Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo

The aminoglycoside antibiotic neomycin B inhibits translation in prokaryote s and interferes with RNA-protein interactions in HIV both in vivo and in v itro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (fd) intron in vivo. All antibiotics tested inhibited splicing, even chlor amphenicol, which does not inhibit splicing in vitro. Splicing of the td in tron in vivo requires translation for proper folding of the pre-mRNA. In th e absence of translation, two interactions between sequences in the upstrea m exon and the 5' and 3' splice sites trap the pre-mRNA in splicing-incompe tent conformations. Their disruption by mutations rendered splicing less de pendent on translation and also less sensitive to neomycin B. Intron splici ng was affected by neither neomycin B nor gentamicin in Escherichia coli st rains carrying antibiotic-resistance genes that modify the ribosomal RNA. T aken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their inter ference with translation. This was further confirmed by assaying splicing o f the Tetrahymena group I intron, which is inserted in the E coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and strep tomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing i s caused by an alternative structural element containing a cryptic 5' splic e site, which serves as a substrate for the ribozyme. Our results demonstra te that aminoglycoside antibiotics display different effects on ribozymes i n vivo and in vitro.