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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, Université de Sherbrooke, Québec, 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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Pab2 shuttles between the nucleus and cytoplasm. The nuclear export assay was performed using S. cerevisiae cells that express the nup49-313 allele as described in the Materials and methods section. The nup49-313 cells expressing either NLS-LacZ-GFP (A–D), Nab2-GFP (E–H) and GFP-Pab2 (I–L) were incubated at 25°C (A, B, E, F, I and J) or shifted to 37°C (C, D, G, H, K and L) before treatment with cycloheximide to block new protein synthesis. Phase contrast (A, C, E, G, I and K) and GFP fluorescence (B, D, F, H, J and L) are shown.
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Figure 6: Pab2 shuttles between the nucleus and cytoplasm. The nuclear export assay was performed using S. cerevisiae cells that express the nup49-313 allele as described in the Materials and methods section. The nup49-313 cells expressing either NLS-LacZ-GFP (A–D), Nab2-GFP (E–H) and GFP-Pab2 (I–L) were incubated at 25°C (A, B, E, F, I and J) or shifted to 37°C (C, D, G, H, K and L) before treatment with cycloheximide to block new protein synthesis. Phase contrast (A, C, E, G, I and K) and GFP fluorescence (B, D, F, H, J and L) are shown.

Mentions: Consistent with previous reports indicating that S. cerevisiae Nab2 is a shuttling protein (40,50), cytoplasmic accumulation of Nab2 was observed at the nonpermissive temperature (Figure 6G and H), whereas Nab2 was predominantly nuclear at the permissive temperature (Figure 6E and F). The expression of GFP-tagged Pab2 in S. cerevisiae resulted in nuclear localization (Figure 6I and J), consistent with the steady-state localization of Pab2 in fission yeast (27). In contrast, significant GFP signal was detected in the cytoplasm of Pab2-expressing cells that were shifted to the nonpermissive temperature (Figure 6K and L). As a control, a GFP-tagged LacZ protein that includes a strong nuclear localization signal remained in the nucleus at both permissive and nonpermissive temperatures (Figure 6A–D). This control also demonstrated that the cycloheximide treatment before imaging prevented de novo synthesis of GFP-tagged proteins in the cytoplasm. The results of the nuclear export assay indicate that Pab2 can shuttle between the nucleus and the cytoplasm.Figure 6.


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)

Pab2 shuttles between the nucleus and cytoplasm. The nuclear export assay was performed using S. cerevisiae cells that express the nup49-313 allele as described in the Materials and methods section. The nup49-313 cells expressing either NLS-LacZ-GFP (A–D), Nab2-GFP (E–H) and GFP-Pab2 (I–L) were incubated at 25°C (A, B, E, F, I and J) or shifted to 37°C (C, D, G, H, K and L) before treatment with cycloheximide to block new protein synthesis. Phase contrast (A, C, E, G, I and K) and GFP fluorescence (B, D, F, H, J and L) are shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 6: Pab2 shuttles between the nucleus and cytoplasm. The nuclear export assay was performed using S. cerevisiae cells that express the nup49-313 allele as described in the Materials and methods section. The nup49-313 cells expressing either NLS-LacZ-GFP (A–D), Nab2-GFP (E–H) and GFP-Pab2 (I–L) were incubated at 25°C (A, B, E, F, I and J) or shifted to 37°C (C, D, G, H, K and L) before treatment with cycloheximide to block new protein synthesis. Phase contrast (A, C, E, G, I and K) and GFP fluorescence (B, D, F, H, J and L) are shown.
Mentions: Consistent with previous reports indicating that S. cerevisiae Nab2 is a shuttling protein (40,50), cytoplasmic accumulation of Nab2 was observed at the nonpermissive temperature (Figure 6G and H), whereas Nab2 was predominantly nuclear at the permissive temperature (Figure 6E and F). The expression of GFP-tagged Pab2 in S. cerevisiae resulted in nuclear localization (Figure 6I and J), consistent with the steady-state localization of Pab2 in fission yeast (27). In contrast, significant GFP signal was detected in the cytoplasm of Pab2-expressing cells that were shifted to the nonpermissive temperature (Figure 6K and L). As a control, a GFP-tagged LacZ protein that includes a strong nuclear localization signal remained in the nucleus at both permissive and nonpermissive temperatures (Figure 6A–D). This control also demonstrated that the cycloheximide treatment before imaging prevented de novo synthesis of GFP-tagged proteins in the cytoplasm. The results of the nuclear export assay indicate that Pab2 can shuttle between the nucleus and the cytoplasm.Figure 6.

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, Université de Sherbrooke, Québec, 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.

Show MeSH