FIG 2.

Ctk1 stimulates translation initiation. (A) Firefly luciferase (F-luc) reporter constructs used to assess initiation-independent translation contain a nonphysiological ApppG cap followed by an inhibitory stem-loop and an IRES. IRESs used correspond to the CrPV IRES or yeast IRESs of the GPR1 or NCE102 gene. A corresponding reporter that carried an m⁷G cap and lacked the inhibitory stem-loop as well as the IRES was used as a control for canonical translation. (B) Loss of Ctk1 causes decreased translation activity of capped mRNA, but not of CrPV IRES-containing mRNA. Translation activity of translation active extracts from mock- and Ctk1-depleted cells for the reporters shown in panel A was analyzed. For each RNA construct, the activity of the mock-depleted extracts was set at 100%. The values for mock-depleted and Ctk1-depleted cells are significantly different (P < 0.05 by Student's t test) as indicated by the brackets and asterisks. (C) Absolute values of luciferase activity of mock- and Ctk1-depleted extracts for the three IRES-containing reporters. The error bars represent the standard deviations from at least three independent experiments. Luciferase activity is shown in arbitrary units (AU). (D and E) Initiation is slightly but significantly impaired in Ctk1-depleted cells in vivo. The 80S peak in polysome profiles of Ctk1-depleted cells (E) is increased compared to mock-depleted cells (D), reflecting a translation initiation defect. The polysome/monosome (80S) (P/M) ratio was calculated by integrating the area under the respective peaks after subtraction of the values of the baseline. Values are means ± SEM from three independent experiments. Changes in the P/M ratio of Ctk1 compared to mock-depleted cells are statistically significant (P < 0.05 by Student's t test).