Limits...
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

RSE inhibition of UPF1 binding is independent of translation.(A) Schematic of GFP reporter mRNA constructs used in immunoprecipitation assays. RNAs containing the GFP ORF and an artificial NMD-inducing 3’UTR comprising a portion of the human GAPDH ORF and the GAPDH 3’UTR (Singh et al., 2008) were modified to contain the RSE (top) or the SMG5-397 sequence (middle). As a control to illustrate the behavior of mRNAs with short 3’UTRs (untreated condition) or shorter overall length (puromycin treatment), we used constructs without added RSE or SMG5-397 sequence in which the GFP termination codon was changed to CAA (bottom), resulting in termination at the downstream GAPDH stop codon. (B) UPF1 association is reduced on 3'UTRs containing the RSE. Plasmids expressing the indicated mRNAs were co-transfected in 293 cells with a construct expressing the GFP ORF followed by the bovine growth hormone (bGH) polyadenylation signal. Endogenous Upf1 was immunoprecipitated from transfected cells grown in the absence or presence of puromycin (100 μg/mL for 3 hr) as indicated, and co-purifying mRNAs were analyzed by northern blot. (C) Quantification of relative RNA recovery upon UPF1 immunoprecipitation, normalized to the recovery of co-transfected GFP-bGH mRNAs. The amount of RSE-containing RNA recovered was set to 1. Error bars indicate ± SD; n ≥ 3 (*p<0.05, **p<0.01 in two-tailed Student’s t-tests when compared to RSE recovery). Under both untreated and puromycin treated conditions, the RSE caused reduced accumulation of UPF1 on mRNAs relative to the SMG5-397 control.DOI:http://dx.doi.org/10.7554/eLife.11155.008
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4764554&req=5

fig2s2: RSE inhibition of UPF1 binding is independent of translation.(A) Schematic of GFP reporter mRNA constructs used in immunoprecipitation assays. RNAs containing the GFP ORF and an artificial NMD-inducing 3’UTR comprising a portion of the human GAPDH ORF and the GAPDH 3’UTR (Singh et al., 2008) were modified to contain the RSE (top) or the SMG5-397 sequence (middle). As a control to illustrate the behavior of mRNAs with short 3’UTRs (untreated condition) or shorter overall length (puromycin treatment), we used constructs without added RSE or SMG5-397 sequence in which the GFP termination codon was changed to CAA (bottom), resulting in termination at the downstream GAPDH stop codon. (B) UPF1 association is reduced on 3'UTRs containing the RSE. Plasmids expressing the indicated mRNAs were co-transfected in 293 cells with a construct expressing the GFP ORF followed by the bovine growth hormone (bGH) polyadenylation signal. Endogenous Upf1 was immunoprecipitated from transfected cells grown in the absence or presence of puromycin (100 μg/mL for 3 hr) as indicated, and co-purifying mRNAs were analyzed by northern blot. (C) Quantification of relative RNA recovery upon UPF1 immunoprecipitation, normalized to the recovery of co-transfected GFP-bGH mRNAs. The amount of RSE-containing RNA recovered was set to 1. Error bars indicate ± SD; n ≥ 3 (*p<0.05, **p<0.01 in two-tailed Student’s t-tests when compared to RSE recovery). Under both untreated and puromycin treated conditions, the RSE caused reduced accumulation of UPF1 on mRNAs relative to the SMG5-397 control.DOI:http://dx.doi.org/10.7554/eLife.11155.008

Mentions: (A) Schematic of reporter mRNA constructs used in immunoprecipitation assays. RNAs containing the GFP ORF and an artificial NMD-inducing 3’UTR comprising a portion of the human GAPDH ORF and the GAPDH 3’UTR (Singh et al., 2008) were modified to contain the RSE or the SMG5-397 sequence 5’ or 3’ of the GAPDH sequence. (B) Upf1 is reduced on 3'UTRs containing a TC-proximal RSE. Plasmids expressing the indicated mRNAs were co-transfected in 293 cells with a construct expressing the GFP ORF followed by the bovine growth hormone (bGH) polyadenylation signal. Endogenous UPF1 was immunoprecipitated from transfected cells, and co-purifying mRNAs were analyzed by northern blot. Bulk goat IgG was used as a non-specific interaction control. (C) Quantification of relative RNA recovery upon UPF1 immunoprecipitation, normalized to the recovery of co-transfected GFP-bGH mRNAs. The amount of RSE-containing RNA recovered was set to 1. Error bars indicate ± SD; n = 3 (*p<0.05; **p<0.01 in two-tailed Student’s t-tests when compared to RSE recovery). See also Figure 2—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)

RSE inhibition of UPF1 binding is independent of translation.(A) Schematic of GFP reporter mRNA constructs used in immunoprecipitation assays. RNAs containing the GFP ORF and an artificial NMD-inducing 3’UTR comprising a portion of the human GAPDH ORF and the GAPDH 3’UTR (Singh et al., 2008) were modified to contain the RSE (top) or the SMG5-397 sequence (middle). As a control to illustrate the behavior of mRNAs with short 3’UTRs (untreated condition) or shorter overall length (puromycin treatment), we used constructs without added RSE or SMG5-397 sequence in which the GFP termination codon was changed to CAA (bottom), resulting in termination at the downstream GAPDH stop codon. (B) UPF1 association is reduced on 3'UTRs containing the RSE. Plasmids expressing the indicated mRNAs were co-transfected in 293 cells with a construct expressing the GFP ORF followed by the bovine growth hormone (bGH) polyadenylation signal. Endogenous Upf1 was immunoprecipitated from transfected cells grown in the absence or presence of puromycin (100 μg/mL for 3 hr) as indicated, and co-purifying mRNAs were analyzed by northern blot. (C) Quantification of relative RNA recovery upon UPF1 immunoprecipitation, normalized to the recovery of co-transfected GFP-bGH mRNAs. The amount of RSE-containing RNA recovered was set to 1. Error bars indicate ± SD; n ≥ 3 (*p<0.05, **p<0.01 in two-tailed Student’s t-tests when compared to RSE recovery). Under both untreated and puromycin treated conditions, the RSE caused reduced accumulation of UPF1 on mRNAs relative to the SMG5-397 control.DOI:http://dx.doi.org/10.7554/eLife.11155.008
© Copyright Policy
Related In: Results  -  Collection

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

fig2s2: RSE inhibition of UPF1 binding is independent of translation.(A) Schematic of GFP reporter mRNA constructs used in immunoprecipitation assays. RNAs containing the GFP ORF and an artificial NMD-inducing 3’UTR comprising a portion of the human GAPDH ORF and the GAPDH 3’UTR (Singh et al., 2008) were modified to contain the RSE (top) or the SMG5-397 sequence (middle). As a control to illustrate the behavior of mRNAs with short 3’UTRs (untreated condition) or shorter overall length (puromycin treatment), we used constructs without added RSE or SMG5-397 sequence in which the GFP termination codon was changed to CAA (bottom), resulting in termination at the downstream GAPDH stop codon. (B) UPF1 association is reduced on 3'UTRs containing the RSE. Plasmids expressing the indicated mRNAs were co-transfected in 293 cells with a construct expressing the GFP ORF followed by the bovine growth hormone (bGH) polyadenylation signal. Endogenous Upf1 was immunoprecipitated from transfected cells grown in the absence or presence of puromycin (100 μg/mL for 3 hr) as indicated, and co-purifying mRNAs were analyzed by northern blot. (C) Quantification of relative RNA recovery upon UPF1 immunoprecipitation, normalized to the recovery of co-transfected GFP-bGH mRNAs. The amount of RSE-containing RNA recovered was set to 1. Error bars indicate ± SD; n ≥ 3 (*p<0.05, **p<0.01 in two-tailed Student’s t-tests when compared to RSE recovery). Under both untreated and puromycin treated conditions, the RSE caused reduced accumulation of UPF1 on mRNAs relative to the SMG5-397 control.DOI:http://dx.doi.org/10.7554/eLife.11155.008
Mentions: (A) Schematic of reporter mRNA constructs used in immunoprecipitation assays. RNAs containing the GFP ORF and an artificial NMD-inducing 3’UTR comprising a portion of the human GAPDH ORF and the GAPDH 3’UTR (Singh et al., 2008) were modified to contain the RSE or the SMG5-397 sequence 5’ or 3’ of the GAPDH sequence. (B) Upf1 is reduced on 3'UTRs containing a TC-proximal RSE. Plasmids expressing the indicated mRNAs were co-transfected in 293 cells with a construct expressing the GFP ORF followed by the bovine growth hormone (bGH) polyadenylation signal. Endogenous UPF1 was immunoprecipitated from transfected cells, and co-purifying mRNAs were analyzed by northern blot. Bulk goat IgG was used as a non-specific interaction control. (C) Quantification of relative RNA recovery upon UPF1 immunoprecipitation, normalized to the recovery of co-transfected GFP-bGH mRNAs. The amount of RSE-containing RNA recovered was set to 1. Error bars indicate ± SD; n = 3 (*p<0.05; **p<0.01 in two-tailed Student’s t-tests when compared to RSE recovery). See also Figure 2—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