Fig 2: Tau Expression and SmB Loss Cause Similar Splicing Errors in Drosophila Brains(A) Key for splicing errors shown in (B).(B) Pan-neuronal expression of TauWT or TauR406W induces differential expression of cryptic splicing errors (left) and intron retention (right), similar to SmBMG/MG, based on RNA-seq in 1-, 10-, or 20-day-old flies (n = 3 per genotype, except n = 2 for same-batch control for TauR406W comparison). See also Figure S6B, Tables S4 and S5, and Data S1, tabs iv and v.(C) Histograms for representative Tau-induced splicing errors in 20-day-old flies, showing RNA-seq reads normalized between control and TauR406W and split reads spanning splice junctions. Transcript structures are indicated below, including reference-annotated (black) versus aberrant exons (gray) resulting from cryptic splice junctions. Transcript orientation (arrows) and the 5′-splice donor sites (green bars) analyzed in (F) are denoted. See also Figure S6C.(D) Genes harboring splicing errors following pan-neuronal expression of TauR406W (union of 1-, 10-, and 20-day results) strongly overlap with SmBMG/MG (10 days). Percentage denotes Tau-associated, differentially spliced genes that also overlap. See also Figures S6D–S6F.(E) Splicing errors in TauR406W transgenic and SmBMG/MG flies occur more commonly in genes with greater numbers of introns (left) or annotated alternatively spliced transcripts (right), compared with all Drosophila genes. ***p < 0.001.(F) Splicing errors occur at exon-intron junctions with splice donor sequences that diverge from the consensus motif for U1 spliceosome binding. The consensus splice donor (5′-splice site) sequence (bold) is shown, along with splice donor sequences corresponding to splicing errors in representative genes (green bars in C). Nucleotides that diverge from the consensus are shown (red), along with the splice site binding strength score. See also Figures S7A and S7B.(G) Many splicing errors in TauR406W flies affect protein coding sequences (blue) or untranslated regions (UTRs: green, 5′-UTR; yellow: 3′-UTR), and are likely to disrupt protein expression. See also Data S1, tabs vi and vii.

Fig 3: Loss of SmB Causes Neurodegeneration(A) Hematoxylin and eosin-stained frontal brain section at the level of mushroom body calyx, highlighting the region of interest for (B) and (C). Scale bar: 25 μm.(B and C) SmBMG/MG adults (B) show progressive loss of cortical nuclei between 2 and 25 days of age (n > 5 for quantification, C). Scale bar: 25 μm.(D) Frontal section at the level of lamina highlighting region of interest for (E) and (F) and DAB-positive cholinergic neurons (ChAT>lacZ reporter). Scale bar: 20 μm.(E and F) SmBMG/MG animals (E) exhibit cholinergic neuron loss between 2 and 25 days (n > 9 for quantification, F). Scale bar: 10 μm.All error bars denote mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.See also Figures S4C–S4F and S5.

Fig 4: Tau Associates and Genetically Interacts with Spliceosomal Factors(A) Following coimmunoprecipitation, 1,065 Tau-associated proteins were identified from AD or control postmortem brain tissue homogenates (n = 4 each). Volcano plot highlights proteins showing >1.5-fold increased (red) or decreased (green) interactions with Tau in AD (all p < 0.05), including numerous ribonucleoproteins (p = 7.7 × 10−5). Spliceosome components are denoted. See also Figure S1, Tables S1, and Data S1, tabs i and ii.(B) Compared with controls (Ctrl, top left), TauV337M expression in the eye (GMR-GAL4) causes reduced eye size and roughened surface (bottom left: 1.98 ± 0.08, mean score ± SEM, n > 35 for all genotypes), and RNAi targeting spliceosome factors enhanced toxicity (bottom row, p < 0.0001: SmB, 3.67 ± 0.06; SmD2, 4.00 ± 0.00; U1–70K, 4.04 ± 0.03; U1C, 3.92 ± 0.09; and SmE, 4.00 ± 0.00). RNAi lines were non-toxic when expressed independently (top row). See also Data S1, tab iii.(C) TauWT expression in the adult retina (Rh1-GAL4) causes reduced electroretinogram (ERG) amplitude in 5-day-old flies, and this phenotype is enhanced in SmBMG heterozygotes (n > 10 for quantification). See also Figures S2A–S2C.(D) Pan-neuronal expression of TauR406W (elav-GAL4) causes progressive neuropil vacuolization (arrows) in hematoxylin and eosin-stained frontal brain sections in 10-day-old adult flies, and this phenotype is enhanced in SmBMG heterozygotes (n > 8 for quantification). Scale bar: 50 μm. See also Figures S2D and S2E. All error bars denote mean ± SEM. **p < 0.01; ***p < 0.001; ns, not significant.

Fig 5: Different sensitivities of MTAP-deleted and MTAP-rescued cells to Type I PRMT inhibition. (A–D) MTAP-deleted cells were reconstituted with the stable re-expression of MTAP. These isogenic cell pairs were cultured for 8 days in the presence of DMSO or 1 μM of MS023 (0.1 μM for MiaPaCa2 cells). Different cell types display differing degrees of sensitivity to MS023, which we established empirically. The cell growth curves are plotted for A549 (A), MiaPaCa2 (B), A172 (C) and MCF-7 (D) cell lines. The expression levels of MTAP are shown for each pair of cell lines. (E) A scatter plot showing the sensitivities of MTAP-deleted cells to PRMT5 knockdown. Each dot in the scatter plot represents a cancer cell line. Data retrieved from the DepMap project (https://depmap.org/portal/). (F) Using total cell lysates from MTAP-deleted and MTAP-rescued cells, the levels of SDMA, ADMA, SmB methylation (Y12) and MTAP were analyzed by western blot.