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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 containing a GAPDH-derived NMD-sensitive 3’UTR.(A) Schematic of tet-regulated β-globin reporter mRNA constructs used in RNA decay assays. RSE or SMG5-397 sequences were inserted in reporter mRNAs upstream of the artificial GAPDH-derived 3’UTR (Singh et al., 2008). (B) Decay assays of reporter mRNAs containing the RSE or the first 397 nt of the SMG5 3’UTR (SMG5-397) sequence in cells expressing either WT UPF1 (left) or an ATPase-dead UPF1 (right). Constructs encoding the tet-regulated transcripts described in (A) were co-transfected with the constitutively expressed wild-type β-globin reporter (pcβWTβ; bottom bands) 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 indicated mRNAs to the SMG5-397 control in the presence of wild-type UPF1).DOI:http://dx.doi.org/10.7554/eLife.11155.007
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fig2s1: The RSE protects reporter mRNAs containing a GAPDH-derived NMD-sensitive 3’UTR.(A) Schematic of tet-regulated β-globin reporter mRNA constructs used in RNA decay assays. RSE or SMG5-397 sequences were inserted in reporter mRNAs upstream of the artificial GAPDH-derived 3’UTR (Singh et al., 2008). (B) Decay assays of reporter mRNAs containing the RSE or the first 397 nt of the SMG5 3’UTR (SMG5-397) sequence in cells expressing either WT UPF1 (left) or an ATPase-dead UPF1 (right). Constructs encoding the tet-regulated transcripts described in (A) were co-transfected with the constitutively expressed wild-type β-globin reporter (pcβWTβ; bottom bands) 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 indicated mRNAs to the SMG5-397 control in the presence of wild-type UPF1).DOI:http://dx.doi.org/10.7554/eLife.11155.007

Mentions: Since UPF1 recruitment is a prerequisite for NMD, we hypothesized that the RSE may employ the simple strategy of preventing UPF1 from associating with the 3’UTR of the protected transcript. To test this idea, we immunopurified endogenous UPF1 and assayed the recovery of reporter mRNAs containing RSE or control sequences placed upstream of the artificial 3’UTR derived from the GAPDH ORF and 3’UTR, in this case lacking additional intronic sequence (Figure 2A). This GAPDH-derived 3’UTR has been previously used for studies of UPF1 association and decay and can be efficiently protected by a TC-proximal RSE (Figure 2—figure supplement 1; Hogg and Goff, 2010; Singh et al., 2008). Because UPF1 associates with mRNAs in a 3’UTR length-dependent manner (Hogg and Goff, 2010; Kurosaki and Maquat, 2013), we used an NMD-permissive 397 nt fragment of the SMG5 3’UTR (SMG5-397) to equalize 3’UTR lengths among the mRNAs studied and thus allow accurate assessment of the influence of the RSE on UPF1 binding. As additional controls, we tested the ability of UPF1 to bind mRNAs in which the RSE or the SMG5-397 fragments were moved to the 3’ end of the mRNA (3’-RSE and 3’-SMG5-397, respectively).10.7554/eLife.11155.006Figure 2.The RSE reduces UPF1 association with 3'UTRs in a position-dependent manner.


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 containing a GAPDH-derived NMD-sensitive 3’UTR.(A) Schematic of tet-regulated β-globin reporter mRNA constructs used in RNA decay assays. RSE or SMG5-397 sequences were inserted in reporter mRNAs upstream of the artificial GAPDH-derived 3’UTR (Singh et al., 2008). (B) Decay assays of reporter mRNAs containing the RSE or the first 397 nt of the SMG5 3’UTR (SMG5-397) sequence in cells expressing either WT UPF1 (left) or an ATPase-dead UPF1 (right). Constructs encoding the tet-regulated transcripts described in (A) were co-transfected with the constitutively expressed wild-type β-globin reporter (pcβWTβ; bottom bands) 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 indicated mRNAs to the SMG5-397 control in the presence of wild-type UPF1).DOI:http://dx.doi.org/10.7554/eLife.11155.007
© Copyright Policy
Related In: Results  -  Collection

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

fig2s1: The RSE protects reporter mRNAs containing a GAPDH-derived NMD-sensitive 3’UTR.(A) Schematic of tet-regulated β-globin reporter mRNA constructs used in RNA decay assays. RSE or SMG5-397 sequences were inserted in reporter mRNAs upstream of the artificial GAPDH-derived 3’UTR (Singh et al., 2008). (B) Decay assays of reporter mRNAs containing the RSE or the first 397 nt of the SMG5 3’UTR (SMG5-397) sequence in cells expressing either WT UPF1 (left) or an ATPase-dead UPF1 (right). Constructs encoding the tet-regulated transcripts described in (A) were co-transfected with the constitutively expressed wild-type β-globin reporter (pcβWTβ; bottom bands) 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 indicated mRNAs to the SMG5-397 control in the presence of wild-type UPF1).DOI:http://dx.doi.org/10.7554/eLife.11155.007
Mentions: Since UPF1 recruitment is a prerequisite for NMD, we hypothesized that the RSE may employ the simple strategy of preventing UPF1 from associating with the 3’UTR of the protected transcript. To test this idea, we immunopurified endogenous UPF1 and assayed the recovery of reporter mRNAs containing RSE or control sequences placed upstream of the artificial 3’UTR derived from the GAPDH ORF and 3’UTR, in this case lacking additional intronic sequence (Figure 2A). This GAPDH-derived 3’UTR has been previously used for studies of UPF1 association and decay and can be efficiently protected by a TC-proximal RSE (Figure 2—figure supplement 1; Hogg and Goff, 2010; Singh et al., 2008). Because UPF1 associates with mRNAs in a 3’UTR length-dependent manner (Hogg and Goff, 2010; Kurosaki and Maquat, 2013), we used an NMD-permissive 397 nt fragment of the SMG5 3’UTR (SMG5-397) to equalize 3’UTR lengths among the mRNAs studied and thus allow accurate assessment of the influence of the RSE on UPF1 binding. As additional controls, we tested the ability of UPF1 to bind mRNAs in which the RSE or the SMG5-397 fragments were moved to the 3’ end of the mRNA (3’-RSE and 3’-SMG5-397, respectively).10.7554/eLife.11155.006Figure 2.The RSE reduces UPF1 association with 3'UTRs in a position-dependent manner.

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