Fig 2: EMC Is Required for Accurate TMD1 Topogenesis of ß1AR(A) 35S-methionine labeled ß1AR-TMD1 (shown in the diagram) was translated in the absence or presence of WT or ?EMC6 (?) hRMs, subjected to PK digestion as indicated, and the products recovered by either immunoprecipitation via the N-terminal HA tag (N-term. IPs) or pull-downs via the C-terminal His6 tag (C-term. pull-downs). The positions of unmodified full-length (FL) product, glycosylated product (+glyc), and N- and C-terminal protease-protected fragments (N-PF and C-PF, respectively) are indicated.(B) 35S-methionine labeled ribosome-nascent chains (stalled 39 residues downstream of the indicated TMDs) produced in reticulocyte lysate were affinity purified via an N-terminal FLAG epitope tag and analyzed by autoradiography to detect the nascent chains or immunoblotting for ribosomal proteins (RPL8 and RPS24) and SRP54. Controls either lacked an epitope tag, TMD, or mRNA.(C) 35S-methionine labeled 116-residue nascent chains of ß1AR were targeted to WT or ?EMC6 hRMs and analyzed by the PK protection assay. The diagram indicates which species are glycosylated and PK-resistant versus non-glycosylated and PK-accessible.(D) 35S-methionine labeled ß1AR nascent chains of the indicated lengths were targeted to WT or ?EMC6 hRMs (top panel), then subjected to sulfhydryl-mediated crosslinking. The crosslinked products were immunoprecipitated using antibodies against Sec61ß and shown in the bottom panel. Controls lacking either mRNA (mock) or a cysteine in the nascent chain showed no Sec61ß immunoprecipitated products.See also Figure S4.

Fig 3: The shaft of the growing axon is scarcely populated by ribosomes.(A) Examples of ribosomes observed in cryo-tomograms of axon shafts from ALI-COs, of other cellular processes from ALI-COs, and of HeLa cells. The bottom panel shows 0.05 µm3 cryo-ET volumes, corresponding to the area shown in the upper panel. Positions of all ribosome-like particles observed in that volume are shown as orange spheres. (B) Comparison of the numbers of ribosome-like particles, normalized to the tomographic volume, observed in axon shafts, other processes and HeLa cells. Individual data points represent individual cryo-tomograms (30, 4, and 5 tomograms, respectively). Mann-Whitney tests were employed for statistical analysis: p < 0.0001 (****); p < 0.05(*). (C) Immunofluorescence images of dissociated neurons from organoids reveal low signal for the ribosomal 60 S component RPL8 in axon processes (identified by SMI312+/MAP2- labeling) in comparison to dendrites (identified by SMI312-/MAP2+ labeling). The yellow box outlines the area magnified in the right panel. The top image of the right panel shows the immunofluorescence signal for the ribosomal subunit RPL8. The bottom image shows the SMI312/MAP2/RPL8 composite. The white dashed line depicts the outline of axons and dendrites and was traced based on the MAP2 and SMI312 signal. The image shown is representative of the data used for quantifications shown in D. (D) Quantification of immunofluorescence images of ALI-CO derived dissociated neurons labeled for five distinct ribosomal proteins. The bars report the mean pixel grey value along axons and dendrites (mean ± SD). Each data point represents a different axon or dendrite. With the exception of quantifications done on the ribosomal protein S6, axons were identified as SMI312+/MAP2- neuronal processes, while dendrites were identified as SMI312-/MAP2+ neuronal processes. Due to antibody incompatibility, in the case of S6, dendrites were identified as MAP2+ processes while axons were identified as GFP+/MAP2- processes. The data pertains to one biological replicate. Mann-Whitney tests were employed for statistical analysis: RPL8, N = 10, p < 0.0001 (****); RPS10, N = 12, p < 0.0001 (****); RPS16, N = 12, p < 0.0001 (****); RPS26, N = 12, p < 0.0001 (****); S6, N = 12, p = 0.0001 (***). Scale bars: 50 nm in A., 20 µm and 2 µm in C.

Fig 4: Evidence for a direct cGAS-ribosome interaction(A) Cytosol from U2OS cells was separated by sucrose gradient sedimentation, and fractions were immunoblotted for cGAS and representative ribosome subunits (ul2 and eS24). Asterisks denote non-specific bands.(B) Purified ribosomes were incubated with Ni-NTA agarose, Ni-NTA agarose with immobilized human recombinant cGAS-8his, or hPrimpol1-8his (control). After washing, the eluate was analyzed by SDS-PAGE and Coomassie staining.(C) Western blot analysis of cGAS, ribosomes (both untreated and DNase-treated ribosomes), or cGAS-ribosome complex, separated by sucrose gradient sedimentation.See also Figure S5.

Fig 5: TAF7 localizes in both the nucleus and the cytoplasm; cytoplasmic TAF7 is associated with RNA polysomes.(A) Immunofluorescence of TAF7 with anti-TAF7 (red) and DAPI (nuclear stain, blue) in HeLa cells. Scale bars, 10 µm. (B) Anti-TAF7 immunoblotting in HeLa nuclear and cytoplasmic fractions. Nuclear TBP and cytoplasmic ß-tubulin assessed purity of respective fractions. (C) Enrichment of cytoplasmic TAF7 in HeLa cells as described in Materials and Methods. Error bars, means ± SD from three independent experiments. (D) FPLC column fractionation of HeLa cytoplasmic TAF7. HeLa TAF7 (WT) cytoplasmic extracts were subjected to Superose 6 gel-filtration chromatography; relative abundance of TAF7 and TBP in each fraction was calculated from results of immunoblotting with anti-TAF7 and anti-TBP. PC: recombinant TAF7 or TBP. (E) Proximity ligation assays (PLA) of TAF7 with RPL5 and RPL8. PLA associations, red spots; nuclear DAPI stain, blue. Scale bars, 10 µm. (F) Quantitation of PLA associations. PLA spots per cell were calculated on 400 to 500 cells. Data are means ± SD, **P < 0.01. (G) Polysome fractionation of HeLa TAF7 (WT) cytoplasmic extracts. Cytoplasmic extracts, treated with RNase inhibitor, were subjected to sucrose gradient centrifugation; fractions were analyzed by immunoblotting with anti-TAF7 and anti-RPL5 (bottom). (H) TAF7 binds RNA in vitro. Recombinant TAF7 was incubated with HeLaS3 total RNA and immunoprecipitated with anti-TAF7. BSA was the control. Precipitated RNAs were 32P 3' end-labeled and resolved in RNA gels. (I) TAF7 binds RNA in cellulo. HeLa TAF7(WT) extracts were immunoprecipitated with anti-TAF7 or control mouse IgG. Precipitated RNA was 3' end-labeled and resolved in RNA gels. Bottom, TAF7 recovery in the IP. (J) Truncation of TAF7 at amino acid 129 abrogates RNA binding in cellulo. Extracts from HeLaS3 cells expressing empty vector, TAF7 (WT), or TAF7 (1 to 129) were immunoprecipitated with anti-FLAG. TAF7-bound RNAs were 3' end-labeled and examined in RNA gels. Arrow, nonspecific band. Bottom, FLAG-TAF7 recovery.