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Updated: Jun 4, 2026
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Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins
Published on: August 9, 2019
U1 snRNA directly interacts with polypyrimidine tract-binding protein during splicing repression.
Shalini Sharma1, Christophe Maris, Frédéric H-T Allain
1Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Molecular Cell
|March 3, 2011
Summary
The polypyrimidine tract-binding protein (PTB) represses c-src N1 exon splicing by directly interacting with U1 snRNA. This interaction inactivates the spliceosome, highlighting novel RNA regulatory mechanisms.
Area of Science:
- Molecular Biology
- RNA Splicing
- Gene Regulation
Background:
- Alternative splicing is a crucial process for generating protein diversity.
- The polypyrimidine tract-binding protein (PTB) is a known regulator of pre-mRNA splicing.
- PTB's role in repressing the c-src N1 exon involves interactions with spliceosomal components.
Purpose of the Study:
- To elucidate the molecular mechanism by which PTB represses c-src N1 exon splicing.
- To investigate the direct interaction between PTB and the U1 small nuclear ribonucleoprotein (snRNP).
- To characterize the binding interface between PTB and U1 snRNA.
Main Methods:
- Nuclease protection assays to study splice site interactions.
- UV crosslinking to identify direct contacts between PTB and pre-mRNA/U1 snRNA.
- Electrophoretic mobility shift assay (EMSA), isothermal titration calorimetry (ITC), and nuclear magnetic resonance (NMR) to characterize binding.
Main Results:
- PTB binding alters the interaction of U1 snRNP with the c-src N1 exon 5' splice site.
- UV crosslinking confirmed a direct contact between PTB and U1 snRNA.
- PTB's RNA recognition motifs (RRMs) 1 and 2 bind to the U1 snRNA stem loop 4, inhibiting spliceosome assembly.
Conclusions:
- PTB directly interacts with U1 snRNA, a novel mechanism for splicing regulation.
- This PTB-U1 snRNA interaction is critical for repressing c-src N1 exon splicing.
- The findings reveal new strategies for how splicing regulatory proteins modulate spliceosome function.
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