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Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway.

Ge Z, Quek BL, Beemon KL, Hogg JR - Elife (2016)

Bottom Line: When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs.PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression.Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, United States.

ABSTRACT
The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD.

No MeSH data available.


Related in: MedlinePlus

The RSE protects reporter mRNAs from EJC-stimulated NMD(A) Schematic of tet-regulated β-globin reporter mRNA constructs used in RNA decay assays. RSE or antisense RSE (AS-RSE) sequences were inserted in reporter mRNAs upstream of a previously-characterized version of the GAPDH-derived 3’UTR engineered to contain the adenovirus major-late intron (AdML intron; Singh et al., 2008), leading to EJC-stimulated NMD. (B) Decay of AdML intron-containing mRNAs in HeLa Tet-off cells. Half-lives and 95% confidence intervals were derived from linear regression of semi-log plots of normalized RNA abundances from three independent experiments (p-values from two-tailed ANCOVA analysis comparing the AS-RSE mRNAs to the RSE mRNAs).DOI:http://dx.doi.org/10.7554/eLife.11155.005
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fig1s2: The RSE protects reporter mRNAs from EJC-stimulated NMD(A) Schematic of tet-regulated β-globin reporter mRNA constructs used in RNA decay assays. RSE or antisense RSE (AS-RSE) sequences were inserted in reporter mRNAs upstream of a previously-characterized version of the GAPDH-derived 3’UTR engineered to contain the adenovirus major-late intron (AdML intron; Singh et al., 2008), leading to EJC-stimulated NMD. (B) Decay of AdML intron-containing mRNAs in HeLa Tet-off cells. Half-lives and 95% confidence intervals were derived from linear regression of semi-log plots of normalized RNA abundances from three independent experiments (p-values from two-tailed ANCOVA analysis comparing the AS-RSE mRNAs to the RSE mRNAs).DOI:http://dx.doi.org/10.7554/eLife.11155.005

Mentions: (A) Schematic of the Rous sarcoma proviral genome. The RSE is located immediately downstream of the gag stop codon. (B) Schematic of tet-regulated β-globin reporter mRNA constructs used in RNA decay assays. The RSE sequence (middle) and a control sequence, the antisense RSE (AS-RSE) sequence (bottom), were inserted into reporter mRNAs containing the β-globin gene and the human SMG5 3’UTR (top). (C) Decay assays of reporter mRNAs containing the wild-type SMG5 3’UTR or variants supplemented with RSE or AS-RSE sequences. 293 Tet-off cells were treated with non-targeting siRNA (siNT; upper panel) or UPF1 siRNA (siUPF1; lower panel). Constructs encoding the indicated tet-regulated transcripts were co-transfected with the constitutively expressed wild-type β-globin reporter (pcβwtβ; bottom bands). Remaining RNA levels at indicated time points were normalized to levels of the wild-type β-globin transfection control. Half-lives and 95% confidence intervals were obtained from 3 independent experiments (***p<0.001; ****p<0.0001 in two-tailed ANCOVA analysis when compared to pcTET2-βwt-SMG5). Rapid decay of AS-RSE mRNAs to background levels in siNT samples precluded accurate quantification of decay rate. See also Figure 1—figure supplements 1 and 2.


Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway.

Ge Z, Quek BL, Beemon KL, Hogg JR - Elife (2016)

The RSE protects reporter mRNAs from EJC-stimulated NMD(A) Schematic of tet-regulated β-globin reporter mRNA constructs used in RNA decay assays. RSE or antisense RSE (AS-RSE) sequences were inserted in reporter mRNAs upstream of a previously-characterized version of the GAPDH-derived 3’UTR engineered to contain the adenovirus major-late intron (AdML intron; Singh et al., 2008), leading to EJC-stimulated NMD. (B) Decay of AdML intron-containing mRNAs in HeLa Tet-off cells. Half-lives and 95% confidence intervals were derived from linear regression of semi-log plots of normalized RNA abundances from three independent experiments (p-values from two-tailed ANCOVA analysis comparing the AS-RSE mRNAs to the RSE mRNAs).DOI:http://dx.doi.org/10.7554/eLife.11155.005
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4764554&req=5

fig1s2: The RSE protects reporter mRNAs from EJC-stimulated NMD(A) Schematic of tet-regulated β-globin reporter mRNA constructs used in RNA decay assays. RSE or antisense RSE (AS-RSE) sequences were inserted in reporter mRNAs upstream of a previously-characterized version of the GAPDH-derived 3’UTR engineered to contain the adenovirus major-late intron (AdML intron; Singh et al., 2008), leading to EJC-stimulated NMD. (B) Decay of AdML intron-containing mRNAs in HeLa Tet-off cells. Half-lives and 95% confidence intervals were derived from linear regression of semi-log plots of normalized RNA abundances from three independent experiments (p-values from two-tailed ANCOVA analysis comparing the AS-RSE mRNAs to the RSE mRNAs).DOI:http://dx.doi.org/10.7554/eLife.11155.005
Mentions: (A) Schematic of the Rous sarcoma proviral genome. The RSE is located immediately downstream of the gag stop codon. (B) Schematic of tet-regulated β-globin reporter mRNA constructs used in RNA decay assays. The RSE sequence (middle) and a control sequence, the antisense RSE (AS-RSE) sequence (bottom), were inserted into reporter mRNAs containing the β-globin gene and the human SMG5 3’UTR (top). (C) Decay assays of reporter mRNAs containing the wild-type SMG5 3’UTR or variants supplemented with RSE or AS-RSE sequences. 293 Tet-off cells were treated with non-targeting siRNA (siNT; upper panel) or UPF1 siRNA (siUPF1; lower panel). Constructs encoding the indicated tet-regulated transcripts were co-transfected with the constitutively expressed wild-type β-globin reporter (pcβwtβ; bottom bands). Remaining RNA levels at indicated time points were normalized to levels of the wild-type β-globin transfection control. Half-lives and 95% confidence intervals were obtained from 3 independent experiments (***p<0.001; ****p<0.0001 in two-tailed ANCOVA analysis when compared to pcTET2-βwt-SMG5). Rapid decay of AS-RSE mRNAs to background levels in siNT samples precluded accurate quantification of decay rate. See also Figure 1—figure supplements 1 and 2.

Bottom Line: When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs.PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression.Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, United States.

ABSTRACT
The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD.

No MeSH data available.


Related in: MedlinePlus