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. 2017 Dec 13;46(3):1457–1469. doi: 10.1093/nar/gkx1237

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© The Author(s) 2017. 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 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 5. — LARP1^497–1019 represses TOP mRNAs through combined recognition of the TOP sequence and adjacent cap structure. (A) The translation of TOP mRNAs is rescued by competition with a TOP motif mimic. A total of 0.5 ng of TOP or non-TOP (nTOP) synthetic mRNAs were translated in extracts from Torin 1-treated cells in the presence of the indicated amounts of a capped pyrimidine RNA competitor and analyzed by luciferase assay. (n = 3, error bars are SD). (B) Rescue of TOP mRNA translation requires a capped TOP competitor. A total of 0.5 ng of synthetic TOP mRNA was translated in extracts from Torin 1-treated cells in the presence of 200 nM of RNA oligonucleotides with the indicated sequences and 5′ modifications and analyzed by luciferase assay. (n = 3, error bars are SD). (C) Purified LARP1^497-1019 selectively binds capped oligopyrimidine RNAs. A total of 100 nM purified LARP1^497–1019 was incubated with 5 nM of the indicated 6-FAM-labeled RNAs and analyzed by EMSA. (D) LARP1^497–1019 selectively represses the translation of TOP mRNAs in vitro. A total of 0.5 ng TOP or non-TOP (nTOP) synthetic mRNAs were translated in control-treated extracts in the presence of the indicated concentrations of LARP1^497–1019 purified from normally growing cells. Reactions were subsequently analyzed by luciferase assay. Values were plotted relative to control reactions with no LARP1^497–1019 added. Significance was calculated by t-test. (n = 3, error bars are SD).