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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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HnRNP H stimulates 3′ end cleavage at the HBB pA signal by binding to a downstream GRS element. (a) Illustration of the HBB region downstream of the cleavage site (CA) and including a GRS located 45 nucleotides downstream of the cleavage site (DGRS). The sequence difference between the wild-type (WT) and mutated DGRS (Mut) is shown. (b) UV crosslinking of NE proteins to the uniformly 32P-labeled r17/HBB pA signal RNA substrate containing the WT or Mut DGRS sequence. The 50-kDa protein crosslinked to the WT but not to the Mut RNA substrate is indicated by the arrow. (c) IP of UV-crosslinked complexes described in panel (b) with the hnRNP H/F antibody followed by SDS–PAGE analysis. (d) NEs depleted of endogenous hnRNP H/F without (mock) or with (depleted) a biotinylated RNA oligonucleotide corresponding to the SVL GRS. NEs were analyzed by SDS–PAGE followed by western blot analysis. (e) In vitro cleavage assays using the 32P-labeled r17/HBB substrate in the presence of mock-depleted NEs (white boxes) or NEs depleted of hnRNP H/F (black boxes), supplemented or not with recombinant hnRNP H (1 pmol). The low cleavage efficiency is due to the depletion procedure. Identities of the uncleaved and cleaved products are shown on the left.
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Figure 4: HnRNP H stimulates 3′ end cleavage at the HBB pA signal by binding to a downstream GRS element. (a) Illustration of the HBB region downstream of the cleavage site (CA) and including a GRS located 45 nucleotides downstream of the cleavage site (DGRS). The sequence difference between the wild-type (WT) and mutated DGRS (Mut) is shown. (b) UV crosslinking of NE proteins to the uniformly 32P-labeled r17/HBB pA signal RNA substrate containing the WT or Mut DGRS sequence. The 50-kDa protein crosslinked to the WT but not to the Mut RNA substrate is indicated by the arrow. (c) IP of UV-crosslinked complexes described in panel (b) with the hnRNP H/F antibody followed by SDS–PAGE analysis. (d) NEs depleted of endogenous hnRNP H/F without (mock) or with (depleted) a biotinylated RNA oligonucleotide corresponding to the SVL GRS. NEs were analyzed by SDS–PAGE followed by western blot analysis. (e) In vitro cleavage assays using the 32P-labeled r17/HBB substrate in the presence of mock-depleted NEs (white boxes) or NEs depleted of hnRNP H/F (black boxes), supplemented or not with recombinant hnRNP H (1 pmol). The low cleavage efficiency is due to the depletion procedure. Identities of the uncleaved and cleaved products are shown on the left.

Mentions: 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.


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)

HnRNP H stimulates 3′ end cleavage at the HBB pA signal by binding to a downstream GRS element. (a) Illustration of the HBB region downstream of the cleavage site (CA) and including a GRS located 45 nucleotides downstream of the cleavage site (DGRS). The sequence difference between the wild-type (WT) and mutated DGRS (Mut) is shown. (b) UV crosslinking of NE proteins to the uniformly 32P-labeled r17/HBB pA signal RNA substrate containing the WT or Mut DGRS sequence. The 50-kDa protein crosslinked to the WT but not to the Mut RNA substrate is indicated by the arrow. (c) IP of UV-crosslinked complexes described in panel (b) with the hnRNP H/F antibody followed by SDS–PAGE analysis. (d) NEs depleted of endogenous hnRNP H/F without (mock) or with (depleted) a biotinylated RNA oligonucleotide corresponding to the SVL GRS. NEs were analyzed by SDS–PAGE followed by western blot analysis. (e) In vitro cleavage assays using the 32P-labeled r17/HBB substrate in the presence of mock-depleted NEs (white boxes) or NEs depleted of hnRNP H/F (black boxes), supplemented or not with recombinant hnRNP H (1 pmol). The low cleavage efficiency is due to the depletion procedure. Identities of the uncleaved and cleaved products are shown on the left.
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Figure 4: HnRNP H stimulates 3′ end cleavage at the HBB pA signal by binding to a downstream GRS element. (a) Illustration of the HBB region downstream of the cleavage site (CA) and including a GRS located 45 nucleotides downstream of the cleavage site (DGRS). The sequence difference between the wild-type (WT) and mutated DGRS (Mut) is shown. (b) UV crosslinking of NE proteins to the uniformly 32P-labeled r17/HBB pA signal RNA substrate containing the WT or Mut DGRS sequence. The 50-kDa protein crosslinked to the WT but not to the Mut RNA substrate is indicated by the arrow. (c) IP of UV-crosslinked complexes described in panel (b) with the hnRNP H/F antibody followed by SDS–PAGE analysis. (d) NEs depleted of endogenous hnRNP H/F without (mock) or with (depleted) a biotinylated RNA oligonucleotide corresponding to the SVL GRS. NEs were analyzed by SDS–PAGE followed by western blot analysis. (e) In vitro cleavage assays using the 32P-labeled r17/HBB substrate in the presence of mock-depleted NEs (white boxes) or NEs depleted of hnRNP H/F (black boxes), supplemented or not with recombinant hnRNP H (1 pmol). The low cleavage efficiency is due to the depletion procedure. Identities of the uncleaved and cleaved products are shown on the left.
Mentions: 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.

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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