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. 2018 Aug 16;46(17):8993–9010. doi: 10.1093/nar/gky733

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 3. — Tethering of 4EIP to an mRNA suppresses expression. (A) Results for 4EIP from the published tethering screen (67). The trypanosomes used inducibly expressed an mRNA encoding the glycolytic enzyme PGKB, which is toxic in bloodstream forms (86). They were transfected with a library for inducible expression of lambdaN fusion proteins created from randomly sheared trypanosome DNA. After induction of both PGKB-boxB and the lamdaN fusions, cells were grown and the polymerase chain reaction products from the integrated lambdaN fusion proteins were sequenced (67). Results from three independent selections (A, B and C) are shown using different symbols. Counts that are higher in the presence of the inducer, tetracycline (magenta symbols), than without tetracycline (cyan symbols) indicate suppression of PGKB expression. Cartoons of 4EIP showing the two conserved regions (cyan and green) and the PQ-rich C-terminal region are below the x-axis. The two downward arrowheads indicate the cleavage positions that would generate the tagged proteins that were observed by western blotting in panel (B) and Figure 1F. (B) Expression of lambdaN…myc fusion proteins for the experiments in panels (C) and (E). The full-length lambdaN-4EIP (upper panel, arrow) was always found to be degraded; the two C-terminal pieces (30 and 20 kDa) will lack the lambda-N portion, so will be inactive in the assay. (C) Tethering assay using CAT reporter. Bloodstream-form trypanosomes expressing a CAT mRNA with five copies of boxB in the 3′-untranslated region (3′-UTR) were used; cells in which the CAT mRNA lacked boxB served as control. In the left-hand panel, cells were transfected with plasmids designed for tetracycline-inducible expression of either LambdaN-4EIP-myc or LambdaN-4EIP-myc with an N-terminal deletion that eliminated the interaction with eIF4E1 (ΔN4EIP). CAT activity was measured in the presence and absence of tetracycline. Results are expressed as arithmetic mean ± standard deviation of at least three measurements. Right-hand panel (with yellow bars in online version) — cells lacking 4EIP were used to test the activity of tethered EIF4E1, with PUF3 as a control. For the EIF4E1, tests were done six times. Results of Student’s t-tests were *P = 0.04; ^〈?P = 0.07; **P = 0.009. Expression of myc-tagged proteins is shown on the right. (D) Time course of the tethering effect in bloodstream forms, using either 4EIP or its C-terminal domain. Results are mean and standard deviation of three measurements. (E) Effect on the CAT reporter mRNA of tethering EIF4E1 in the presence or absence of 4EIP. Results are mean and standard deviation of three measurements. In the paired bars, mRNA measurements are on the left and CAT activity on the right.