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Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism
J E Mermoud1, P T Cohen, A I Lamond
1European Molecular Biology Laboratory.
Abstract:
Splicing of mRNA precursors (pre-mRNA) is preceded by assembly of the pre-mRNA with small nuclear ribonucleoprotein particles (snRNPs) and protein factors to form a splicesome. Here we show that stimulating Ser/Thr-specific protein dephosphorylation selectively inhibits an early step during mammalian spliceosome assembly. Treatment of HeLa nuclear splicing extracts with human protein phosphatase 1 (PP1) expressed in Escherichia coli, or PP1 purified from rabbit skeletal muscle, prevents pre-spliceosome E complex (early complex) formation and stable binding of U2 and U4/U6.U5 snRNPs to the pre-mRNA. PP1 does not inhibit splicing catalysis if added after spliceosome assembly has taken place. Addition of purified SR protein splicing factors restores spliceosome formation and splicing to PP1-inhibited extracts, consistent with SR proteins being targets regulated by phosphorylation. These data extend earlier observations showing that splicing catalysis, but not spliceosome assembly, is blocked by inhibiting protein phosphatases. It therefore appears that pre-mRNA splicing, in common with other biological processes, can be regulated both positively and negatively by reversible protein phosphorylation.
Insights
Stimulating protein dephosphorylation with protein phosphatase 1 (PP1) inhibits early spliceosome assembly, preventing pre-mRNA binding. Restoring SR proteins rescues spliceosome formation, highlighting phosphorylation
Area of Science:
- Molecular Biology
- RNA Processing
- Gene Expression Regulation
Background:
- Pre-mRNA splicing requires assembly of spliceosomes, involving snRNPs and protein factors.
- Protein phosphorylation plays a critical role in regulating biological processes, including splicing.
Purpose of the Study:
- To investigate the role of protein dephosphorylation in mammalian spliceosome assembly.
- To determine the specific step of spliceosome assembly affected by protein phosphatase 1 (PP1).
Main Methods:
- Treatment of HeLa nuclear splicing extracts with purified human or rabbit PP1.
- Analysis of spliceosome assembly intermediates, including E complex formation and snRNP binding.
- Assessing the effect of SR protein addition on PP1-inhibited splicing extracts.
Main Results:
- PP1 treatment inhibited early spliceosome assembly, specifically preventing E complex formation.
- PP1 blocked the stable binding of U2 and U4/U6.U5 snRNPs to pre-mRNA.
- PP1 did not inhibit splicing catalysis once spliceosomes were assembled.
- Addition of SR proteins restored spliceosome formation and splicing in PP1-treated extracts.
Conclusions:
- Ser/Thr-specific protein dephosphorylation, mediated by PP1, selectively inhibits early spliceosome assembly.
- SR proteins are likely targets of phosphorylation-dependent regulation during spliceosome assembly.
- Pre-mRNA splicing is regulated by reversible protein phosphorylation, impacting both assembly and catalysis.