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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

Identification of RSE-interacting proteins by tandem mass spectrometry.(A) Schematic of β-globin reporter mRNA constructs used for PP7-based affinity purification. The RSE sequence (top) and a control sequence, the antisense RSE sequence (AS-RSE; bottom) were inserted into reporter mRNAs containing the β-globin gene and the GAPDH 3’UTR. (B) mRNP profiles of the transcripts described in (A). Proteins were separated on a 4–12% SDS-PAGE gel and visualized by Krypton Infrared Protein Stain (Pierce). (C) Purified mRNPs were subjected to trypsin digestion and tandem mass spectrometry. Spectral counts from selected proteins enriched in either the RSE-containing sample or the antisense RSE sample are shown. Complete mass spectrometry data are compiled in Figure 3—source data 1. (D) Control for post-lysis reassortment. Extracts from cells in which protein A-tagged UPF1 was co-expressed with the indicated GFP reporter mRNAs or extracts from cells separately expressing protein A-tagged UPF1 and the exogenous mRNAs were used for affinity purification. mRNAs containing the GAPDH artificial 3’UTR were used as recovery controls. Top panels: Northern blots of co-transfected reporter mRNAs. Identical settings were used to image northern blots of input extracts and purified material from co-expressed and mixed samples, respectively. Bottom panels: immunoblotting of mixed extracts with an α-UPF1 antibody indicates equal purification efficiency across all conditions. (E) Relative recoveries of the indicated mRNAs with affinity purified UPF1 were determined, normalized to recovery of mRNAs containing the GAPDH 3’UTR. Error bars indicate ± SD; n = 3 (**p<0.01 in two-tailed Student’s t-test comparing the recovery of RSE-GAPDH to RSE-∆PTB-GAPDH).DOI:http://dx.doi.org/10.7554/eLife.11155.011
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fig3s1: Identification of RSE-interacting proteins by tandem mass spectrometry.(A) Schematic of β-globin reporter mRNA constructs used for PP7-based affinity purification. The RSE sequence (top) and a control sequence, the antisense RSE sequence (AS-RSE; bottom) were inserted into reporter mRNAs containing the β-globin gene and the GAPDH 3’UTR. (B) mRNP profiles of the transcripts described in (A). Proteins were separated on a 4–12% SDS-PAGE gel and visualized by Krypton Infrared Protein Stain (Pierce). (C) Purified mRNPs were subjected to trypsin digestion and tandem mass spectrometry. Spectral counts from selected proteins enriched in either the RSE-containing sample or the antisense RSE sample are shown. Complete mass spectrometry data are compiled in Figure 3—source data 1. (D) Control for post-lysis reassortment. Extracts from cells in which protein A-tagged UPF1 was co-expressed with the indicated GFP reporter mRNAs or extracts from cells separately expressing protein A-tagged UPF1 and the exogenous mRNAs were used for affinity purification. mRNAs containing the GAPDH artificial 3’UTR were used as recovery controls. Top panels: Northern blots of co-transfected reporter mRNAs. Identical settings were used to image northern blots of input extracts and purified material from co-expressed and mixed samples, respectively. Bottom panels: immunoblotting of mixed extracts with an α-UPF1 antibody indicates equal purification efficiency across all conditions. (E) Relative recoveries of the indicated mRNAs with affinity purified UPF1 were determined, normalized to recovery of mRNAs containing the GAPDH 3’UTR. Error bars indicate ± SD; n = 3 (**p<0.01 in two-tailed Student’s t-test comparing the recovery of RSE-GAPDH to RSE-∆PTB-GAPDH).DOI:http://dx.doi.org/10.7554/eLife.11155.011

Mentions: To identify possible protein cofactors of the RSE, we isolated mRNPs containing the RSE in the sense or antisense orientations by an RNA-based affinity purification technique (Hogg and Goff, 2010). Reporter mRNAs tagged with a single copy of the 25 nt Pseudomonas phage 7 coat protein (PP7CP) RNA hairpin binding site (Figure 3—figure supplement 1A) were expressed in 293T cells, and the resulting endogenously assembled mRNPs were purified from cell extracts using the PP7CP tagged with tandem Staphylococcus aureus protein A domains. The isolated mRNPs containing either the RSE sequence or the control sequence displayed a similar profile of co-purifying proteins, while mock purifications from cell extracts containing no tagged RNA yielded very few contaminating proteins (Figure 3—figure supplement 1B, additional data not shown). Comprehensive analysis of the composition of the purified mRNP complexes by tandem mass spectrometry revealed a large number of proteins that were equally represented in the RSE-containing mRNPs and the control mRNPs, many of which are common RNA binding proteins, ribosomal proteins, and translation factors (Figure 3—source data 1). UPF1 was enriched in the control mRNP (Figure 3—figure supplement 1C), consistent with our finding that the RSE inhibits UPF1 association with the 3’UTR (Figure 2). Several proteins were over-represented in mRNPs containing the RSE sequence, including polypyrimidine tract binding protein 1 (PTBP1), heterogeneous nuclear ribonucleoprotein L (hnRNP L), MATRIN 3, and splicing factor, arginine and serine-rich 14 (SFRS14; Figure 3—figure supplement 1C). We were able to confirm the preferential interaction between these proteins and the RSE by immunoblotting of purified mRNPs or co-immunoprecipitation experiments (Figure 3 and additional data not shown).10.7554/eLife.11155.009Figure 3.Accumulation of PTBP1 on the 3‘UTR prevents UPF1 binding.


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)

Identification of RSE-interacting proteins by tandem mass spectrometry.(A) Schematic of β-globin reporter mRNA constructs used for PP7-based affinity purification. The RSE sequence (top) and a control sequence, the antisense RSE sequence (AS-RSE; bottom) were inserted into reporter mRNAs containing the β-globin gene and the GAPDH 3’UTR. (B) mRNP profiles of the transcripts described in (A). Proteins were separated on a 4–12% SDS-PAGE gel and visualized by Krypton Infrared Protein Stain (Pierce). (C) Purified mRNPs were subjected to trypsin digestion and tandem mass spectrometry. Spectral counts from selected proteins enriched in either the RSE-containing sample or the antisense RSE sample are shown. Complete mass spectrometry data are compiled in Figure 3—source data 1. (D) Control for post-lysis reassortment. Extracts from cells in which protein A-tagged UPF1 was co-expressed with the indicated GFP reporter mRNAs or extracts from cells separately expressing protein A-tagged UPF1 and the exogenous mRNAs were used for affinity purification. mRNAs containing the GAPDH artificial 3’UTR were used as recovery controls. Top panels: Northern blots of co-transfected reporter mRNAs. Identical settings were used to image northern blots of input extracts and purified material from co-expressed and mixed samples, respectively. Bottom panels: immunoblotting of mixed extracts with an α-UPF1 antibody indicates equal purification efficiency across all conditions. (E) Relative recoveries of the indicated mRNAs with affinity purified UPF1 were determined, normalized to recovery of mRNAs containing the GAPDH 3’UTR. Error bars indicate ± SD; n = 3 (**p<0.01 in two-tailed Student’s t-test comparing the recovery of RSE-GAPDH to RSE-∆PTB-GAPDH).DOI:http://dx.doi.org/10.7554/eLife.11155.011
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fig3s1: Identification of RSE-interacting proteins by tandem mass spectrometry.(A) Schematic of β-globin reporter mRNA constructs used for PP7-based affinity purification. The RSE sequence (top) and a control sequence, the antisense RSE sequence (AS-RSE; bottom) were inserted into reporter mRNAs containing the β-globin gene and the GAPDH 3’UTR. (B) mRNP profiles of the transcripts described in (A). Proteins were separated on a 4–12% SDS-PAGE gel and visualized by Krypton Infrared Protein Stain (Pierce). (C) Purified mRNPs were subjected to trypsin digestion and tandem mass spectrometry. Spectral counts from selected proteins enriched in either the RSE-containing sample or the antisense RSE sample are shown. Complete mass spectrometry data are compiled in Figure 3—source data 1. (D) Control for post-lysis reassortment. Extracts from cells in which protein A-tagged UPF1 was co-expressed with the indicated GFP reporter mRNAs or extracts from cells separately expressing protein A-tagged UPF1 and the exogenous mRNAs were used for affinity purification. mRNAs containing the GAPDH artificial 3’UTR were used as recovery controls. Top panels: Northern blots of co-transfected reporter mRNAs. Identical settings were used to image northern blots of input extracts and purified material from co-expressed and mixed samples, respectively. Bottom panels: immunoblotting of mixed extracts with an α-UPF1 antibody indicates equal purification efficiency across all conditions. (E) Relative recoveries of the indicated mRNAs with affinity purified UPF1 were determined, normalized to recovery of mRNAs containing the GAPDH 3’UTR. Error bars indicate ± SD; n = 3 (**p<0.01 in two-tailed Student’s t-test comparing the recovery of RSE-GAPDH to RSE-∆PTB-GAPDH).DOI:http://dx.doi.org/10.7554/eLife.11155.011
Mentions: To identify possible protein cofactors of the RSE, we isolated mRNPs containing the RSE in the sense or antisense orientations by an RNA-based affinity purification technique (Hogg and Goff, 2010). Reporter mRNAs tagged with a single copy of the 25 nt Pseudomonas phage 7 coat protein (PP7CP) RNA hairpin binding site (Figure 3—figure supplement 1A) were expressed in 293T cells, and the resulting endogenously assembled mRNPs were purified from cell extracts using the PP7CP tagged with tandem Staphylococcus aureus protein A domains. The isolated mRNPs containing either the RSE sequence or the control sequence displayed a similar profile of co-purifying proteins, while mock purifications from cell extracts containing no tagged RNA yielded very few contaminating proteins (Figure 3—figure supplement 1B, additional data not shown). Comprehensive analysis of the composition of the purified mRNP complexes by tandem mass spectrometry revealed a large number of proteins that were equally represented in the RSE-containing mRNPs and the control mRNPs, many of which are common RNA binding proteins, ribosomal proteins, and translation factors (Figure 3—source data 1). UPF1 was enriched in the control mRNP (Figure 3—figure supplement 1C), consistent with our finding that the RSE inhibits UPF1 association with the 3’UTR (Figure 2). Several proteins were over-represented in mRNPs containing the RSE sequence, including polypyrimidine tract binding protein 1 (PTBP1), heterogeneous nuclear ribonucleoprotein L (hnRNP L), MATRIN 3, and splicing factor, arginine and serine-rich 14 (SFRS14; Figure 3—figure supplement 1C). We were able to confirm the preferential interaction between these proteins and the RSE by immunoblotting of purified mRNPs or co-immunoprecipitation experiments (Figure 3 and additional data not shown).10.7554/eLife.11155.009Figure 3.Accumulation of PTBP1 on the 3‘UTR prevents UPF1 binding.

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