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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

PTPB1 protects reporter mRNAs from NMD.Decay assays of pcTET2 -βwt-RSE-ΔPTB-SMG5 in HEK293 Tet-off cells treated with non-targeting or anti-UPF1 siRNAs. The reporter constructs (upper band) were co-transfected with the constitutively expressed wild-type β-globin reporter (pcβwtβ; bottom bands) into cells 24 hrs after the siRNAs were introduced to the cells. Levels of tet-regulated reporter mRNAs were normalized to levels of the wild-type β-globin transfection control. Relative remaining RNA levels at indicated time points from three independent experiments were used to calculate half-lives and 95% confidence intervals (***p<0.001 in two-tailed ANCOVA analysis, compared to the half-life of βwt-RSE-ΔPTB-SMG5 in cells treated with non-targeting siRNAs).DOI:http://dx.doi.org/10.7554/eLife.11155.013
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fig4s1: PTPB1 protects reporter mRNAs from NMD.Decay assays of pcTET2 -βwt-RSE-ΔPTB-SMG5 in HEK293 Tet-off cells treated with non-targeting or anti-UPF1 siRNAs. The reporter constructs (upper band) were co-transfected with the constitutively expressed wild-type β-globin reporter (pcβwtβ; bottom bands) into cells 24 hrs after the siRNAs were introduced to the cells. Levels of tet-regulated reporter mRNAs were normalized to levels of the wild-type β-globin transfection control. Relative remaining RNA levels at indicated time points from three independent experiments were used to calculate half-lives and 95% confidence intervals (***p<0.001 in two-tailed ANCOVA analysis, compared to the half-life of βwt-RSE-ΔPTB-SMG5 in cells treated with non-targeting siRNAs).DOI:http://dx.doi.org/10.7554/eLife.11155.013

Mentions: (A) Schematic of unspliced RSV RNA expression constructs used for RNA accumulation assays in chicken fibroblasts. (B) RNase protection assays of RSV unspliced viral RNA containing the wt RSE or RSE variants. Experimental and control constructs were co-transfected into CEFs, and total cellular RNAs were harvested 43–48 hr post-transfection. Top band: protected fragment of the probe corresponding to the unspliced viral RNAs from the experimental constructs. Bottom band: stable viral loading control that protects a different sized fragment of the same probe due to a small in-frame deletion. (C) Quantification of RNase protection assays. Levels of the experimental unspliced RSV RNAs (top band) were normalized to levels of the transfection control (bottom band). RNA levels are reported as a fraction of RSV RNA containing wt RSE. Error bars indicate ± SD; n ≥ 5 (*p<0.05; ****p<0.0001 in two-tailed Student’s t-tests when compared to RSE constructs). (D) Schematic of reporter mRNAs containing the β-globin gene, RSE variants (RSE, RSE-ΔPTB, or RSE-ΔPTB+6xPTBBS), and the full-length human SMG5 3‘UTR. (E) Decay assays of the indicated reporter mRNAs. Tet-regulated transcripts (upper bands) were co-transfected with the constitutively expressed wild-type β globin reporter (pcβwtβ; bottom bands) in HeLa Tet-off cells. Levels of tet-regulated reporter mRNAs 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-values from two-tailed ANCOVA analyses when compared to pcTET2-βwt-SMG5). See also Figure 4—figure supplement 1.


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)

PTPB1 protects reporter mRNAs from NMD.Decay assays of pcTET2 -βwt-RSE-ΔPTB-SMG5 in HEK293 Tet-off cells treated with non-targeting or anti-UPF1 siRNAs. The reporter constructs (upper band) were co-transfected with the constitutively expressed wild-type β-globin reporter (pcβwtβ; bottom bands) into cells 24 hrs after the siRNAs were introduced to the cells. Levels of tet-regulated reporter mRNAs were normalized to levels of the wild-type β-globin transfection control. Relative remaining RNA levels at indicated time points from three independent experiments were used to calculate half-lives and 95% confidence intervals (***p<0.001 in two-tailed ANCOVA analysis, compared to the half-life of βwt-RSE-ΔPTB-SMG5 in cells treated with non-targeting siRNAs).DOI:http://dx.doi.org/10.7554/eLife.11155.013
© Copyright Policy
Related In: Results  -  Collection

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

fig4s1: PTPB1 protects reporter mRNAs from NMD.Decay assays of pcTET2 -βwt-RSE-ΔPTB-SMG5 in HEK293 Tet-off cells treated with non-targeting or anti-UPF1 siRNAs. The reporter constructs (upper band) were co-transfected with the constitutively expressed wild-type β-globin reporter (pcβwtβ; bottom bands) into cells 24 hrs after the siRNAs were introduced to the cells. Levels of tet-regulated reporter mRNAs were normalized to levels of the wild-type β-globin transfection control. Relative remaining RNA levels at indicated time points from three independent experiments were used to calculate half-lives and 95% confidence intervals (***p<0.001 in two-tailed ANCOVA analysis, compared to the half-life of βwt-RSE-ΔPTB-SMG5 in cells treated with non-targeting siRNAs).DOI:http://dx.doi.org/10.7554/eLife.11155.013
Mentions: (A) Schematic of unspliced RSV RNA expression constructs used for RNA accumulation assays in chicken fibroblasts. (B) RNase protection assays of RSV unspliced viral RNA containing the wt RSE or RSE variants. Experimental and control constructs were co-transfected into CEFs, and total cellular RNAs were harvested 43–48 hr post-transfection. Top band: protected fragment of the probe corresponding to the unspliced viral RNAs from the experimental constructs. Bottom band: stable viral loading control that protects a different sized fragment of the same probe due to a small in-frame deletion. (C) Quantification of RNase protection assays. Levels of the experimental unspliced RSV RNAs (top band) were normalized to levels of the transfection control (bottom band). RNA levels are reported as a fraction of RSV RNA containing wt RSE. Error bars indicate ± SD; n ≥ 5 (*p<0.05; ****p<0.0001 in two-tailed Student’s t-tests when compared to RSE constructs). (D) Schematic of reporter mRNAs containing the β-globin gene, RSE variants (RSE, RSE-ΔPTB, or RSE-ΔPTB+6xPTBBS), and the full-length human SMG5 3‘UTR. (E) Decay assays of the indicated reporter mRNAs. Tet-regulated transcripts (upper bands) were co-transfected with the constitutively expressed wild-type β globin reporter (pcβwtβ; bottom bands) in HeLa Tet-off cells. Levels of tet-regulated reporter mRNAs 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-values from two-tailed ANCOVA analyses when compared to pcTET2-βwt-SMG5). See also Figure 4—figure supplement 1.

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