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In this paper, they measured de novo mitochondrial protein synthesis of the 13 mtDNA-encoded respiratory complex subunits and showed a dramatic loss of translation in the Ptcd1 knockout mice compared to controls in both tissues (Figures 3A and S1A), indicating that PTCD1 is essential for mitochondrial protein synthesis.

HeLa cells were transfected with control siRNA, DDX49 siRNA, pEGFP-C1 vector or pEGFP-DDX49 construct as indicated and the cell numbers were counted at 48, 72 and 96 hours post-transfection. The colony forming potential was analyzed in cells treated with control siRNA, DDX49 siRNA, pEGFP-C1 vector or pEGFP-DDX49 construct as indicated(Figure 5C, D). They observed a very strong reduction in colony forming ability of DDX49 depleted cells suggesting that DDX49 is essential for proper cell growth and proliferation (Figure 5C)

They assessed the stability of 47S rRNA by actinomycin D pulse chase assay and found that the siRNA mediated depletion of DDX49 substantially reduced the stability of 47S rRNA, while the overexpression of DDX49 did not show any alterations (Figure 4B). This suggests that DDX49 is required to stabilize the 47S rRNA.

They depleted Hrp48 from SL2 cells and tested the ability of exogenous SXL to inhibit the translation of a reporter containing full-length msl-2 5′ and 3′ UTRs (Figure 3A). Depletion of Hrp48 indeed reduced the capacity of SXL to inhibit translation of the reporter (Figure 3B). The results of this paper support a model where a repressor complex composed of SXL, UNR and Hrp48 assembles at the 3′ UTR of msl-2 mRNA; Hrp48 then contributes to inhibit ribosome recruitment by targeting eIF3d.(Figure 8)

Luciferase reporter mRNA is sensitive to competition with cap analog and translation in mTOR-inhibited extracts is less. The 5'end of mRNA was truncated by encoding hammerhead ribozyme upstream of 5'UTR. Primer extension assays proved ligation efficiency of 90%. Translation of TOP reporter is more repressed in mTOR inhibited extracts. This means no/less production of luciferase.

figure 1 D,E and Sup. figure 2

Depletion of eIF3d from SL2 cells reduced translation of an msl-2 reporter, while depletion of other subunits (eIF3e, eIF3h) had no effect (Figure 5DE)

ARPP21 localizes to the cytosol and overlaps staining with stress granule. ARPP21 used FLAG-tagg expression in HeLa cells and treated with arsenite Figure 2A,B