Journal: bioRxiv

Article Title: CAKUT variants in PRPF8, DYRK2 , and CEP78 : implications for splicing and ciliogenesis

doi: 10.1101/2025.07.16.665151

Figure Lengend Snippet: A) The graph provides an overview of the splicing process. PRPF8 is a core component of the U5 snRNP and the spliceosome. B) Ubiquitination and degradation of CP110 regulates ciliogenesis. CP110 caps distal end of the mother centriole and thereby inhibits ciliogenesis. EDD-DYRK2-DDB1 VprBP complex is constitutively located at the (sub)distal end of the mother centriole. CEP350 recruits CEP78, and CEP78 activates VPRBP and the EDD-DYRK2-DDB1 VprBP complex. Phosphorylation of CP110 by DYRK2 enables recognition of CP110, which is brought close to EDD that transfers ubiquitin to CP110. PRPF8 functions as receptor for ubiquitin chains of CP110. Ubiquitination causes CP110-disassembly and removal from mother centriole, initiating ciliogenesis. C) Summary of renal and extra-renal manifestations in patients with PRPF8 variants. The patient with the de novo PRPF8 R1681W variant displayed the most severe phenotype with multiple malformations. None of the patients presented with RP symptoms. D) Examples of sequence conservation of PRPF8 amino acids mutated in CAKUT. E) Protein domain structure of human PRPF8 showing the position of de novo variants (red), heterozygous CAKUT variants (black) and RP mutations (magenta). Green arrow depicts a missense mutation ( Prpf8 +/N1531S ) in a mouse model exhibiting a ciliopathy phenotype. PRO8NT: PRP8 N-terminal domain or Bromodomain; PROCN PRO8 central domain; RT reverse transcriptase domain; RNaseH-like Ribonuclease H domain; Jab1/MPN Jun activation domain-binding protein 1/Mpr1, Pad1 N-terminal domain; RP Retinitis Pigmentosa.

Article Snippet: WT and mutant plasmids for human PRPF8, CEP78, EDD, and DDB1 were obtained from GenScript.

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Variant Assay, Sequencing, Mutagenesis, Reverse Transcription, Activation Assay, Binding Assay

Journal: bioRxiv

Article Title: CAKUT variants in PRPF8, DYRK2 , and CEP78 : implications for splicing and ciliogenesis

doi: 10.1101/2025.07.16.665151

Figure Lengend Snippet: A) Exon and domain structure of human DYRK2 . Arrowheads show positions of heterozygous CAKUT variants. B) Alphafold-predicted 3D structure of the DYRK2–CEP78 complex. The red square in the PAE plot suggests a potential direct interaction. Variants are scattered and do not cluster at the interface. C) DYRK2 kinase activity assessed via NDEL1 phosphorylation. Immunoblotting shows comparable phospho-NDEL1S336 levels for all CAKUT variants relative to WT, indicating preserved kinase function. K251R served as a kinase-dead control; empty vector as negative control. D) Co-IP of DYRK2 variants with EDD, DDB1, and VprBP. Two variants (p.Arg326Cys, p.Arg326His) showed reduced complex formation (n=3).

Article Snippet: WT and mutant plasmids for human PRPF8, CEP78, EDD, and DDB1 were obtained from GenScript.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Control, Plasmid Preparation, Negative Control, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: CAKUT variants in PRPF8, DYRK2 , and CEP78 : implications for splicing and ciliogenesis

doi: 10.1101/2025.07.16.665151

Figure Lengend Snippet: A) The graph provides an overview of the splicing process. PRPF8 is a core component of the U5 snRNP and the spliceosome. B) Ubiquitination and degradation of CP110 regulates ciliogenesis. CP110 caps distal end of the mother centriole and thereby inhibits ciliogenesis. EDD-DYRK2-DDB1 VprBP complex is constitutively located at the (sub)distal end of the mother centriole. CEP350 recruits CEP78, and CEP78 activates VPRBP and the EDD-DYRK2-DDB1 VprBP complex. Phosphorylation of CP110 by DYRK2 enables recognition of CP110, which is brought close to EDD that transfers ubiquitin to CP110. PRPF8 functions as receptor for ubiquitin chains of CP110. Ubiquitination causes CP110-disassembly and removal from mother centriole, initiating ciliogenesis. C) Summary of renal and extra-renal manifestations in patients with PRPF8 variants. The patient with the de novo PRPF8 R1681W variant displayed the most severe phenotype with multiple malformations. None of the patients presented with RP symptoms. D) Examples of sequence conservation of PRPF8 amino acids mutated in CAKUT. E) Protein domain structure of human PRPF8 showing the position of de novo variants (red), heterozygous CAKUT variants (black) and RP mutations (magenta). Green arrow depicts a missense mutation ( Prpf8 +/N1531S ) in a mouse model exhibiting a ciliopathy phenotype. PRO8NT: PRP8 N-terminal domain or Bromodomain; PROCN PRO8 central domain; RT reverse transcriptase domain; RNaseH-like Ribonuclease H domain; Jab1/MPN Jun activation domain-binding protein 1/Mpr1, Pad1 N-terminal domain; RP Retinitis Pigmentosa.

Article Snippet: WT and mutant plasmids for human PRPF8, CEP78, EDD, and DDB1 were obtained from GenScript.

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Variant Assay, Sequencing, Mutagenesis, Reverse Transcription, Activation Assay, Binding Assay

Journal: bioRxiv

Article Title: CAKUT variants in PRPF8, DYRK2 , and CEP78 : implications for splicing and ciliogenesis

doi: 10.1101/2025.07.16.665151

Figure Lengend Snippet: A) Growth of yeast containing different Prp8 variants at various temperatures. A strain harboring the de novo variant PRPF8 R1681W ( Prp8 R1753W ) is inviable at 37 °C. B) CAKUT variants do not rescue the prp28-1 cold sensitive phenotype at 16 °C. C) Prp8 variants do not suppress the U4-cs1 phenotype at 16 °C, however PRPF8 R1414H (Prp8 R1468H ) shows a weak suppression of U4-cs1 at 23°C. D) Multiple Prp8 variants show genetic interactions with brr2-1 . E) Schematic overview of the ACT-1CUP1 assay, including the locations of non-consensus substitutions. F-H) ACT1-CUP1 assay results for the A3C (panel F), BS-C (panel G), BS-G (panel H), and UUG reporters (panel I).

Article Snippet: WT and mutant plasmids for human PRPF8, CEP78, EDD, and DDB1 were obtained from GenScript.

Techniques: Variant Assay

Journal: bioRxiv

Article Title: CAKUT variants in PRPF8, DYRK2 , and CEP78 : implications for splicing and ciliogenesis

doi: 10.1101/2025.07.16.665151

Figure Lengend Snippet: A) No difference between ability to increase Gli1 and Ptch expression in Prpf8 +/N1531S mouse embryonic fibroblast treated with SAG. B) GLI1 and PTCH1 expression of RPE-1 cells transfected with plasmids endoding CAKUT variants ( red = de novo , green = Prpf8 N1531S , grey =CAKUT heterozygous), and RP variants (magenta) . Cells were stimulated with SAG and GLI1 and PTCH1 expression analyzed by RT-qPCR, using endogenous PRPF8 mRNA and 18S RNA levels for normalization. P13L and S1722G serve as negative controls.

Article Snippet: WT and mutant plasmids for human PRPF8, CEP78, EDD, and DDB1 were obtained from GenScript.

Techniques: Expressing, Transfection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: CAKUT variants in PRPF8, DYRK2 , and CEP78 : implications for splicing and ciliogenesis

doi: 10.1101/2025.07.16.665151

Figure Lengend Snippet: RNA in situ hybridization analysis of Prpf8 expression on sagittal sections of mouse kidney (first row), transverse sections of the ureter (second row) and sagittal sections of the bladder (third row) of wildtype embryos from E11.5 to E18.5. Note that all sections were developed for the same time, except the ones from the last column (*) for which color development was prolonged to detect weak expression domains. n =4 for each stage and tissue. Size bars represent 100 µm. ble, bladder epithelium; blm, bladder mesenchyme; cl, cloaca; k, kidney; u, ureter; ue, ureteric epithelium; um, ureteric mesenchyme; us, ureteric stalk, ut, ureteric tip.

Article Snippet: WT and mutant plasmids for human PRPF8, CEP78, EDD, and DDB1 were obtained from GenScript.

Techniques: RNA In Situ Hybridization, Expressing