In vitro processing of Drosophila melanogaster 5 S ribosomal RNA: 3′ end effects and requirement for internal domains of mature 5 S RNA

5 S RNA processing in Drosophila melanogaster, the removal of 15 nucleotides from the 3′ end of the 135-nucleotide (nt) primary transcript, may play an important role in the regulation of 5 S RNA transport and ribosome assembly. We have uncoupled processing from transcription using gel purified primary transcripts processed in vitro by a cellular S100 extract. The RNA was generated by a homologous transcription system or by a T7 RNA polymerase reaction using a constructed 5 S RNA gene linked to a T7 promoter. In vitro D. melanogaster 5 S RNA processing is heat- and EDTA-sensitive, suggesting a requirement for protein, and produces a 3′ end characteristic of mature 5 S RNA. Processing of substrate RNAs with altered 3′ ends shows that the 3′-U₅ tail (nt 131-135) inhibits the reaction. 30 nt, including all of domain IV and most of domain V, are dispensible for processing, whereas deletions including the base of stem V and all or part of stem III severely inhibit it. Several possible mechanisms are discussed.