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A physical and functional link between splicing factors promotes pre-mRNA 3' end processing.

Millevoi S, Decorsière A, Loulergue C, Iacovoni J, Bernat S, Antoniou M, Vagner S - Nucleic Acids Res. (2009)

Bottom Line: We identify PTB as the protein factor whose binding to the human beta-globin (HBB) 3' UTR is abrogated by a 3' end processing-inactivating mutation.We show that PTB promotes both in vitro 3' end cleavage and polyadenylation and recruits directly the splicing factor hnRNP H to G-rich sequences associated with several pA signals.Therefore, our results provide evidence of a concerted regulation of pA signal recognition by splicing factors bound to auxiliary polyadenylation sequence elements.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U563, Toulouse, Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan, Toulouse, F-31300, France. stefania.millevoi@inserm.fr

ABSTRACT
Polypyrimidine tract-binding protein (PTB) is a splicing regulator that also plays a positive role in pre-mRNA 3' end processing when bound upstream of the polyadenylation signal (pA signal). Here, we address the mechanism of PTB stimulatory function in mRNA 3' end formation. We identify PTB as the protein factor whose binding to the human beta-globin (HBB) 3' UTR is abrogated by a 3' end processing-inactivating mutation. We show that PTB promotes both in vitro 3' end cleavage and polyadenylation and recruits directly the splicing factor hnRNP H to G-rich sequences associated with several pA signals. Increased binding of hnRNP H results in stimulation of polyadenylation through a direct interaction with poly(A) polymerase. Therefore, our results provide evidence of a concerted regulation of pA signal recognition by splicing factors bound to auxiliary polyadenylation sequence elements.

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PTB promotes polyadenylation by increasing the binding of hnRNP H to a GRS located upstream of the HBB pA signal. (a) Illustration of the r17/HBB pre-cleaved substrate containing a GRS element upstream of the pA signal (named UGRS). (b) In vitro polyadenylation assays using the 32P-labeled r17/HBB pre-cleaved substrate and NEs in the absence or presence of R17–U2AF65 (10 pmol) or increasing amounts of R17–PTB (2.5, 5 and 10 pmol). Normalized pA efficiency is shown. (c) UV crosslinking of NE proteins using the 32P-labeled r17/HBB pre-cleaved substrate with (5 pmol) or without R17–PTB. The arrow indicates the 50-kDa protein whose binding to the HBB pre-cleaved RNA is increased upon R17–PTB addition. (d) IP of NE proteins UV crosslinked to the 32P-labeled r17/HBB pre-cleaved substrate in the absence or presence of R17–PTB (5 pmol) or R17 (5 pmol) using the hnRNP H/F antibody. (e) Reconstituted polyadenylation assays using the 32P-labeled r17/HBB pre-cleaved substrate, PAP (0.1 pmol) and PABPN1 (1.2 pmol), in the presence of increasing amounts of hnRNP H (2 and 4 pmol) or hnRNP F (4 pmol). Lane 1: input RNA; arrow: non-polyadenylated RNA substrate. (f) Reconstituted polyadenylation assays using the 32P-labeled r17/HBB pre-cleaved substrate and PAP (0.1 pmol), in the presence of R17–PTB (4 pmol) and/or hnRNP H (4 pmol). Arrow: non-polyadenylated RNA substrate. (g) GST pull-down assay to test the interaction between GST-tagged PTB and PAP in the absence or presence of hnRNP H. The input lane accounts for 10% of hnRNP H and PAP used in the assay. (h) IP pull down assay with hnRNP H and PAP using the hnRNP H/F antibody. The input lane accounts for 10% of PAP used in the assay.
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Figure 6: PTB promotes polyadenylation by increasing the binding of hnRNP H to a GRS located upstream of the HBB pA signal. (a) Illustration of the r17/HBB pre-cleaved substrate containing a GRS element upstream of the pA signal (named UGRS). (b) In vitro polyadenylation assays using the 32P-labeled r17/HBB pre-cleaved substrate and NEs in the absence or presence of R17–U2AF65 (10 pmol) or increasing amounts of R17–PTB (2.5, 5 and 10 pmol). Normalized pA efficiency is shown. (c) UV crosslinking of NE proteins using the 32P-labeled r17/HBB pre-cleaved substrate with (5 pmol) or without R17–PTB. The arrow indicates the 50-kDa protein whose binding to the HBB pre-cleaved RNA is increased upon R17–PTB addition. (d) IP of NE proteins UV crosslinked to the 32P-labeled r17/HBB pre-cleaved substrate in the absence or presence of R17–PTB (5 pmol) or R17 (5 pmol) using the hnRNP H/F antibody. (e) Reconstituted polyadenylation assays using the 32P-labeled r17/HBB pre-cleaved substrate, PAP (0.1 pmol) and PABPN1 (1.2 pmol), in the presence of increasing amounts of hnRNP H (2 and 4 pmol) or hnRNP F (4 pmol). Lane 1: input RNA; arrow: non-polyadenylated RNA substrate. (f) Reconstituted polyadenylation assays using the 32P-labeled r17/HBB pre-cleaved substrate and PAP (0.1 pmol), in the presence of R17–PTB (4 pmol) and/or hnRNP H (4 pmol). Arrow: non-polyadenylated RNA substrate. (g) GST pull-down assay to test the interaction between GST-tagged PTB and PAP in the absence or presence of hnRNP H. The input lane accounts for 10% of hnRNP H and PAP used in the assay. (h) IP pull down assay with hnRNP H and PAP using the hnRNP H/F antibody. The input lane accounts for 10% of PAP used in the assay.

Mentions: GST pull-down assays were performed by incubating 1 μg of purified GST–R17 or GST–R17–PTB bound to 20 μl of glutathione agarose beads with 200 μg of HeLa NEs (Figure 3a) in NETN buffer (20 mM Tris at pH 8.0, 100 mM NaCl, 0.5% NP-40, 0.5 mM EDTA) for 60 min at 4°C. Beads were then washed five times, treated with 10 μg/ml RNAse A at room temperature for 30 min and washed again. Protein elution was performed by adding SDS loading buffer to the beads. Eluted proteins were resolved by 10% SDS–polyacrylamide gel electrophoresis (PAGE) and analyzed by western blot. GST pull-down assays shown in Figures 3b and 6g and h were performed as described above except that it was done with 1 μg of His-tagged hnRNP H, hnRNP F or PAP, and bound proteins were visualized by Coomassie blue staining.Figure 1.


A physical and functional link between splicing factors promotes pre-mRNA 3' end processing.

Millevoi S, Decorsière A, Loulergue C, Iacovoni J, Bernat S, Antoniou M, Vagner S - Nucleic Acids Res. (2009)

PTB promotes polyadenylation by increasing the binding of hnRNP H to a GRS located upstream of the HBB pA signal. (a) Illustration of the r17/HBB pre-cleaved substrate containing a GRS element upstream of the pA signal (named UGRS). (b) In vitro polyadenylation assays using the 32P-labeled r17/HBB pre-cleaved substrate and NEs in the absence or presence of R17–U2AF65 (10 pmol) or increasing amounts of R17–PTB (2.5, 5 and 10 pmol). Normalized pA efficiency is shown. (c) UV crosslinking of NE proteins using the 32P-labeled r17/HBB pre-cleaved substrate with (5 pmol) or without R17–PTB. The arrow indicates the 50-kDa protein whose binding to the HBB pre-cleaved RNA is increased upon R17–PTB addition. (d) IP of NE proteins UV crosslinked to the 32P-labeled r17/HBB pre-cleaved substrate in the absence or presence of R17–PTB (5 pmol) or R17 (5 pmol) using the hnRNP H/F antibody. (e) Reconstituted polyadenylation assays using the 32P-labeled r17/HBB pre-cleaved substrate, PAP (0.1 pmol) and PABPN1 (1.2 pmol), in the presence of increasing amounts of hnRNP H (2 and 4 pmol) or hnRNP F (4 pmol). Lane 1: input RNA; arrow: non-polyadenylated RNA substrate. (f) Reconstituted polyadenylation assays using the 32P-labeled r17/HBB pre-cleaved substrate and PAP (0.1 pmol), in the presence of R17–PTB (4 pmol) and/or hnRNP H (4 pmol). Arrow: non-polyadenylated RNA substrate. (g) GST pull-down assay to test the interaction between GST-tagged PTB and PAP in the absence or presence of hnRNP H. The input lane accounts for 10% of hnRNP H and PAP used in the assay. (h) IP pull down assay with hnRNP H and PAP using the hnRNP H/F antibody. The input lane accounts for 10% of PAP used in the assay.
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Figure 6: PTB promotes polyadenylation by increasing the binding of hnRNP H to a GRS located upstream of the HBB pA signal. (a) Illustration of the r17/HBB pre-cleaved substrate containing a GRS element upstream of the pA signal (named UGRS). (b) In vitro polyadenylation assays using the 32P-labeled r17/HBB pre-cleaved substrate and NEs in the absence or presence of R17–U2AF65 (10 pmol) or increasing amounts of R17–PTB (2.5, 5 and 10 pmol). Normalized pA efficiency is shown. (c) UV crosslinking of NE proteins using the 32P-labeled r17/HBB pre-cleaved substrate with (5 pmol) or without R17–PTB. The arrow indicates the 50-kDa protein whose binding to the HBB pre-cleaved RNA is increased upon R17–PTB addition. (d) IP of NE proteins UV crosslinked to the 32P-labeled r17/HBB pre-cleaved substrate in the absence or presence of R17–PTB (5 pmol) or R17 (5 pmol) using the hnRNP H/F antibody. (e) Reconstituted polyadenylation assays using the 32P-labeled r17/HBB pre-cleaved substrate, PAP (0.1 pmol) and PABPN1 (1.2 pmol), in the presence of increasing amounts of hnRNP H (2 and 4 pmol) or hnRNP F (4 pmol). Lane 1: input RNA; arrow: non-polyadenylated RNA substrate. (f) Reconstituted polyadenylation assays using the 32P-labeled r17/HBB pre-cleaved substrate and PAP (0.1 pmol), in the presence of R17–PTB (4 pmol) and/or hnRNP H (4 pmol). Arrow: non-polyadenylated RNA substrate. (g) GST pull-down assay to test the interaction between GST-tagged PTB and PAP in the absence or presence of hnRNP H. The input lane accounts for 10% of hnRNP H and PAP used in the assay. (h) IP pull down assay with hnRNP H and PAP using the hnRNP H/F antibody. The input lane accounts for 10% of PAP used in the assay.
Mentions: GST pull-down assays were performed by incubating 1 μg of purified GST–R17 or GST–R17–PTB bound to 20 μl of glutathione agarose beads with 200 μg of HeLa NEs (Figure 3a) in NETN buffer (20 mM Tris at pH 8.0, 100 mM NaCl, 0.5% NP-40, 0.5 mM EDTA) for 60 min at 4°C. Beads were then washed five times, treated with 10 μg/ml RNAse A at room temperature for 30 min and washed again. Protein elution was performed by adding SDS loading buffer to the beads. Eluted proteins were resolved by 10% SDS–polyacrylamide gel electrophoresis (PAGE) and analyzed by western blot. GST pull-down assays shown in Figures 3b and 6g and h were performed as described above except that it was done with 1 μg of His-tagged hnRNP H, hnRNP F or PAP, and bound proteins were visualized by Coomassie blue staining.Figure 1.

Bottom Line: We identify PTB as the protein factor whose binding to the human beta-globin (HBB) 3' UTR is abrogated by a 3' end processing-inactivating mutation.We show that PTB promotes both in vitro 3' end cleavage and polyadenylation and recruits directly the splicing factor hnRNP H to G-rich sequences associated with several pA signals.Therefore, our results provide evidence of a concerted regulation of pA signal recognition by splicing factors bound to auxiliary polyadenylation sequence elements.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U563, Toulouse, Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan, Toulouse, F-31300, France. stefania.millevoi@inserm.fr

ABSTRACT
Polypyrimidine tract-binding protein (PTB) is a splicing regulator that also plays a positive role in pre-mRNA 3' end processing when bound upstream of the polyadenylation signal (pA signal). Here, we address the mechanism of PTB stimulatory function in mRNA 3' end formation. We identify PTB as the protein factor whose binding to the human beta-globin (HBB) 3' UTR is abrogated by a 3' end processing-inactivating mutation. We show that PTB promotes both in vitro 3' end cleavage and polyadenylation and recruits directly the splicing factor hnRNP H to G-rich sequences associated with several pA signals. Increased binding of hnRNP H results in stimulation of polyadenylation through a direct interaction with poly(A) polymerase. Therefore, our results provide evidence of a concerted regulation of pA signal recognition by splicing factors bound to auxiliary polyadenylation sequence elements.

Show MeSH
Related in: MedlinePlus