Fig 1: ZNF598 Engages a Sub-population of Poly(A)-Stalled Ribosomes In Vitro(A) Strategy to analyze poly(A)-stalled ribosome-nascent chain complexes produced by in vitro translation (IVT). VHP is a small autonomously folding three-helix bundle from the villin head piece.(B) Poly(A)-stalled translation complexes were affinity purified via the nascent chain and separated by sucrose gradient sedimentation. The affinity-purified products (input) and each gradient fraction were analyzed by autoradiography (to detect nascent chains) or immunoblotting for recombinant ZNF598 or ribosomal proteins uL2 and eS24. Mono- and di-ribosome fractions are indicated.(C) Poly(A)-stalled translation complexes from reactions lacking or containing 5 nM FLAG-tagged ZNF598 were immunopurified via the FLAG tag, and the nascent chains were detected by autoradiography. 1° and 2° indicate the position of nascent chains from the first and second ribosome of the stalled complexes (see diagram).(D) The input and affinity-purified samples prepared as in (C) were separated by sucrose gradient and the nascent chains detected by autoradiography. The graph below the autoradiograms depicts the distribution of nascent chains corresponding to mono-ribosomes (1°, black) or di-ribosomes (2°, red) in the input sample or the nascent chains recovered by affinity purification (”IP ZNF598” - blue).See also Figure S1.
Fig 2: EMC Is Required for Accurate TMD1 Topogenesis of β1AR(A) 35S-methionine labeled β1AR-TMD1 (shown in the diagram) was translated in the absence or presence of WT or ΔEMC6 (Δ) 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 β1AR were targeted to WT or ΔEMC6 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 β1AR nascent chains of the indicated lengths were targeted to WT or ΔEMC6 hRMs (top panel), then subjected to sulfhydryl-mediated crosslinking. The crosslinked products were immunoprecipitated using antibodies against Sec61β and shown in the bottom panel. Controls lacking either mRNA (mock) or a cysteine in the nascent chain showed no Sec61β immunoprecipitated products.See also Figure S4.
Fig 3: The Ribosome Queue at a Site of Ribosome Stalling Is Relieved by ASCC(A) Elution fractions of purified recombinant human ASCC from insect cells.(B) Schematic of collided polysome production and analysis.(C) Sucrose gradient fractions from rabbit reticulocyte lysate (RRL) analyzed directly (no IVT) or after 45 min of in vitro translation (IVT) without or with 0.8 µM eRF1AAQ. The migration of ribosomes was detected using anti-eS24 (small subunit) and anti-uL2 (large subunit). The background products seen in fractions 1 and 2 in many blots is due to a large amount of hemoglobin from RRL.(D and E) 45-min translation reactions containing the indicated recombinant proteins were analyzed by sucrose gradient fractionation and immunoblotting for eS24. The following proteins were used: 0.8 µM eRF1AAQ, 75 nM ZNF598, and 50 nM ASCC or 50 nM ASCCAA lacking helicase activity.(F) 45-min translation reactions containing 0.8 µM eRF1AAQ and 75 nM ZNF598 were subsequently incubated for 30 min without or with 12.5 U/mL apyrase (to deplete ATP), then supplemented with 50 nM ASCC for another 30 min before sucrose gradient analysis.(G) 15-min translation reactions containing 0.8 µM eRF1AAQ without or with 330 µM E1 inhibitor PYR-41 were supplemented with ZNF598 and ASCC and continued for another 30 min followed by sucrose gradient analysis.See also Figure S2.
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: Subcellular localization of Pdcd4 and translational control.A Immunofluorescence of HEK293T cells shows that the Pdcd4 (green) is mainly colocalized in nucleus (blue) under normal growth, which migrates to cytoplasm under nutrient deprivation, resulting in a lower polysome/monosome ratio. The scale bar is 25 μm. Error bars represent the mean ± SEM (n = 3). B Sucrose gradient fractionation showing an enrichment of Pdcd4 comigrating with the ribosomal complexes under glucose starvation. The western blot indicated that Pdcd4 co-migrates mainly with the 40S (ribosomal protein eS24) and 80S ribosome (ribosomal proteins eS24 and uL2). The experiment was repeated at least three times with similar results.
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