Analysis of RNA products formed in an in vitro splicing reaction. A nuclear extract from HeLa cells was incubated with a 497-nucleotide radiolabeled RNA that contained portions of two exons (numbered 1 and 2) from human beta-globin mRNA separated by a 130-nucleotide intron. After incubation for various times, the RNA was purified and subjected to electrophoresis and autoradiography, along with RNA markers (lane M). The number of nucleotides in the various species is indicated. Much of the slower-migrating starting RNA (497) was correctly spliced, yielding a 367-nucleotide product. The excised intron (130*) migrated slower than expected based on its size, indicating that it is not a linear molecule. Likewise, one of the reaction intermediates (339*) exhibited an anomalously slow mobility. The 252* band, an aberrant product of the in vitro reaction, is greatly reduced in reactions in which the RNA is capped.