The quantitation and distribution of splicing intermediates in HeLa cells and adenovirus RNAs

The steady state level of splicing intermediates in HeLa cells and in adenovirus RNA made late in the infectious cycle has been measured by a branch point analysis. About one in ten poly A(+) nuclear RNAs contained a branch point, but only 1/3 as many adenovirus RNAs were branched. Fewer branches were found in the poly A(-) RNAs of the nucleus and of late adenovirus transcripts suggesting that excised lariat introns do not accumulate in vivo. Branched RNAs were found in the poly A(+) RNAs from a nuclear matrix fraction, but several experiments failed to show an enrichment in these splicing intermediates in this matrix fraction. Branches were found in all size classes of poly A(+) nuclear RNA and were not exclusively associated with either the 3' or 5' regions, but were randomly distributed within RNA molecules. These results as well as the base and sequence data on branch points (1,18) are consistent with the conclusion that branched poly A(+) RNAs are splicing intermediates.