EMBO/SourceData huggingface dataset:
Figure 2(A) Volcano plots representing transcript down- (red) or up-regulation (yellow) after 48 hours PTX in the somatic and process compartment. Representative transcripts are highlighted and labelled. Linear model was used to fit transcriptome data and subsequently contrasted using likelihood ratio testing for PTX-treatment effects across compartments.
Figure 3(A) Real-time quantitative PCR (RT-qPCR) of transcripts changing differentially between compartments using the compartmentalized cultures after 48 hours PTX-treatment. n=8 independent biological replicates (except for Plk2 (n=7), where one measurement couldn't be performed due to insufficient cDNA); PTX-effect was assessed by three-way ANOVA followed by Tukey's post-hoc multiple comparison test; *p < 0.05; **p < 0.01.(B) Representative images of single-molecule FISH (smFISH) in either control or 48 h PTX-treated rat hippocampal neurons (DIV20) using probes specific for Add2, Dnajc6 and Sort1 (green). MAP2 immunostaining (red) was used to visualize neuronal somata and dendrites. Inserts at higher magnification illustrate PTX-dependent changes in dendritic RNA puncta. Scale bar = 10 μm. (C) Quantification of B. (n=3-4 independet biological replicates with 8-10 cells averaged per condition and replicate; two-sample Student's t-test; *p < 0.05; **p < 0.01). B
Figure 4(D) Representative plots of genes changing after PTX at the transcript and protein level. (Upper row: Scatterplots of transcript level; n=2; crossbar represents mean. Lower row: Boxplot of protein levels; n=4). Boxplots: central line: median; box: 25th to 75th percentile; whiskers: until last data point within 1.5x interquartile range (IQR).
Figure 5(A) Representative pictures of nascent Camk2a-peptides in mock- and PTX-treated neurons. Upper Panels: Representative overview images with merged channels (grey: MAP2-staining, red: Duolink-PLA signal, blue: Hoechst nuclear staining). Middle Panels: Magnifications of boxed areas marked in (A). MAP2 signal was used to outline somata and dendritic processes (white lines). Lower Panels: Negative control of puromycin-untreated cells with representative Duolink-signal in dendrite and soma, respectively. (Scale bars: in overview images 20 μm, in inserts of higher magnification 5 μm). (B) Same as A with nascent Synapsin1-peptides. (
Figure 6(C) Enrichment analysis for RBP motifs in down- and up-regulated genes in either compartment. Fisher's exact test was used to perform enrichment analysis; FDR-corrected.
Figure 1Immunostaining from Daam2LacZ/+ animals with Aldh1l1-EGFP reporter indicates that Daam2 (LacZ+, Red) is expressed in astrocytes (Green). (A) Daam2 expression is observed in multiple brain regions at postnatal day 21.Immunostaining from Daam2LacZ/+ animals with Aldh1l1-EGFP reporter indicates that Daam2 (LacZ+, Red) is expressed in astrocytes (Green). (B) Expression of Daam2 in cortex persists throughout development (P7 to adult). C. Quantification of Daam2-expressing astrocytes (LacZ+) with Aldh1l1-EGFP labeled astrocytes in cortex during development. Data is presented as mean ± SEM. Two brain sections, N=4 for each age.G. Representative images of astrocytes labeled with Aldh1l1-EGFP reporter in cortex and OB of Daam2 cHet and cKO mice at postnatal day 28 (P28). Scale bar: 50 μm. H-K. Overall complexity of astrocytes with Aldh1l1-EGFP reporter was measured by Sholl analysis (Two-way ANOVA) and total process length (Student's t-test). Data is presented as mean ± SEM. n= 6-10 cells from N=3-5 mice per genotype, *p<0.05, p****<0.0001
Figure 2A. Representative images of GFAP immunostaining in Daam2 cKO mice compared to controls in cortex and OB at P28. Scale bar: 20 μm.D. Western blot analysis showed a significant increase in the amount of GFAP protein in Daam2 KO primary astrocytes. N=2F. Overexpression of Daam2 via injection of AAV-GfaABC1D-myc-Daam2 in wild type P1 pups revealed reduced GFAP intensity compared to animals injected with control virus (AAV-GfaABC1D-GFP). AAV-GfaABC1D-myc-Daam2 was injected into Aldh1l1-GFP reporter mice for morphological complexity analysis and infected cells (Myc positive) were compared with non-infected cells. Dotted white line indicates virus-infected cells. Filled triangles and empty triangles indicate astrocytic primary branches with the control and reduced level of GFAP or Aldh1l1-GFP, respectively. Scale bar: 20 μm.
Figure 3E. Schematics of astrocytic calcium imaging and representative images of spontaneous calcium signaling (green) of Daam2 cHet and cKO mice injected with AAV-GfaABC1D-GCaMP6f virus in OB. Approximate territory of astrocytes including soma and multiple microdomains is outlined in yellow. Representative traces of GCaMP6f signal in Daam2 cHet and cKO astrocytes are shown in black and red respectively. F-H. Quantification of amplitude (F), frequency (G), and area under curve (AUC) (H) of ∆F/F GCaMP6f signal events in OB. Amplitude and AUC of Daam2-deficient astrocytes are significantly increased in OB. Data is presented as mean ± SEM. Total number of cells is n=16, 15 (F-H) from N=3-4 mice of each genotype. Student's t-test was used for statistics, *p<0.05.
Figure 4A-F. (A, D) Schematics of whole cell recording in ex vivo brain slices and representative traces of spontaneous EPSC and IPSC in neurons in cortex from Daam2 cHet (top, black) and cKO (bottom, red) mice. (B-C, E-F) Quantification of amplitude and frequency of sEPSC and sIPSC in each genotype. Data is presented as mean ± SEM. Total number of cells is n=19, 20 (B-C) and 17, 13 (E-F) from N=7 mice for each genotype. Student's t-test was used for comparison, *p<0.05.
Figure 5A. Astrocyte-specific Daam2 cKO mice show delays in buried food finding. N=13, 15 mice per genotype. Data is presented as mean ± SEM. Student's t-test was used, *p<0.05.
Figure 6E-F. qRT-PCR (E) and immunostaining (F) confirm differential expression of genes found in MS profiling. qRT-PCR is repeated as triplicate and is presented as mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. Student's t-test was used. Scale bar: 20 μm.
Figure 7F-J. Representative images of GFAP immunostaining (F) and quantification (G-J) in controls, Daam2 icKO, Slc4a4 icKO, and double icKO 6 weeks after tamoxifen injection. Scale bar: 20 μm. Data is presented as mean ± SEM. n=10-24 images, N=3-4 mice per genotype. Scale bar: 20 μm. Two-way ANOVA is used for statistics. [*] and [#] indicates comparison with control and double icKO, respectively. **p<0.01, p***<0.001, ##p<0.01.
Figure 1(A) Chromosome 7 of S. bayanus is incompatible with the S. cerevisiae genome. The chromosome 7 replacement stain (Sc+Sb-chr7) exhibited serious growth defects when grown on the non-fermentable carbon source (glycerol). The respiration defects could be partially rescued when a whole set of S. bayanus chromosomes were provided (Sc+Sb-chr7 x Sb-ρ⁰), or fully rescued with the addition of S. bayanus mtDNA (Sc+Sb-chr7-ρ⁰ x Sb). Cell cultures were serially-diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed. Sc, S. cerevisiae. Sb, S. bayanus. ρ⁰, mtDNA-less strains.(B) Ectopically-expressing Sc-CCM1 rescues the respiration defect of Sc+Sb-chr7. Spot assays for the wild-type S. cerevisiae (Sc) and the chromosome 7 replacement stain (Sc+Sb-chr7) carrying an empty plasmid (+ vector) or the plasmid with the S. cerevisiae CCM1 gene (+ Sc-CCM1).(C) Sb-CCM1 incompatibility could only be rescued in the presence of S. bayanus mtDNA. The native CCM1 ORF in S. cerevisiae was replaced by either Sb-CCM1 (Sc+Sb-CCM1) or Sc-CCM1 (Sc+Sc-CCM1, as a control) coding regions. Crossing the Sc+Sb-CCM1 strain with the mtDNA-less Sb-ρ⁰ strain (Sc+Sb-CCM1 x Sb-ρ⁰) could not rescue the growth defect.(D) CCM1 incompatibility is symmetric between S. cerevisiae and S. bayanus. The CCM1 ORF in S. bayanus was replaced by either Sc-CCM1 (Sb+Sc-CCM1) or Sb-CCM1 (Sb+Sb-CCM1, as a control) coding regions. Only the Sb+Sc-CCM1 strain exhibited respiration defects and the defect could not be rescued even when a whole set of S. cerevisiae chromosomes were provided (Sb+Sc-CCM1 x Sc-ρ⁰).(E) CCM1 incompatibility probably occurred during the divergence between S. bayanus and the common ancestor of S. cerevisiae, S. paradoxus, S. mikatae and S. kudriavzevii. The endogenous CCM1 in S. cerevisiae was replaced with its orthologous alleles from other Saccharomyces sensu stricto yeasts. Only the strain carrying S. bayanus CCM1 displayed growth defects on glycerol-containing plates.
Figure 2(A) Sb-Ccm1 protein is transported efficiently to S. cerevisiae mitochondria. Total cell extracts (total), cytosolic fractions (cyto) or mitochondrial fractions (mito) were hybridized with different antibodies to detect the proteins. G6PDH (glucose-6-phosphate dehydrogenase) and Cox2 served as cytosolic and mitochondrial markers, respectively. Both Sc-Ccm1 and Sb-Ccm1 were fused with c-Myc at the C-terminus and expressed from a single-copy plasmid. The Myc-tagged Ccm1 proteins consistently appeared as double bands, which were probably due to some unknown protein modifications.(B) The level of 15S rRNA is reduced in Sc+Sb-CCM1 cells. RNA isolated from Sc+Sb-CCM1 (labeled as Sb) or Sc+Sc-CCM1 (labeled as Sc) cells was hybridized with gene-specific probes using Northern blots.(C) Sb-Ccm1 interacts weakly with S. cerevisiae 15S rRNA. The level of Ccm1-bound 15S rRNA was measured using quantitative PCR analysis following mitochondrial RNA immunoprecipitation of S. cerevisiae cells expressing Sc-CCM1-13Myc or Sb-CCM1-13Myc. Data was normalized to the control group without the Myc tag to obtain the relative fold enrichment. Graph was plotted using data from three independent repeats for each strain and p value was calculated by unpaired, two-sided student t-test. Error bars indicate S.D. ***, p value < 0.001.(D) Overexpressing Sb-CCM1 rescues the respiration defect of Sc+Sb-CCM1 cells. Cell cultures were serially-diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed.(E) The steady-state level of mtDNA-encoded proteins is reduced in Sc+Sb-CCM1 cells. Immunoblotting for the mitochondrial proteins in Sc+Sb-CCM1 (labeled as Sb) or Sc+Sc-CCM1 (labeled as Sc) cells. Cox1, Cox2 and Cox3 are mtDNA-encoded complex IV subunits. Tom20 is a nucleus-encoded mitochondrial protein.
Figure 3(A) Tetrad analysis reveals that the respiration defect of Sc+Sb-CCM1 cells can be rescued by mutations in mitochondrial or nuclear genomes. The suppressor clones were crossed with the Sc+Sb-CCM1-ρ⁰ strain and the tetrads from the cross were analyzed. A non-Mendelian 4:0 segregation pattern for improved respiration is expected if the suppressor mutation is in mtDNA (left panel). Otherwise, a 2:2 segregation pattern would be observed if the rescue effect involved a single nuclear mutation (right panel).
Figure 4(A) Reconstituted Sb-CCM1 mutants can rescue the incompatibility of Sb-CCM1. Among 94 suppressor clones, 20 of them carry mutations in the Sb-CCM1 gene. These mutants were reconstructed in Sc+Sb-CCM1 cells to test their function. Superscripts next to Sb-CCM1 indicate the respective amino acid substitutions and numbers in parentheses indicate the number of independent clones in our suppressor screen. Sc, wild-type S. cerevisiae.(C) The 400th residue plays a determinant role in the S. cerevisiae-S. bayanus incompatibility. Both Sb-CCM1E375Q and Sb-CCM1D400N mutants relieved the incompatibility in S. cerevisiae, but only Sb-CCM1D400N caused respiration defects in S. bayanus. The Sb-CCM1E375Q mutant was selected as a control in this experiment since it was a charged-to-polar substitution similar to Sb-CCM1D400N and also appeared three times in our screen. Sb, wild-type S. bayanus.(D) Protein structural analysis shows that all intramolecular suppressor mutations were located on the predicted PPR motifs. The predicted protein structure of Ccm1 is shown at left. Blue and green colors indicate the two α-helices in each predicted PPR motif and white color indicates additional α-helices in the N-terminus and the middle region. Red color indicates the positions of the suppressor mutations. Predicted PPR motifs occupy around 52% of the whole protein (453 of 871 residues). The box at right displays a magnified version of some PPR motifs where the substitutions are located.
Figure 5(A) 15S rRNA levels are increased in the suppressor clones. Total RNA was isolated from the replacement strains (Sc+Sc-CCM1 and Sc+Sb-CCM1), two nuclear suppressor strains (Sc+Sb-CCM1D400N and Sc+Sb-CCM1E375Q) and one mitochondrial suppressor strain and then subjected to quantitative PCR analyses. Relative mRNA abundance was calculated by normalizing the 15S rRNA level to the ATP6 mRNA level. Graph was plotted using data from five independent repeats for each strain and p values were calculated by unpaired, two-sided student t-test. Error bars indicate S.D. ***, p value < 0.001. **, p value < 0.01. Asterisks in black, comparing Sc+Sc-CCM1 with others. Asterisks in gray, comparing Sc+Sb-CCM1 with others.(B) The steady-state level of mtDNA-encoded proteins is increased in the suppressor clones. Protein extracts from the replacement and suppressor strains were hybridized with antibodies against G6PDH, Cox1, Cox2 or Cox3.
Figure 6(B) Seven of 12 tested PPR genes exhibit incompatibility between S. cerevisiae and at least one other Saccharomyces sensu stricto species. The doubling time of S. cerevisiae cells carrying the endogenous PPR genes (Sc) or orthologs of other yeast species (Sp, Sm, Sk and Sb) were measured in YPD (glucose) or YPG (glycerol) media. All the measurements were then normalized to the doubling time of Sc to obtain the relative doubling time. Graphs were plotted using data from at least three independent repeats for each strain. Sc, S. cerevisiae. Sp, S. paradoxus. Sm, S. mikatae. Sk, S. kudriavzevii. Sb, S. bayanus. n.d., growth not detected. p values were calculated by unpaired, two-sided student t-test. Error bars indicate S.D. *, p value < 0.05. **, p value < 0.01. ***, p value < 0.001.(B) Seven of 12 tested PPR genes exhibit incompatibility between S. cerevisiae and at least one other Saccharomyces sensu stricto species. The doubling time of S. cerevisiae cells carrying the endogenous PPR genes (Sc) or orthologs of other yeast species (Sp, Sm, Sk and Sb) were measured in YPD (glucose) or YPG (glycerol) media. All the measurements were then normalized to the doubling time of Sc to obtain the relative doubling time. Graphs were plotted using data from at least three independent repeats for each strain. Sc, S. cerevisiae. Sp, S. paradoxus. Sm, S. mikatae. Sk, S. kudriavzevii. Sb, S. bayanus. n.d., growth not detected. p values were calculated by unpaired, two-sided student t-test. Error bars indicate S.D. *, p value < 0.05. **, p value < 0.01. ***, p value < 0.001.
Figure 1(A) The expression of AR was determined using Western blots (overexpressing AR in J82 cells and knocking down AR in UMUC3 cells).(B) Relative expression of the top 20 up-regulated circRNAs from the GEO dataset GSE92675 (BCa tumor tissues compared to para-tumor tissues) in response to AR modulation in J82 and UMUC3 cells. The # indicates an RNA with a relatively low expression (qRT-PCR cycle number > 35), thus the circRNA was excluded for further analysis.(E) Real-time quantitative PCR (qRT-PCR) results show the knock down efficiency for these 4 circRNAs in J82 oe-AR (J82-AR) cells.(F) Transwell invasion assays showed that knock down of hsa_circ_0084171 (circ_oo84171), but not the other circRNAs, can partly reverse oeAR-increased invasion in J82 cells. Scale bar, 100 μm.(G) MTT assays showed that shcirc_0084171 can partly reverse oeAR-increased cisplatin chemo-resistance (2 μg/ml) in J82 cells. To calculate the mean, the value of 0 µg/ml cisplatin groups were set as 1.(H) The qRT-PCR results showed that the overexpression (oe) efficacy for hsa_circ_0084171 (oecirc_0084171) in UMUC3-shAR cells.(I) Transwell invasion assays showed that oecirc_0084171 can partly rescue shAR induced invasion deduction in UMUC3 cells. Scale bar, 100 μm.(J) MTT assays showed that oecirc_0084171 can partly overcome shAR effect for cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.
Figure 2B) The relative expression of circFNTA was elevated in multiple BCa cell lines (T24, J82, UMUC3, and 5637) compared to normal bladder epithelial cell line SVHUC as shown by qRT-PCR.C) The expression of circFNTA and GAPDH mRNA in J82 and UMUC3 cells treated with or without RNase R was detected by qRT-PCR.D) RNA Fluorescence in situ hybridization (FISH) demonstrated that circFNTA was predominantly localized to the cytoplasm of J82 and UMUC3 cells. DAPI was mainly localized to the cytoplasm. Scale bar, 20 μm.E) Two shcircFNTA plasmids using PLKO.1 vector were transfected into UMUC3 cells. The knock down efficacy was measured by qRT-PCR.G) The qRT-PCR results show oecircFNTA efficacy in J82 cells compared with PWPI vector (left panel) and oecircFNTA or oelinearFNTA in J82 cells (right panel).Transwell invasion assays showed that shcircFNTA can decrease UMUC3 cell invasion (H) more profoundly than oelinearFNTA as compared with the oevector group. Scale bar, 100 μm.Transwell invasion assays showed that shcircFNTA can increase J82 cells invasion (I) more profoundly than oelinearFNTA as compared with the oevector group. Scale bar, 100 μm.J) MTT assays showed that oecircFNTA enhances J82 cells chemo-resistance to cisplatin (0, 1, 2, 5, 10 μg/ml were tested). This effect was more obvious in the oecircFNTA group as in the oelinearFNTA group (upper panel). MTT assays showed that shcircFNTA increases UMUC3 cells chemo-sensitivity to cisplatin (lower panel).K) FISH analyses revealed that circFNTA was significantly upregulated in BCa tissues as compared with the adjacent para-tumor tissues (N=41 patients) and representative images are shown. Scale bar, 250 μm.L) The qRT-PCR assays also demonstrated that circFNTA is significantly elevated in fresh BCa tissues compared to para-tumor tissues (N=23 patients).
Figure 3(A) The protein expression of AR and FNTA (circFNTA host gene) were determined using Western blot and showed that shAR can decrease FNTA protein expression in UMUC3 cells and oeAR can increase FNTA protein expression in J82 cells.(B) qRT-PCR results showed that mRNA levels of FNTA are not changed dramatically in UMUC3 cells with shAR and J82 cells with oeAR.(C) The mRNA levels of 4 RNA editing genes (ADAR, ADAR2, DHX9, and QKI) were tested with manipulated AR (UMUC3 cells in left panel and J82 cells in right panel). Results showed ADAR2 mRNA is negatively correlated with AR expression.(D) Western blot analysis also confirmed that ADAR2 can be negatively regulated by AR at the protein level.(E) Knock down efficiency of AR and ADAR2 (shADAR2#1 and shADAR#2) in UMUC3 cells, as determined by qRT-PCR.(F) shADAR2#1 and shADAR#2 could both reverse shAR-dependent circFNTA reduction in UMUC3 cells.(G) Transwell invasion assays confirmed that both shADAR2 constructs can partly reverse shAR-dependent UMUC3 cells invasion reduction. Scale bar, 100 μm.(H) MTT assays confirmed that both shADAR2 constructs can partly overcome shAR-dependent effects on cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.(J) ChIP assays confirmed that AR can directly bind to ADAR2 AREI/II (-572nt to -555nt).(K) ChIP pull-down DNA products were amplified by qRT-PCR reaction. The IgG antibody was used as a negative control.(M) Co-transfection of ARE Wild type (WT) or Mutant (Mut) ADAR2 promoter pGL3-Luciferase constructs into UMUC3 cells with/without shAR (left panel), and J82 cells with/without oeAR (right panel). The luciferase assay was used to detect promoter activity.
Figure 4(A) Western blot analysis showed that shcircFNTA can decrease FNTA expression in UMUC3 cells (upper panel) and oecircFNTA can increase FNTA expression, and that this was more profound than using oelinearFNTA in J82 cells (lower panel).(B) A RNA pull-down assay was used to test the binding of circFNTA to 11 miRNAs (predicted by online database) in UMUC3 cells. The results showed that miR-328-5p, miR-370-3p and miR-920 can be pulled-down by circFNTA.(C) Relative miRNA expressions were quantified by qRT-PCR to test miRNA overexpression efficacy.Transwell invasion assay (D) confirmed that only oemiR-370-3p can reverse oecircFNTA induced invasion in J82 cells,MTT assays (E) confirmed that oemiR-370-3p can reverse oecircFNTA induced cisplatin chemo-resistance (2 μg/ml) in J82 cells. Scale bar, 100 μm.Transducing the miR-370-3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Western blot (F)Transducing the miR-370-3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Transwell assay (G).(H) MTT assays also confirmed that transducing with a miR-370-3p inhibitor in UMUC3 cells can partly reverse cisplatin chemo-resistance (2 μg/ml) reduction by shcircFNTA.(J) The transfection efficacy of oecircFNTA WT and oecircFNTA Mut was tested by qRT-PCR.(K) Transfection with oecircFNTA WT could increase J82 cells invasion, while oecircFNTA Mut cannot. Scale bar, 100 μm.(L) Tramsfection with oecircFNTA WT could increase J82 cells cisplatin chemo-resistance (2 μg/ml), while oecircFNTA Mut did not have this effect.(N) Luciferase reporter activity after transfection of WT and Mut FNTA 3'UTR reporter constructs into UMUC3 cells (left panel), comparing transduced miR-370-3p inhibitor vs Ctrl and J82 cells (right panel), and comparing oemiR-370-3p vs control PLV.
Figure 5(A) Western blots show the knock-down efficiency of shFNTA in J82 and UMUC3 cells.(B) Transwell assays indicated that shFNTA can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Quantifications are shown on the right. Scale bar, 100 μm.(C) MTT assays also confirmed that shFNTA can reverse oecircFNTA-increased chemo-resistance to cisplatin (2 μg/ml) in J82 and UMUC3 cells.Transwell assays (D, with quantitations at the right) indicated that treatment with Tipifarnib (farnesyltransferase inhibitor) can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Scale bar, 100 μm.MTT assays (E) indicated that treatment with Tipifarnib (farnesyltransferase inhibitor) can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Scale bar, 100 μm.(F) Western blots showed that shcircFNTA in UMUC3 or oecircFNTA in J82 cells did not change KRAS protein level significantly.(G) Raf-1 RBD GST-pull-down assays confirmed that shcircFNTA can decease KRAS activity in UMUC3 cells (upper panel), and oecircFNTA can increase KRAS activity in J82 cells (lower panel).(H) Western blots showed that shcircFNTA can decrease ERK1/2 and MEK1/2 phosphorylation in UMUC3 cells (left panel), and oecircFNTA can increase their phosphorylation in J82 cells (right panel).