Abstract
Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions. The network accurately captures known physical and functional associations and identifies new ones, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during spliceosome assembly. In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF, and on targets of antitumor drugs, and can be widely used to identify mechanisms of splicing regulation.
Highlights
Pre-mRNA splicing is carried out by the spliceosome, one of the most complex molecular machineries of the cell, composed of five small nuclear ribonucleoprotein particles (U1, U2, U4/5/6 snRNP) and about 150 additional polypeptides
We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions
In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties
Summary
Pre-mRNA splicing is carried out by the spliceosome, one of the most complex molecular machineries of the cell, composed of five small nuclear ribonucleoprotein particles (U1, U2, U4/5/6 snRNP) and about 150 additional polypeptides (reviewed by Wahl et al, 2009). U2 snRNP binding involves interactions of pre-mRNA sequences with U2 snRNA as well as with U2 proteins (e.g., SF3B1). Transition between spliceosomal subcomplexes involves profound dynamic changes in protein composition as well as extensive rearrangements of base-pairing interactions between snRNAs and between snRNAs and splice site sequences (Wahl et al, 2009). RNA structures contributed by base-pairing interactions between U2 and U6 snRNAs serve to coordinate metal ions critical for splicing catalysis (Fica et al, 2013), implying that the spliceosome is an RNA enzyme whose catalytic center is only established upon assembly of its individual components
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