Fig 2: Molecular and phenotypic characterization of rbbp4off and rbbp4on alleles.(A) Diagram of the rbbp4off allele. (B) Plot of RT-qPCR results from wild type +/+ (n=3), heterozygous rbbp4off/+(n=3), and homozygous rbbp4off/off (n=3) larvae showing the relative level of rbbp4 mRNA transcript using reference gene rps6kb1b. Primer pairs were located in exons 4 and 5, or downstream exons 11 and 12. (C – E) Gross phenotype of rbbp4Δ4/+ (C), rbbp4off/Δ4 (D), and rbbp4Δ4/Δ4 (E) 5 dpf larvae. Arrowhead in (D) points to overlap of rbbp4off 2A-mRFP primary reporter and gcry1:BFP secondary reporter expression in the lens, which appears purple. (F – H) Caspase-3 and HuC/D labeling of sectioned head tissue from 2 dpf rbbp4Δ4/+ (F) rbbp4off/Δ4 (G) and rbbp4Δ4/Δ4 (H) embryos. (I) Diagram of the rbbp4on allele. (J) Plot of RT-qPCR results from wild type +/+ (n=3), heterozygous rbbp4on/+ (n=3), and homozygous rbbp4on/on (n=3) larvae showing the relative level of rbbp4 mRNA transcript using reference gene rps6kb1b. Primer pairs were located in exons 4 and 5, or downstream exons 11 and 12. (K, L) Gross phenotype of rbbp4on/+ (K) and rbbp4on/Δ4 (L) 5 dpf larvae. The rbbp4on allele secondary marker gcry1:BFP expression is visible in the lens. Caspase-3 and HuC/D labeling of sectioned head tissue from 2 dpf rbbp4Δ4/+ (M) and rbbp4off/Δ4 (N) embryos. OT, optic tectum; R, retina; Th, thalamic region. Error bars represent mean ± s.e.m. Scale bars: 200 μm (C–E, K, L), 50 μm (F–H, M,N). Figure 4—source data 1.Source data for rbbp4 RT-quantitative PCR analysis in wildtype, heterozygous and homozygous embryos from rbbp4off/+ and rbbp4on/+ incrosses.

Fig 3: rbbp4 and rb1 intronic gRNA efficiency and F1 UFlip allele junction analysis.(A) rbbp4 gene model with sequence of the intron 4 reverse strand gRNA. Gel image of PCR amplicons surrounding the target site from 8 Cas9 plus gRNA injected and 1 uninjected (U) embryo. Amplicons from embryo #3 and the uninjected embryo were sequenced and analyzed with Synthego’s ICE software, and indicate 50% indel efficiency at the target site. Plot shows the range and percentage of indels present in the sequences. PAM sequence shown in bold and underlined. (B) rb1 gene model with sequence of the intron 6 reverse strand gRNA. Gel image of PCR amplicons surrounding the target site from eight embryos injected with Cas9 and the gRNA (1-8), and two uninjected embryos (U). Amplicons from embryo #1 and an uninjected embryo were sequenced and analyzed with Synthego’s ICE software, and indicate 95% indel efficiency at the target site. Plots show the range and percentage of indels present in the sequences. PAM sequences shown in bold and underlined. (C) 5’ and 3’ genomic-UFlip integration junctions were PCR amplified from F1 transgenic zebrafish fin clip genomic DNA. The PCR products were sequenced and aligned to the reference sequence expected for a precise integration at the genomic target site. Capitalized red nucleotides represent 48 bp homology arms. Lowercase green nucleotides represent random inserted sequences.

Fig 4: LIN9 and LIN37 scaffold the MuvB complex.a Domain architecture of human LIN9 and LIN37, with regions of predicted and validated structure shown as blocks. The conserved LIN37 CRAW domain and LIN9 DIRP domain structures are determined here. MBD is the MYB-binding domain. b Schematic model for subunit interactions within MuvB. c The indicated tagged subunits or MuvB complex (GST-LIN52, Strep-RBAP48, His-LIN54504–749, GST-LIN37, and GST-LIN994–542) were expressed in Sf9 cells and extracts were precipitated with resin capturing the indicated tag. Proteins were visualized with coomassie staining. *Indicates impurities or degradation observed in some RBAP48 expressions. These bands are not pulled out from the tandem purification. The experiment was repeated three times with similar results. d HCT116 cells were transfected with plasmids encoding the indicated FLAG-tagged mouse protein. FLAG-tagged proteins were precipitated from extracts using anti-FLAG antibody and visualized with anti-FLAG immunoblotting and immunoblotting with antibodies that recognize RBAP48, LIN37, LIN54, and p130. A biological replicate of the experiment was performed, and results were similar.

Fig 5: LIN37 CRAW domain binds both LIN9 and RBAP48.a Interactions of LIN9 and LIN37 with the RBAP48 insertion loop. b Alignment of LIN37 sequences from organisms as in Fig. 2b. Residues that contact LIN9 (.), RBAP48 (*), and both LIN9 and RBAP48 (@) are indicated. c Close-up view of the LIN9-LIN37 interface. d The indicated FLAG-GFP control and FLAG-LIN9 WT and mutants were expressed by transient transfection in arrested HCT116 cells. Proteins were immunoprecipitated using anti-FLAG beads and the indicated proteins visualized by western blot. The 3x LIN9 mutant is E125A/W126A/F127A and the 4x LIN9 mutant is R174A/R175A/F180A/F181A. e Same as (d) but expressing the indicated RBAP46 and RBAP48 WT and mutant proteins. These immunoprecipitation experiments were performed each with a biological replicate, and results were similar.