B. Pilch et al., Specific inhibition of serine- and arginine-rich splicing factors phosphorylation, spliceosome assembly, and splicing by the antitumor drug NB-506, CANCER RES, 61(18), 2001, pp. 6876-6884
Specific phosphorylation of serine- and arginine-rich pre-m RNA splicing fa
ctors (SR proteins) is one of the key determinants regulating splicing even
ts. Several kinases involved in SR protein phosphorylation have been identi
fied and characterized, among which human DNA topoisomerase I is known to h
ave DNA-relaxing activity. In this study, we have investigated the mechanis
m of splicing inhibition by a glycosylated indolocarbazole derivative (NB-5
06), a potent inhibitor of both kinase and relaxing activities of topoisome
rase 1. NB-506 completely inhibits the capacity of topoisomerase I to phosp
horylate, in vitro, the human splicing factor 2/alternative splicing factor
(SF2/ASF). This inhibition is specific, because NB-506 does not demonstrat
e activity against other kinases known to phosphorylate SF2/ASF such as SR
protein kinase 1 and cdc2 kinase. Importantly, HeLa nuclear extracts compet
ent in splicing but not splicing-deficient cytoplasmic S100 extracts treate
d with the drug fail to phosphorylate SF2/ASF and to support splicing of pr
e-mRNA substrates containing SF2/ASF-target sequences. Native gel analysis
of splicing complexes revealed that the drug affects the formation of the s
pliceosome. a dynamic ribonucleoprotein structure where splicing takes plac
e. In the presence of the drug, neither pre-spliceosome nor spliceosome is
formed, demonstrating that splicing inhibition occurs at early steps of spl
iceosome assembly. Splicing inhibition can be relieved by adding phosphoryl
ated SF2/ASF, showing that extracts treated with NB-506 lack a phosphorylat
ing activity required for splicing. Moreover, NB-506 has a cytotoxic effect
on murine P388 leukemia cells but not on P388CPT5 camptothecin-resistant c
ells that carry two point mutations in conserved regions of topoisomerase I
gene (Gly361Val and Asp709Tyr). After drug treatment, P388 cells accumulat
ed hypophosphorylated forms of SR proteins and polyadenylated RNA in the nu
cleus. In contrast, neither SR protein phosphorylation nor polyadenylated m
RNA distribution was affected in P388 CPT5-treated cells. Consistently, NB5
06 treatment altered the mRNA levels and/or splicing pattern of several tes
ted genes (Bel-X, CD 44, SC35, and Sty) in P388 cells but not in P388 CPT5
cells. The study shows for the first time that indolocarbazole drugs target
ing topoisomerase I can affect gene expression by modulating pre-mRNA splic
ing through inhibition of SR proteins phosphorylation.