Incorporation of 5-fluorouracil into U2 and U6 snRNA inhibits mRNA precursor splicing.
The splicing activities of 5-fluorouracil (FUra)-substituted U2 and U6 small nuclear RNAs (snRNAs) were examined in an in vitro splicing system. Yeast splicing extracts were specifically depleted of endogenous U2 and U6 snRNAs by antisense oligonucleotide-directed RNase H hydrolysis. Splicing activity was recovered when the extracts were reconstituted with synthetic U2 and U6 snRNAs. However, U2 snRNA with all uracils substituted with FUra (FU2) did not restore any splicing activity. Nondenaturing gel electrophoresis showed that FU2 failed to promote the assembly of spliceosome complexes. The ability of U2 snRNA to restore splicing in U2-depleted extracts increased as FUra content decreased but was still only 60% of control activity at 25% substitution of uracils with FUra. Addition of FU2 to nondepleted extracts caused strong inhibition of splicing accompanied by increased degradation of the pre-mRNA, suggesting that FU2 forms an inactive complex with a protein splicing factor that normally binds to the pre-mRNA. FU6 restored full splicing activity to U6-depleted extracts, but at a 5-fold higher concentration than U6 snRNA. These results demonstrate that the incorporation of FUra can impair the functions of catalytic RNA molecules.
Lenz
H J
HJ
Department of Biochemistry and Molecular Biology, University of Southern California School of Medicine, Los Angeles 90033.
Manno
D J
DJ
Danenberg
K D
KD
Danenberg
P V
PV
D001483:Base Sequence; D002474:Cell-Free System; D017931:DNA Primers / Q000737:chemistry; D005472:Fluorouracil / Q000737:chemistry*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000737:chemistry*; D012441:Saccharomyces cerevisiae; D017381:Spliceosomes / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D017931:DNA Primers; D046911:Macromolecular Substances; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D005472:Fluorouracil