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, 6 (4), e92

Posttranscriptional Gene Regulation by Spatial Rearrangement of the 3' Untranslated Region

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Posttranscriptional Gene Regulation by Spatial Rearrangement of the 3' Untranslated Region

Andrea B Eberle et al. PLoS Biol.

Abstract

Translation termination at premature termination codons (PTCs) triggers degradation of the aberrant mRNA, but the mechanism by which a termination event is defined as premature is still unclear. Here we show that the physical distance between the termination codon and the poly(A)-binding protein PABPC1 is a crucial determinant for PTC recognition in human cells. "Normal" termination codons can trigger nonsense-mediated mRNA decay (NMD) when this distance is extended; and vice versa, NMD can be suppressed by folding the poly(A) tail into proximity of a PTC or by tethering of PABPC1 nearby a PTC, indicating an evolutionarily conserved function of PABPC1 in promoting correct translation termination and antagonizing activation of NMD. Most importantly, our results demonstrate that spatial rearrangements of the 3' untranslated region can modulate the NMD pathway and thereby provide a novel mechanism for posttranscriptional gene regulation.

Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The Distance between the TC and the Poly(A) Tail Is Crucial for NMD
(A) Relative miniμ mRNA levels, normalized to the mRNA levels of the cotransfected β-globin WT gene, were determined by RT-qPCR from miniμ constructs with PTCs at the indicated amino acid positions. PTC positions on the mRNA are plotted on the x-axis, and average values and SD of four qPCR runs are shown. The miniμ mRNA is schematically shown below the plot with the positions of the PTCs in the different constructs indicated. In all figures, 5′ and 3′ UTRs are depicted as black lines, ORFs as gray boxes, and exon-exon junctions as vertical white lines. (B) Northern blot and RT-qPCR analysis of miniμ mRNA with a 3′ UTR extension by 600 or 1,200 nucleotides. RT-qPCR data were obtained and normalized as in (A). (C) mRNA decay kinetics of the control miniμ C3/C4 WT construct and the +1,200 construct illustrated in (B) were determined in HeLa Tet-Off cells. Relative miniμ mRNAs, normalized to cotransfected β-globin, were analyzed 0, 1, 2, 4, 6, and 8 h after doxycycline addition by RT-qPCR. Average values and SD of two independent experiments with three RT-qPCR runs each are shown. (D) Effect of RNAi-mediated depletion of Upf1, Upf2, or Upf3b on the relative miniμ mRNA levels of the control and the +1,200 construct. The ratio of the normalized miniμ mRNA levels between the indicated Upf knockdown and the control knockdown (scrambled) is represented. Average and SD of three qPCR runs are shown in (B) and (D). The efficacy of the Upf1, Upf2, and Upf3b knockdown was monitored by Western blotting (lower panel). Detection of SmB/B' served as loading control.
Figure 2
Figure 2. Suppression of EJC-Independent NMD by Poly(A) Tail FB
(A) Schematic illustration of the mRNAs expressed by the indicated constructs. The 26-nucleotide sequence located 42 nucleotides downstream of codon 440 is depicted in red, and the insertion upstream of the poly(A) tail of this sequence (red) or of the complementary sequence (green) is indicated. WT = construct with full-length ORF; ter440 = construct with PTC at codon 440. (B) Half-lives of the FB mRNAs were measured as described in Figure 1C. (C) Relative miniμ mRNA levels from the EJC-independent FB constructs shown in (A) normalized to β-globin WT mRNA from a cotransfected plasmid, were measured by RT-qPCR from Upf1-depleted cells (light gray bars) or from control cells expressing a scrambled shRNA (dark gray bars). (D) The efficacy of the Upf1 knockdown was assessed by Western blotting. Detection of lamin A/C served as loading control.
Figure 3
Figure 3. The Distance between the PTC and the Poly(A) Tail Is an Important Criterion for mRNA Stability
(A) Schematic illustration of the miniμ C3/H4 ter440 FB mRNAs expressed by the indicated constructs. Complementary sequences to the regions FB, FB2, FB3, FB4, and FB5 were inserted as double-stranded oligonucleotides at the position marked by the arrow. The distance (in number of nucleotides) between the PTC (ter440) and the first base of the respective base pairing region are indicated below. (B) Half-lives of the FB mRNAs were measured as described in Figure 1C. Average values and SD from one experiment with three RT-qPCR runs are shown. (C) Half-lives of the indicated mRNAs (data from Figures 2B and 3B) were plotted against the distance between ter440 and the first base of the base-pairing region.
Figure 4
Figure 4. Suppression of EJC-Enhanced NMD by Poly(A) Tail FB
(A) Schematic illustration of the mRNAs expressed by the indicated constructs. The 26-nucleotide sequence located 28 nucleotides downstream of codon 310 is depicted in red, and the insertion upstream of the poly(A) tail of this sequence (red) or of the complementary sequence (green) is indicated. Predicted EJCs in the 3′ UTR are shown by yellow ovals. WT = construct with full-length ORF; ter310 = construct with PTC at codon 310; SL = stemloop control construct with complementary sequence inserted in exon C2. (B) Half-lives of the FB mRNAs were measured as described in Figure 1C. (C) Relative miniμ mRNA levels from FB constructs with 3′ UTR introns expressed in cells with (+) or without (−) Upf1 knockdown were measured as in Figure 2C and are shown below the histogram. The histogram depicts the fold increase of miniμ mRNA upon Upf1 knockdown. (D) The Upf1 knockdown efficacy was assessed by Western blotting as in Figure 2D. Detection of Sm B/B' served as loading control.
Figure 5
Figure 5. Tethering PABPC1 Nearby a PTC Suppresses NMD
(A and B, upper panels) Shows schematic illustration of PTC-containing reporter genes miniμ (A) and TCRβ (B). A cassette comprising six MS2 binding sites (marked in red) was inserted about 50 nucleotides downstream of a PTC in construct A or further away in construct B. (A and B, middle panels) Relative mRNA levels of construct A (dark bars) and construct B (light bars), normalized to a cotransfected rGPx-1 gene, were measured by RT-qPCR. Average mRNA levels and SD were derived from two independent experiments with two qPCR runs each. (A and B, lower panels) The expression of the fusion proteins were analyzed by immunoblotting using monoclonal mouse α-HA antibody. Detection of SmB/B' served as loading control.
Figure 6
Figure 6. Model for Posttranscriptional Gene Regulation by Spatial Rearrangement of the 3′ UTR
Proper translation termination requires a termination-promoting signal from the poly(A) tail. When the ribosome terminates close enough to the poly(A) tail to receive this signal no NMD occurs, and the mRNA remains intact. If the physical distance between the stop codon and the poly(A) tail is too large to allow transmission of the termination-promoting signal, NMD ensues, resulting in a short half-life and low steady-state level of the mRNA. The physical distance between the stop codon and the poly(A) tail depends on the 3-D structure of the 3′ UTR. The 3′ UTR structure can be reconfigured by altering (i) intramolecular base pairing, (ii) interaction of the mRNA with RNA-binding proteins, and (iii) interactions among the involved proteins through posttranslational modifications.

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