscriptional and complex context of the protein across organisms, connecting it to splicing-factor recruitment and a pre-organized PRP19-related complex.

    Evidence Co-IP/MS and ChIP-Seq with Rat1 in budding yeast; affinity purification, sedimentation and snRNA co-precipitation of the trypanosome PRC; bacterial two-hybrid with fission yeast Nrl1

    PMID:33978753 PMID:34209806 PMID:34850936

    Open questions at the time
    • Nrl1 interaction rests on a single positive method not confirmed by TAP
    • Generality of the PRC pre-organization to mammalian spliceosomes untested
  8. 2025 High

    Established CRNKL1 as a human disease gene required for brain development, connecting its molecular function to a Mendelian neurodevelopmental disorder.

    Evidence De novo variant cohort identifying recurrent Arg267 mutations; zebrafish mRNA microinjection with imaging, proliferation/p53 immunostaining, and RNA-seq

    PMID:40857589

    Open questions at the time
    • How Arg267 substitutions impair CRNKL1 molecular function (dominant-negative vs loss-of-function) not resolved
    • Link between splicing/nuclear-retention activity and the neurodevelopmental phenotype not mechanistically established

Open questions

Synthesis pass · forward-looking unresolved questions
  • How CRNKL1's spliceosomal scaffolding activity mechanistically produces nuclear retention of intron-retaining RNAs and brain-specific developmental requirements remains unresolved.
  • No structural or biochemical model linking splicing function to export control in human cells
  • No demonstration that the disease variants alter specific splicing or retention events in neural lineages

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0008092 cytoskeletal protein binding 3 GO:0060090 molecular adaptor activity 3 GO:0003723 RNA binding 1
Localization
GO:0005634 nucleus 1
Pathway
R-HSA-8953854 Metabolism of RNA 3 R-HSA-1640170 Cell Cycle 2 R-HSA-1266738 Developmental Biology 1 R-HSA-69306 DNA Replication 1
Partners
CEF1ISY1MUD2NTC20ORC2PRP22PRP40SYF1
Complex memberships
NTC/Prp19-associated complexspliceosome (U4/U6.U5 tri-snRNP-containing)trypanosome PRP19-related complex (PRC)

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1999 Yeast CLF1 (ortholog of Drosophila crooked neck / human CRNKL1) encodes an essential TPR-repeat protein required for pre-mRNA 5' splice site cleavage (first step of splicing). Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 tri-snRNP association, establishing Clf1p as a scaffolding factor required for the prespliceosome-to-spliceosome transition. In vitro splicing assays with Clf1p-depleted extracts; spliceosome assembly analysis; yeast two-hybrid and in vitro binding studies RNA (New York, N.Y.) High 10445879
1999 Clf1p interacts specifically with the U1 snRNP protein Prp40p and the yeast U2AF65 homolog Mud2p, as shown by yeast two-hybrid and in vitro binding assays, suggesting Clf1p bridges U1 and U2 snRNP components during spliceosome assembly. Yeast two-hybrid; in vitro binding studies RNA (New York, N.Y.) Medium 10445879
2000 Yeast Syf3p (same protein as Clf1p/CRNKL1 ortholog) and Syf1p are spliceosome-associated proteins that associate weakly or transiently with U6 and U5 snRNAs; depletion of Syf1p or Syf3p blocks pre-mRNA splicing and causes cell cycle arrest at the G2/M transition, demonstrating dual roles in splicing and cell cycle progression. Spliceosome association assays (snRNA co-precipitation); in vivo depletion with splicing and cell cycle phenotype readouts RNA (New York, N.Y.) High 11105756
2000 Genetic screen in S. cerevisiae identified SYF1, SYF2, and SYF3 (CLF1) as synthetically lethal with prp17/cdc40Δ; two-hybrid and biochemical analyses showed SYF gene products interact with each other and with Isy1p, Cef1p, Prp22p, and Ntc20p, placing them in a protein complex that assembles into spliceosomes. Synthetic lethality screen; yeast two-hybrid; biochemical co-immunoprecipitation Genetics High 11102353
2002 Clf1p (CRNKL1 ortholog) plays a direct role in the initiation of DNA replication in S. cerevisiae: temperature-sensitive clf1 mutants show delayed S-phase entry that cannot be suppressed by deletion of the CDK inhibitor SIC1; Clf1p interacts with the DNA replication initiation protein Orc2p by two-hybrid and co-immunoprecipitation; and Clf1p preferentially associates with origins of DNA replication in an Orc2p-dependent manner. Temperature-sensitive mutant analysis; cell cycle block/release; yeast two-hybrid; co-immunoprecipitation; chromatin association assays at replication origins Genetics High 11973290
2005 The NMR solution structure of the first FF domain of yeast Prp40 revealed a novel ligand-binding site that directly contacts the N-terminal crooked-neck TPR (crn-TPR1) motif of Clf1p; this binding site is structurally distinct from the CTD-RNAPII binding pocket of the related FF domain of FBP11. No interaction was detected between Prp40 FF1 and CTD-RNAPII peptides, indicating domain-specific and protein-specific binding of the Clf1p–Prp40 interaction. NMR structure determination; chemical shift perturbation assays mapping the binding interface The Journal of biological chemistry High 16253993
2009 CLF1/SYF3 genetically interacts with the spliceosomal NTC component Prp45/SNW1: a synthetic lethality screen showed that the prp45(1-169) allele is lethal in combination with clf1/syf3 alleles, and Cwc2-associated spliceosomal complexes from prp45 mutant cells show altered Prp22 stoichiometry, placing Clf1p/Syf3p in the same functional network as Prp45 and the second-step helicase Prp22. Synthetic lethality screen; spliceosomal complex purification and stoichiometry analysis by Cwc2-pulldown Journal of cellular biochemistry Medium 19016306
2020 Molecular dynamics simulations and community network analysis of the yeast spliceosome C complex identified Clf1p (CRNKL1 ortholog) as a critical signal conveyor in cross-communication pathways across the spliceosome, functioning alongside Cwc2 and specific Prp8 domains to direct conformational rearrangements required for splicing fidelity. Molecular dynamics simulations (800,000-atom model); community network analysis Journal of the American Chemical Society Low 32275149
2021 CRNKL1 (human ortholog) suppresses cytoplasmic levels of unspliced HIV-1 RNA and a subset of intron-retaining cellular mRNAs; CRISPR/Cas knockdown of CRNKL1 causes more than 20-fold enhancement of unspliced HIV-1 RNA cytoplasmic levels, predominantly by affecting nuclear export efficiency rather than splicing per se, identifying CRNKL1-dependent nuclear retention as a cellular mechanism regulating intron-retaining RNAs. Genome-wide CRISPR/Cas screen; targeted CRNKL1 depletion; cytoplasmic/nuclear RNA fractionation; transcriptomic analysis (RNA-seq) mBio High 33468685
2021 In fission yeast, the C-terminal region of Nrl1 interacts with the spliceosome component Syf3 (CRNKL1 ortholog), as demonstrated by bacterial two-hybrid assay; however, Syf3 did not co-purify with the N-terminal region of Nrl1 by tandem affinity purification, indicating the interaction is domain-specific. Bacterial two-hybrid (BACTH); tandem affinity purification International journal of molecular sciences Low 34209806
2021 In budding yeast, Rat1 (5'→3' exoribonuclease) co-immunoprecipitates and was identified by mass spectrometry to interact with Clf1p among other splicing factors; recruitment of splicing factors to introns was compromised in Rat1 mutants, placing Clf1p in the cotranscriptional splicing recruitment pathway. Mass spectrometry; co-immunoprecipitation; ChIP-Seq; splicing assays in Rat1 mutants Nucleic acids research Medium 33978753
2021 In trypanosomes, SYF3 (CRNKL1 ortholog) forms a distinct PRP19-related complex (PRC) together with SYF1, ISY1, and trypanosomatid-specific proteins PRC3 and PRC5; PRC co-precipitates U4 snRNA, indicating it enters the spliceosome prior to the PRP19 complex, establishing a unique pre-organization of these factors. Gene silencing; affinity purification; sucrose gradient sedimentation; snRNA co-precipitation Nucleic acids research Medium 34850936
2025 Recurrent de novo missense variants in human CRNKL1 (specifically at Arg267) cause severe microcephaly with pontocerebellar hypoplasia and seizures. Microinjection of mRNA encoding mutant Crnkl1 into zebrafish caused severe lack of brain development, significant reduction of proliferating cells, widespread cellular stress (p53 staining), and broad transcriptomic changes including altered expression of neuronal and cell cycle genes, establishing CRNKL1 as required for brain development. Human genetics (de novo variant cohort); zebrafish mRNA microinjection; immunostaining (p53, proliferation markers); RNA sequencing American journal of human genetics High 40857589
2025 ASFV protein pI73R overexpression causes downregulation of CRNKL1 (a core spliceosome component) in host cells, suggesting a mechanism by which the viral protein modulates host pre-mRNA splicing through suppression of CRNKL1. Overexpression of viral pI73R; mass spectrometry interactome; Western blot/protein abundance measurement International journal of molecular sciences Low 41465200
2025 CRNKL1 was validated as a repressor of unspliced HIV-1 RNA nuclear export using a dual-fluorescent HIV-1 reporter system; targeted depletion of CRNKL1 enhanced Rev-independent (unspliced) transcript levels, confirming its role in nuclear retention of unspliced RNA. Dual-fluorescent HIV-1 reporter system; flow cytometry; targeted CRNKL1 depletion Microbiology spectrum Medium 39902983

Source papers

Stage 0 corpus · 52 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2013 Mutational landscape of basal cell carcinomas by whole-exome sequencing. The Journal of investigative dermatology 171 23774526
2008 Variation in WNT genes is associated with non-syndromic cleft lip with or without cleft palate. Human molecular genetics 140 18413325
2000 Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae. Genetics 105 11102353
2005 Novel regulators of kidney development from the tips of the ureteric bud. Journal of the American Society of Nephrology : JASN 100 15917337
2006 Wnt9b is the mutated gene involved in multifactorial nonsyndromic cleft lip with or without cleft palate in A/WySn mice, as confirmed by a genetic complementation test. Birth defects research. Part A, Clinical and molecular teratology 98 16998816
2010 Studies with Wnt genes and nonsyndromic cleft lip and palate. Birth defects research. Part A, Clinical and molecular teratology 73 20890934
1999 Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition. RNA (New York, N.Y.) 66 10445879
1998 Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family. Journal of immunology (Baltimore, Md. : 1950) 63 9686600
2005 Investigations of the genomic region that contains the clf1 mutation, a causal gene in multifactorial cleft lip and palate in mice. Birth defects research. Part A, Clinical and molecular teratology 50 15690355
2000 Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae. RNA (New York, N.Y.) 49 11105756
2001 Unravelling the complex genetics of cleft lip in the mouse model. Mammalian genome : official journal of the International Mammalian Genome Society 47 11353389
1995 The major locus for multifactorial nonsyndromic cleft lip maps to mouse chromosome 11. Mammalian genome : official journal of the International Mammalian Genome Society 43 7767007
2020 Decrypting the Information Exchange Pathways across the Spliceosome Machinery. Journal of the American Chemical Society 40 32275149
2014 Epigenetic mechanism causes Wnt9b deficiency and nonsyndromic cleft lip and palate in the A/WySn mouse strain. Birth defects research. Part A, Clinical and molecular teratology 34 25257647
2011 The clf2 gene has an epigenetic role in the multifactorial etiology of cleft lip and palate in the A/WySn mouse strain. Birth defects research. Part A, Clinical and molecular teratology 34 21384535
2009 Prp45 affects Prp22 partition in spliceosomal complexes and splicing efficiency of non-consensus substrates. Journal of cellular biochemistry 31 19016306
2005 The structure of Prp40 FF1 domain and its interaction with the crn-TPR1 motif of Clf1 gives a new insight into the binding mode of FF domains. The Journal of biological chemistry 30 16253993
2004 Novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/BSF-3)/cardiotrophin-like cytokine (CLC)--a novel gp130 cytokine with pleiotropic functions. Cytokine & growth factor reviews 27 15450249
2021 Curled Flag Leaf 2, Encoding a Cytochrome P450 Protein, Regulated by the Transcription Factor Roc5, Influences Flag Leaf Development in Rice. Frontiers in plant science 26 33643332
2021 Novel Cecropin-4 Derived Peptides against Methicillin-Resistant Staphylococcus aureus. Antibiotics (Basel, Switzerland) 24 33401476
2004 A digenic cause of cleft lip in A-strain mice and definition of candidate genes for the two loci. Birth defects research. Part A, Clinical and molecular teratology 24 15329828
2016 Cytokine-Like Factor 1, an Essential Facilitator of Cardiotrophin-Like Cytokine:Ciliary Neurotrophic Factor Receptor α Signaling and sorLA-Mediated Turnover. Molecular and cellular biology 23 26858303
2021 CRNKL1 Is a Highly Selective Regulator of Intron-Retaining HIV-1 and Cellular mRNAs. mBio 21 33468685
2017 Abundance of Secreted Proteins of Trichoderma reesei Is Regulated by Light of Different Intensities. Frontiers in microbiology 21 29375497
2002 Evidence that the pre-mRNA splicing factor Clf1p plays a role in DNA replication in Saccharomyces cerevisiae. Genetics 17 11973290
2011 Cytokine-like factor 1 (CLF1): life after development? Cytokine 13 21715184
2003 Cryptococcus neoformans with a mutation in the tetratricopeptide repeat-containing gene, CCN1, causes subcutaneous lesions but fails to cause systemic infection. Infection and immunity 13 12654817
1999 BSMAP, a novel protein expressed specifically in the brain whose gene is localized on chromosome 19p12. Biochemical and biophysical research communications 13 10527841
2004 Saccharomyces cerevisiae CSM1 gene encoding a protein influencing chromosome segregation in meiosis I interacts with elements of the DNA replication complex. Experimental cell research 12 15023545
2024 Brassica rapa CURLY LEAF is a major H3K27 methyltransferase regulating flowering time. Planta 8 38865018
2021 Identification of Nrl1 Domains Responsible for Interactions with RNA-Processing Factors and Regulation of Nrl1 Function by Phosphorylation. International journal of molecular sciences 7 34209806
2024 Carryover effects of maternal late-gestation heat stress on granddaughters' growth and mammary gland development. Journal of dairy science 6 38642652
2021 A termination-independent role of Rat1 in cotranscriptional splicing. Nucleic acids research 6 33978753
2021 Neuroprotective Effects of Coreopsis lanceolata Flower Extract against Oxidative Stress-Induced Apoptosis in Neuronal Cells and Mice. Antioxidants (Basel, Switzerland) 6 34204667
2021 A distinct complex of PRP19-related and trypanosomatid-specific proteins is required for pre-mRNA splicing in trypanosomes. Nucleic acids research 5 34850936
2025 RANBP1 Regulates NOTCH3-Mediated Autophagy in High Glucose-Induced Vascular Smooth Muscle Cells. Frontiers in bioscience (Landmark edition) 4 40018934
1988 Cell-mediated responses and protection elicited by a carbohydrate-lipid-containing fraction extracted from Leishmania major promastigotes. Cellular immunology 4 3338094
2024 Cigarette smoking combined with genetic variation regulates the m6A methylation of CRNKL1 and is associated with bladder cancer risk. Environmental toxicology 3 38270278
2021 Antiproliferative activity on Trypanosoma cruzi (Y strain) of the triterpene 3β,6β,16β-trihidroxilup-20 (29)-ene isolated from Combretum leprosum. Journal of biomolecular structure & dynamics 3 34436980
2025 Characterization of the antagonistic secondary metabolites of Paenibacillus polymyxa MEZ6 against Staphylococcus aureus. Frontiers in microbiology 2 40822391
2025 Transcriptomic and flavor metabolomic exploration of the genetic basis of meat quality and flavor in Tibetan sheep. BMC genomics 2 41023800
2024 Environmentally relevant concentrations of 2,3,7,8-TCDD induced inhibition of multicellular alternative splicing and transcriptional dysregulation. The Science of the total environment 2 38346650
2023 Antimicrobial, Antibiofilm Activities and Synergic Effect of Triterpene 3β,6β,16β-trihydroxyilup-20(29)-ene Isolated from Combretum leprosum Leaves Against Staphylococcus Strains. Current microbiology 2 37029832
2025 Recurrent de novo variants in the spliceosomal factor CRNKL1 are associated with severe microcephaly and pontocerebellar hypoplasia with seizures. American journal of human genetics 1 40857589
2025 Structural and Functional Analysis of ASFV pI73R Reveals GNB1 Binding and Host Gene Modulation. International journal of molecular sciences 1 41465200
2022 Hypoglycemic effect on adult zebrafish (Danio rerio) of the 3β-6β-16β-trihydroxylup-20(29)-ene triterpene isolated from Combretum leprosum leaves in vivo and in silico approach. Fundamental & clinical pharmacology 1 35261066
2017 SorLA and CLC:CLF-1-dependent Downregulation of CNTFRα as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry. Journal of visualized experiments : JoVE 1 28117780
1998 The region 3' to Calpha1 gene of human IG heavy chain displays a polymorphic duplicated sequence and encodes an RNA associated with polysomes. Gene 1 9756987
2025 Development and characterization of a double-fluorescent HIV-1 reporter cellular model to tackle the Rev-dependent export pathway. Microbiology spectrum 0 39902983
2025 Multi-Omics Analysis of Survival-Related Splicing Factors and Identifies CRNKL1 as a Therapeutic Target in Esophageal Cancer. Genes 0 40282339
2025 Robust fluorine-rich YF3 artificial interfacial layer for providing uniform Zn2+ flux and enhancing cycling stability of Zn anodes. Nanoscale 0 40843544
2025 Transgenerational skin adaptations to late-gestation heat stress in great-granddaughters. JDS communications 0 40922959

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