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. 2023 Mar 13;51(7):3452–3464. doi: 10.1093/nar/gkad151

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© The Author(s) 2023. 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 (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Ribosomes redistribution. (A) Predicted steady-state concentration levels of the free ribosomes varying the miRNA binding constant in different design conditions. (B) Predicted steady-state ribosomal densities (Materials and Methods) for both the miTarget and capacity monitor genes varying the miRNA binding constant in different design conditions. Design conditions include different location and number of the miRNA target sites within the UTRs of the miTarget gene. The miRNA binding constant is considered as an independent variable and thus is not set to a fixed value. Instead, the values considered for span a range of reasonable characteristic values. An increase in the miRNA binding constant is correlated to an increase in the pool of free ribosomes and the ribosomal densities, and supports the hypothesis of ribosomes reallocation upon miRNA-driven regulation. (C, D) Relative distribution of transcripts and co-sedimentation analysis in polysomes profiles in H1299 cells transfected with a bidirectional promoter plasmid encoding for EGFP (capacity monitor) and mKate (miTarget) with miR-31 TS either at the 5’ or 3’ UTR. As control, we used the same plasmid lacking the miR-31 target sites. We analysed both synthetic (C) and endogenous (D) genes. Polysome profiles were obtained 48 hours post transfection. Means of the relative percentage of transcript sedimentation along the profile ± SE. SE: standard errors are shown. N = 3 biological replicates. Unpaired two-sided t-test. P-value: * < 0.05, ** < 0.005.

Inline graphic — Ribosomes redistribution. (A) Predicted steady-state concentration levels of the free ribosomes varying the miRNA binding constant in different design conditions. (B) Predicted steady-state ribosomal densities (Materials and Methods) for both the miTarget and capacity monitor genes varying the miRNA binding constant in different design conditions. Design conditions include different location and number of the miRNA target sites within the UTRs of the miTarget gene. The miRNA binding constant is considered as an independent variable and thus is not set to a fixed value. Instead, the values considered for span a range of reasonable characteristic values. An increase in the miRNA binding constant is correlated to an increase in the pool of free ribosomes and the ribosomal densities, and supports the hypothesis of ribosomes reallocation upon miRNA-driven regulation. (C, D) Relative distribution of transcripts and co-sedimentation analysis in polysomes profiles in H1299 cells transfected with a bidirectional promoter plasmid encoding for EGFP (capacity monitor) and mKate (miTarget) with miR-31 TS either at the 5’ or 3’ UTR. As control, we used the same plasmid lacking the miR-31 target sites. We analysed both synthetic (C) and endogenous (D) genes. Polysome profiles were obtained 48 hours post transfection. Means of the relative percentage of transcript sedimentation along the profile ± SE. SE: standard errors are shown. N = 3 biological replicates. Unpaired two-sided t-test. P-value: * < 0.05, ** < 0.005.