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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 facilitates hnRNP H binding to the HBB pA signal. (a) UV crosslinking using NEs with the 32P-labeled r17/HBB substrate in the absence or presence of R17–PTB. The identity of the 50-kDa protein whose binding to the RNA is increased upon R17–PTB addition (indicated by an arrow) was tested in (b) by IP of the UV-crosslinked complexes with the antibody against hnRNP H/F. SDS–PAGE analysis of the immunoprecipitated complexes UV crosslinked to the 32P-labeled r17/HBB substrate in the absence or presence of R17–PTB or R17. (c) UV crosslinking/IP as described in (b) except for lane 3 in which PTB replaced R17. The poor quality of the migration of the crosslinking reaction is due to the presence of polyvinylalcohol in the reaction. (d) UV crosslinking/IP as described in (b) but using a 32P-labeled r17/HBB substrate containing the DGRS WT or Mut (as described in Figure 4). (e) UV crosslinking of recombinant hnRNP H and/or hnRNP F to the 32P-labeled r17/HBB substrate in the absence or presence of the R17–PTB protein as indicated (upper panel). (f) (Left panel) UV crosslinking/IP of hnRNP H/F from NEs to 32P-labeled RNA substrates containing the r17 moiety upstream of the L3, F2 or C2 pA signals (illustrated in the upper part) in the absence or presence of R17–PTB. (Right panel) UV crosslinking of hnRNP H with or without R17–PTB and using the 32P-labeled r17/HBB, C2 or F2 pA signals.
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Figure 5: PTB facilitates hnRNP H binding to the HBB pA signal. (a) UV crosslinking using NEs with the 32P-labeled r17/HBB substrate in the absence or presence of R17–PTB. The identity of the 50-kDa protein whose binding to the RNA is increased upon R17–PTB addition (indicated by an arrow) was tested in (b) by IP of the UV-crosslinked complexes with the antibody against hnRNP H/F. SDS–PAGE analysis of the immunoprecipitated complexes UV crosslinked to the 32P-labeled r17/HBB substrate in the absence or presence of R17–PTB or R17. (c) UV crosslinking/IP as described in (b) except for lane 3 in which PTB replaced R17. The poor quality of the migration of the crosslinking reaction is due to the presence of polyvinylalcohol in the reaction. (d) UV crosslinking/IP as described in (b) but using a 32P-labeled r17/HBB substrate containing the DGRS WT or Mut (as described in Figure 4). (e) UV crosslinking of recombinant hnRNP H and/or hnRNP F to the 32P-labeled r17/HBB substrate in the absence or presence of the R17–PTB protein as indicated (upper panel). (f) (Left panel) UV crosslinking/IP of hnRNP H/F from NEs to 32P-labeled RNA substrates containing the r17 moiety upstream of the L3, F2 or C2 pA signals (illustrated in the upper part) in the absence or presence of R17–PTB. (Right panel) UV crosslinking of hnRNP H with or without R17–PTB and using the 32P-labeled r17/HBB, C2 or F2 pA signals.

Mentions: To investigate whether PTB was able to recruit hnRNP H to HBB transcripts, we analyzed hnRNP H RNA binding activity upon addition of R17–PTB using UV crosslinking/IP experiments. The 32P-labeled r17/HBB pA signal RNA was incubated in NEs followed by the addition of the recombinant R17–PTB. The UV crosslinking pattern showed an increase in the binding of a 55-kDa species upon addition of R17–PTB (Figure 5a). This band was identified as hnRNP H/F by IP of the crosslinked proteins with the hnRNP H/F antibody (Figure 5b). Addition of R17–PTB (Figure 5b), but not of R17 (Figure 5b) or of PTB alone (Figure 5c), resulted in an enhanced hnRNP H crosslinking only in the presence of a WT DGRS (Figure 5d), suggesting that the physical interaction between PTB and hnRNP H creates a molecular link across the HBB pA signal.


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 facilitates hnRNP H binding to the HBB pA signal. (a) UV crosslinking using NEs with the 32P-labeled r17/HBB substrate in the absence or presence of R17–PTB. The identity of the 50-kDa protein whose binding to the RNA is increased upon R17–PTB addition (indicated by an arrow) was tested in (b) by IP of the UV-crosslinked complexes with the antibody against hnRNP H/F. SDS–PAGE analysis of the immunoprecipitated complexes UV crosslinked to the 32P-labeled r17/HBB substrate in the absence or presence of R17–PTB or R17. (c) UV crosslinking/IP as described in (b) except for lane 3 in which PTB replaced R17. The poor quality of the migration of the crosslinking reaction is due to the presence of polyvinylalcohol in the reaction. (d) UV crosslinking/IP as described in (b) but using a 32P-labeled r17/HBB substrate containing the DGRS WT or Mut (as described in Figure 4). (e) UV crosslinking of recombinant hnRNP H and/or hnRNP F to the 32P-labeled r17/HBB substrate in the absence or presence of the R17–PTB protein as indicated (upper panel). (f) (Left panel) UV crosslinking/IP of hnRNP H/F from NEs to 32P-labeled RNA substrates containing the r17 moiety upstream of the L3, F2 or C2 pA signals (illustrated in the upper part) in the absence or presence of R17–PTB. (Right panel) UV crosslinking of hnRNP H with or without R17–PTB and using the 32P-labeled r17/HBB, C2 or F2 pA signals.
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Figure 5: PTB facilitates hnRNP H binding to the HBB pA signal. (a) UV crosslinking using NEs with the 32P-labeled r17/HBB substrate in the absence or presence of R17–PTB. The identity of the 50-kDa protein whose binding to the RNA is increased upon R17–PTB addition (indicated by an arrow) was tested in (b) by IP of the UV-crosslinked complexes with the antibody against hnRNP H/F. SDS–PAGE analysis of the immunoprecipitated complexes UV crosslinked to the 32P-labeled r17/HBB substrate in the absence or presence of R17–PTB or R17. (c) UV crosslinking/IP as described in (b) except for lane 3 in which PTB replaced R17. The poor quality of the migration of the crosslinking reaction is due to the presence of polyvinylalcohol in the reaction. (d) UV crosslinking/IP as described in (b) but using a 32P-labeled r17/HBB substrate containing the DGRS WT or Mut (as described in Figure 4). (e) UV crosslinking of recombinant hnRNP H and/or hnRNP F to the 32P-labeled r17/HBB substrate in the absence or presence of the R17–PTB protein as indicated (upper panel). (f) (Left panel) UV crosslinking/IP of hnRNP H/F from NEs to 32P-labeled RNA substrates containing the r17 moiety upstream of the L3, F2 or C2 pA signals (illustrated in the upper part) in the absence or presence of R17–PTB. (Right panel) UV crosslinking of hnRNP H with or without R17–PTB and using the 32P-labeled r17/HBB, C2 or F2 pA signals.
Mentions: To investigate whether PTB was able to recruit hnRNP H to HBB transcripts, we analyzed hnRNP H RNA binding activity upon addition of R17–PTB using UV crosslinking/IP experiments. The 32P-labeled r17/HBB pA signal RNA was incubated in NEs followed by the addition of the recombinant R17–PTB. The UV crosslinking pattern showed an increase in the binding of a 55-kDa species upon addition of R17–PTB (Figure 5a). This band was identified as hnRNP H/F by IP of the crosslinked proteins with the hnRNP H/F antibody (Figure 5b). Addition of R17–PTB (Figure 5b), but not of R17 (Figure 5b) or of PTB alone (Figure 5c), resulted in an enhanced hnRNP H crosslinking only in the presence of a WT DGRS (Figure 5d), suggesting that the physical interaction between PTB and hnRNP H creates a molecular link across the HBB pA signal.

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