Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro.

We have identified six distinct factors necessary for pre-mRNA splicing in vitro by selective inactivation and complementation studies, and by fractionation procedures. Splicing factor 1 (SF1) is sensitive to micrococcal nuclease, and appears to consist of at least U1 and U2 snRNPs, since splicing is inhibited when the 5' termini of U1 and U2 snRNAs are removed by site-directed cleavage with RNAase H. SF2 is a micrococcal nuclease-resistant factor present in the nuclear extract but absent from an S100 extract.

Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro.

To study the mechanisms of RNA splicing we have analyzed the products generated by in vitro processing of a truncated 32P-labeled human beta-globin RNA precursor that contains the first two exons and the first intervening sequence (IVS1). Six major RNA products were detected and characterized. The first detectable RNA processing event is cleavage at the 5' GT of IVS1.

Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro.

Human beta-globin mRNA precursors (pre-mRNAs) synthesized in vitro from a bacteriophage SP6 promoter/beta-globin gene fusion are accurately and efficiently spliced when added to a HeLa cell nuclear extract. Under optimal conditions, the first intervening sequence (IVS 1) is removed by splicing in up to 90% of the input pre-mRNA. Splicing requires ATP and in its absence the pre-mRNA is neither spliced nor cleaved at splice junctions.

Nucleotide sequence at the end of the gene for the RNA polymerase beta' subunit (rpoC).

We have determined the DNA sequence surrounding the transcription terminator following rpoC, the gene that codes for the beta' subunit of RNA polymerase in E. coli K12. The 2044 bp sequence obtained contains the distal 335 codons of rpoC followed by a 212 bp non-coding region and a second open reading frame (ORFa) of 179 codons.