Fig 2: Hypothesis of CDR2L and CDR2 involvement in protein synthesis in Purkinje neurons. CDR2 localizes to the nucleus and directly interacts with nuclear speckle protein eIF4A3. eIF4A3, in conjugation with other cytoplasmic initiation factors, facilitates mRNA binding to the 40S ribosomal subunit. This event is important for mRNA maturation and translation, ultimately resulting in the synthesis of new proteins. CDR2L interacts with ribosomal subunit protein rpS6; therefore, we propose that CDR2L and CDR2 are both involved in the process of protein synthesis. Furthermore, Yo antibody (green) binding to CDR2L in Purkinje neurons of PCD patients may, therefore, interfere with the function of the ribosomal machinery, resulting in disrupted mRNA translation and/or protein synthesis.

Fig 3: TRAP1 associates with the mitochondrial protein import machinery and favors localized translation. (A) Representative images of PLA positivity between TRAP1 and TOM40. Positive signals of interaction are shown as red dots, nuclei are stained with DAPI (blue), mitochondria are marked by the mitochondria-directed YFP (green). Scale bar: 10 µm. The graph shows the average number of PLA spots/cell, with a two-tailed p-value representing the statistical significance based on the Student’s t-test (n=5). (B) Immunoprecipitation of unfused GFP and TRAP1-GFP performed in HeLa cells following 24 hrs induction of GFP and TRAP1-GFP. Total lysates were incubated with GFP-trap beads to isolate the proteins and the resulting samples were immunoblotted with indicated antibodies. (C) Representative image of PLA showing the interaction of TRAP1 with EF-TuMT in HCT116 cells. Positive signals of interaction are shown as red dots, nuclei are stained with DAPI (blue). Negative control has been obtained by hybridizing cells with TRAP1 antibody only. (D-E) Immunoprecipitation of unfused GFP and TRAP1-GFP performed in HeLa cells following 24 hrs induction of GFP and TRAP1-GFP. Where indicated, cells were treated for 15 min with emetine (100 µg /mL) or harringtonine (2 µg/mL), or for 1 hr with chloramphenicol (200 µg /mL) or Linezolid (30 µM). Total lysates were incubated with GFP-trap beads to isolate the proteins and the resulting samples were immunoblotted with indicated antibodies. (F) Representative image of PLA showing the interaction of TOM20 with phosphorylated (active) ribosomal protein eS6 in HeLa cells following 24hrs induction of TRAP1-GFP or unfused GFP. Positive signals of interaction are shown as red dots, nuclei are stained with DAPI (blue). Scale bar: 10 µm. The graph shows the average number of PLA spots/cell, with a p-value representing the statistical significance based on the Student’s t-test (n=3). (G) Cytosolic, MAM, ER, fractions isolated from HCT116 cells probed with the indicated antibodies.

Fig 4: Proteomic analysis of VLPs.(A) The composition of MLV Gag induced VLPs. (B) Correlation of protein abundances (LFQ intensity) between VLPs carrying MAGEA4 antigen with VLPs without recombinant antigen (MLV Gag). Pearson correlation coefficient is shown on the blot. (C) Differently expressed proteins in MLV Gag VLPs and VLPs carrying melanoma antigens. (D) Comparison of LFQ intensities of VLPs carrying different cancer antigens. (E) Western blot analysis of VLPs using antibodies against MLV Gag and cellular proteins α-tubulin, Rps6, Pabp, histone H3 and Mcm6. The equal amount of total protein (100 ng for MLV Gag and 2 μg for others) was loaded on each lane.

Fig 5: Poly-GR mainly co-localizes with nucleolar ribosomes.(A) HEK293 cells were co-transfected with GFP, GFP-(GR)149, or (PR)175-GFP expression vectors and RFP-STAU1 as in Fig 3A and endogenous RPS6 detected by immunofluorescence. Note that the large cytoplasmic poly-GR/PR granules induced by STAU1 expression are not clearly stained with the ribosomal subunit compared with the cytoplasm. Merge shows additionally DAPI in gray. Single confocal planes are shown. Scale bar denotes 20 µm. (B) Frontal cortex of a C9orf72 patient and a control brain were co-stained for poly-GR and ribosomal proteins as in Fig 5A. RPS25 and RPL36A are partially sequestered into poly-GR inclusions. DAPI visualizes nuclei. Single confocal planes are shown. Scale bar depicts 20 µm. (C) Quantification of GR aggregates co-localizing with ribosomal proteins (n = 3 cortical sections with 100 GR aggregates counted each from two different patients; mean ± SEM is shown).