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THE IRE RNA. |
Current
Hall Lab Projects
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Mode of alternative splicing by
Polypyrimidine Tract Binding Protein (PTB). (Dr. Caroline Clerte) Recently, our PTB1 binding studies
have been our PTB1 binding studies have been extended to include the c-src
pre-mRNA. The excluded N1 exon in c-src pre-mRNA is flanked by PTB binding
sites in upstream and downstream introns, and both sites are required for
efficient repression (Chan & Black, 1997). This RNA differs from that of
GABA pre-mRNA in the length of its polypyrimidine tracts, but also in their
context. While the secondary structure of the GABAA receptor γ2
pre-mRNA is predominantly single-stranded, that of c-src contains many
stem-loops with internal bulges of polypyrimidine tracts. We hypothesize that
this difference in structural context of the two RNAs will be a major
determinant of the binding mode of PTB on these two targets. The
predicted secondary structure of c-src pre-mRNA is shown in Figure 1. This is
the complete sequence of the mouse pre-mRNA used in experiments of the Black
lab, and now also by us.
Figure 1. Predicted secondary structure (lowest free energy
conformations from mfold) of the c-src sequence used in our experiments. The
hairpin loop and internal loop in the 5′ intron contain CUCUCUCU where
RNA:PTB crosslinks were observed (Amir-Ahmady et al., 2005). Crosslinks to
PTB were also observed from the CUCUCUCU sequence in the 3′
intron. Our experiments show
that five PTB1 proteins bind to c-src RNA. In addition to the complete sequence shown in Figure 1, we have synthesized several shorter sequences: the 5′ half of the RNA, from nucleotide 1 to nucleotide 78 (includes N1); the 5′ end from nucleotide 1 to 41, and the 3′ half, from nucleotide 79 to 224. These RNAs have been used in binding and stoichiometry assays with PTB and its truncated constructs.
The 5′ half from nt 1-78. Three PTB1 molecules bind
here.
The 5′ end from nt 1-41. Three PTB1 molecules bind. These RNAs have been used
in EMSA and stoichiometry experiments.
PTB1, PTB1:12, and PTB1:34 bind to the RNAs, although with different
affinities. Both RBD1 and RBD4 have been crosslinked to this RNA (Amir-Ahmady
et al., 2005), so PTB1:12 and PTB1:34 are expected to bind, although here
PTB1:34 binding is much weaker than that of PTB1:12. Our hypothesis is that
the conformation of PTB1 on this RNA differs from its arrangement on the GABA
RNA, and that this difference is important for its role in c-src splicing
(Chou et al., 2000). Amir-Ahmady, B., Boutz, P.L.,
Markovtsov, V., Phillips, M.L., Black, D.L. 2005. Exon repression by
polypyrimidine tract binding protein. RNA 11, 699-716. Chan, R.C., Black, D.l.
1997. The polypyrimidine tract binding protein binds upstream of neural
cell-specific c-src exon N1 to repress the splicing of the intron downstream.
Mol Cell Biol 17, 4667-4676. Chou, M-Y., Underwood,
J.G., Nikolic, J., Luu, M.H.T., Black, D.L. 2000. Multisite RNA binding and
release of polypyrimidine tract binding protein during the regulation of
c-src neural-specific splicing. Molecular Cell 5:949-957.
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Dr. Kathleen B. Hall (hall(at)biochem.wustl.edu)
Department of Biochemistry and
660
office: 314-362-4196
lab: 314-362-4197
or 314-747-8079
FAX: 314-362-7183
send email to kathleenhal(at)gmail.com
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