Inhibition of mammalian spliceosome assembly and pre-mRNA splicing by peptide inhibitors of protein kinases.

Four peptides are shown to block mammalian spliceosome assembly and pre-mRNA splicing in vitro. Previously, these peptides have been shown to inhibit Ca2+-dependent calmodulin kinase II (CaMK II) via distinct mechanisms. One is a competitive inhibitor of the kinase, two interfere with autophosphorylation events, and one competes for binding to calmodulin, a CaMK II-activating protein. However, because EGTA does not inhibit splicing, the involvement of CaMK II itself in splicing is unlikely; rather, a protein similar to CaMK II may be involved in spliceosome assembly and splicing. Two of the inhibitory peptides, the calmodulin binding domain (CBD) and glycogen synthase (GS) fragment, block assembly of spliceosomal complex C. These peptides inhibited splicing if they were added to reactions any time within the first 10 min of splicing assays. No inhibition of spliceosome assembly or splicing occurred in the presence of randomized versions of the CBD or GS peptide. Additionally, the GS peptide inhibited splicing when added to assays at later time points, despite the fact that spliceosomal complex C had formed. Cumulatively, these analyses suggest that the peptides inhibit at least two distinct events in the spliceosomal cycle. The first event occurs early during in vitro splicing. For this event, prolonged incubations of splicing reactions do not result in a recovery of splicing activity. The second event occurs later and represents a slowing of an essential step, because splicing activity can be recovered in prolonged incubations. Peptides known to inhibit protein kinase A and protein kinase C had no effect on pre-mRNA splicing, underscoring the specificity of the observed inhibitory effects.