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Cotranscriptional recruitment of the nuclear poly(A)-binding protein Pab2 to nascent transcripts and association with translating mRNPs.

Lemieux C, Bachand F - Nucleic Acids Res. (2009)

Bottom Line: Tandem affinity purification coupled with mass spectrometry also revealed that Pab2 associates with several ribosomal proteins as well as general translation factors.Importantly, whereas previous results suggest that the nuclear poly(A)-binding protein is not present on cytoplasmic mRNAs, we show that fission yeast Pab2 is associated with polysomes.Our findings suggest that Pab2 is recruited to nascent mRNPs during transcription and remains associated with translated mRNPs after nuclear export.

View Article: PubMed Central - PubMed

Affiliation: RNA Group, Department of Biochemistry, UniversitƩ de Sherbrooke, QuƩbec, Canada.

ABSTRACT
Synthesis of the pre-mRNA poly(A) tail in the nucleus has important consequences on the translational activity of the mature mRNA in the cytoplasm. In most eukaryotes, nuclear polyadenylation of pre-mRNAs is thought to require the nuclear poly(A)-binding protein (PABP2/PABPN1) for poly(A) tail synthesis and ultimate length control. As yet, however, the extent of the association between PABP2 and the exported mRNA remains poorly understood. Here, we used chromatin immunoprecipitation (ChIP) assays to show that the fission yeast ortholog of mammalian PABP2 (Pab2) is cotranscriptionally recruited to active genes. Notably, the association of Pab2 to genes precedes that of a typical 3'-processing/polyadenylation factor, suggesting that Pab2 recruitment during the transcription cycle precedes polyadenylation. The inclusion of an RNase step in our ChIP and immunoprecipitation assays suggests that Pab2 is cotranscriptionally recruited via nascent mRNA ribonucleoprotein (mRNPs). Tandem affinity purification coupled with mass spectrometry also revealed that Pab2 associates with several ribosomal proteins as well as general translation factors. Importantly, whereas previous results suggest that the nuclear poly(A)-binding protein is not present on cytoplasmic mRNAs, we show that fission yeast Pab2 is associated with polysomes. Our findings suggest that Pab2 is recruited to nascent mRNPs during transcription and remains associated with translated mRNPs after nuclear export.

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Purification of proteins associated with TAP-tagged Pab2. (A) Proteins copurified with Pab2 by tandem affinity purification (lane 1) were resolved using a Bisā€“Tris 4ā€“12% gradient SDSā€“PAGE and analyzed by silver staining. The result for an identically treated extract from control S. pombe is shown in lane 2. Molecular weight markers are shown on the right in kilodaltons (kDa). The position of Pab2 is indicated on the left. (B) Summary of the nonribosomal proteins identified by mass spectrometry from the Pab2-TAP purification.
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Figure 5: Purification of proteins associated with TAP-tagged Pab2. (A) Proteins copurified with Pab2 by tandem affinity purification (lane 1) were resolved using a Bisā€“Tris 4ā€“12% gradient SDSā€“PAGE and analyzed by silver staining. The result for an identically treated extract from control S. pombe is shown in lane 2. Molecular weight markers are shown on the right in kilodaltons (kDa). The position of Pab2 is indicated on the left. (B) Summary of the nonribosomal proteins identified by mass spectrometry from the Pab2-TAP purification.

Mentions: To date, the protein interaction network of the nuclear poly(A)-binding protein remains largely unknown. To get further insights into the mechanism by which Pab2 is cotranscriptionally recruited to nascent mRNPs, a tandem affinity purification approach was used to identify proteins that associate with fission yeast Pab2. Extracts were prepared from 8ā€‰l of cells that expressed a TAP-tagged version of Pab2 as well as from untagged control cells. Following two rounds of purification over IgG-sepharose and calmodulin-bound resins, the eluted proteins were resolved by SDSā€“PAGE and visualized by silver staining. As can be seen in FigureĀ 5A, all of the associated proteins were in substoichiometric amounts relative to Pab2. Analysis of the eluted proteins by mass spectrometry identified unique peptides that corresponded to gene products that are involved in a wide range of RNA-related functions (FigureĀ 5B and TableĀ 2). Notably, peptides from 32 ribosomal proteins, 4 general translation factors, as well as from the cytosolic poly(A)-binding protein (Pab1) were identified (TableĀ 2 and FigureĀ 5B). Peptides that correspond to the evolutionarily conserved nuclear poly(A) polymerase were also detected (FigureĀ 5B), consistent with the role of Pab2 in polyadenylation (27). Other proteins identified that participate in RNA metabolism included the RNA helicase Mtr4, the 5ā€²-to-3ā€² exonuclease Exo2, the mRNA decapping subunit Dcp2 and the nuclear cap-binding protein Cbp80 (FigureĀ 5B). Protein factors for which the S. cerevisiae orthologs are functionally implicated in mRNA export were also coprecipitated with Pab2; these included the orthologs of the S. cerevisiae proteins Thp1, Mtr10 and Mtr2. Consistent with the aforementioned results (FigureĀ 4), RNA could be responsible for the copurification of some of these proteins. Importantly, with the exception of one ribosomal protein for which some peptides were detected in the control purification, no peptides were detected in the control for any of the proteins indicated in FigureĀ 5B and TableĀ 2. In conclusion, our proteomic analysis suggests that Pab2 is associated with factors involved in several steps of the mRNA life cycle.FigureĀ 5.


Cotranscriptional recruitment of the nuclear poly(A)-binding protein Pab2 to nascent transcripts and association with translating mRNPs.

Lemieux C, Bachand F - Nucleic Acids Res. (2009)

Purification of proteins associated with TAP-tagged Pab2. (A) Proteins copurified with Pab2 by tandem affinity purification (lane 1) were resolved using a Bisā€“Tris 4ā€“12% gradient SDSā€“PAGE and analyzed by silver staining. The result for an identically treated extract from control S. pombe is shown in lane 2. Molecular weight markers are shown on the right in kilodaltons (kDa). The position of Pab2 is indicated on the left. (B) Summary of the nonribosomal proteins identified by mass spectrometry from the Pab2-TAP purification.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
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Figure 5: Purification of proteins associated with TAP-tagged Pab2. (A) Proteins copurified with Pab2 by tandem affinity purification (lane 1) were resolved using a Bisā€“Tris 4ā€“12% gradient SDSā€“PAGE and analyzed by silver staining. The result for an identically treated extract from control S. pombe is shown in lane 2. Molecular weight markers are shown on the right in kilodaltons (kDa). The position of Pab2 is indicated on the left. (B) Summary of the nonribosomal proteins identified by mass spectrometry from the Pab2-TAP purification.
Mentions: To date, the protein interaction network of the nuclear poly(A)-binding protein remains largely unknown. To get further insights into the mechanism by which Pab2 is cotranscriptionally recruited to nascent mRNPs, a tandem affinity purification approach was used to identify proteins that associate with fission yeast Pab2. Extracts were prepared from 8ā€‰l of cells that expressed a TAP-tagged version of Pab2 as well as from untagged control cells. Following two rounds of purification over IgG-sepharose and calmodulin-bound resins, the eluted proteins were resolved by SDSā€“PAGE and visualized by silver staining. As can be seen in FigureĀ 5A, all of the associated proteins were in substoichiometric amounts relative to Pab2. Analysis of the eluted proteins by mass spectrometry identified unique peptides that corresponded to gene products that are involved in a wide range of RNA-related functions (FigureĀ 5B and TableĀ 2). Notably, peptides from 32 ribosomal proteins, 4 general translation factors, as well as from the cytosolic poly(A)-binding protein (Pab1) were identified (TableĀ 2 and FigureĀ 5B). Peptides that correspond to the evolutionarily conserved nuclear poly(A) polymerase were also detected (FigureĀ 5B), consistent with the role of Pab2 in polyadenylation (27). Other proteins identified that participate in RNA metabolism included the RNA helicase Mtr4, the 5ā€²-to-3ā€² exonuclease Exo2, the mRNA decapping subunit Dcp2 and the nuclear cap-binding protein Cbp80 (FigureĀ 5B). Protein factors for which the S. cerevisiae orthologs are functionally implicated in mRNA export were also coprecipitated with Pab2; these included the orthologs of the S. cerevisiae proteins Thp1, Mtr10 and Mtr2. Consistent with the aforementioned results (FigureĀ 4), RNA could be responsible for the copurification of some of these proteins. Importantly, with the exception of one ribosomal protein for which some peptides were detected in the control purification, no peptides were detected in the control for any of the proteins indicated in FigureĀ 5B and TableĀ 2. In conclusion, our proteomic analysis suggests that Pab2 is associated with factors involved in several steps of the mRNA life cycle.FigureĀ 5.

Bottom Line: Tandem affinity purification coupled with mass spectrometry also revealed that Pab2 associates with several ribosomal proteins as well as general translation factors.Importantly, whereas previous results suggest that the nuclear poly(A)-binding protein is not present on cytoplasmic mRNAs, we show that fission yeast Pab2 is associated with polysomes.Our findings suggest that Pab2 is recruited to nascent mRNPs during transcription and remains associated with translated mRNPs after nuclear export.

View Article: PubMed Central - PubMed

Affiliation: RNA Group, Department of Biochemistry, UniversitƩ de Sherbrooke, QuƩbec, Canada.

ABSTRACT
Synthesis of the pre-mRNA poly(A) tail in the nucleus has important consequences on the translational activity of the mature mRNA in the cytoplasm. In most eukaryotes, nuclear polyadenylation of pre-mRNAs is thought to require the nuclear poly(A)-binding protein (PABP2/PABPN1) for poly(A) tail synthesis and ultimate length control. As yet, however, the extent of the association between PABP2 and the exported mRNA remains poorly understood. Here, we used chromatin immunoprecipitation (ChIP) assays to show that the fission yeast ortholog of mammalian PABP2 (Pab2) is cotranscriptionally recruited to active genes. Notably, the association of Pab2 to genes precedes that of a typical 3'-processing/polyadenylation factor, suggesting that Pab2 recruitment during the transcription cycle precedes polyadenylation. The inclusion of an RNase step in our ChIP and immunoprecipitation assays suggests that Pab2 is cotranscriptionally recruited via nascent mRNA ribonucleoprotein (mRNPs). Tandem affinity purification coupled with mass spectrometry also revealed that Pab2 associates with several ribosomal proteins as well as general translation factors. Importantly, whereas previous results suggest that the nuclear poly(A)-binding protein is not present on cytoplasmic mRNAs, we show that fission yeast Pab2 is associated with polysomes. Our findings suggest that Pab2 is recruited to nascent mRNPs during transcription and remains associated with translated mRNPs after nuclear export.

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