, 1986) has suggested that differences in mRNA splicing processes exist between plants and animals. To gain more information about the specificity of plant pre-mRNA processing, we have compared the splicing of the soybean leghemoglobin pre-mRNA with that of the human I-globin pre-mRNA in transfected plant (Orychophragmus violaceus and Nicotiana tabacum) protoplasts and mammalian (HeLa) cells. Of the three introns of leghemoglobin pre-mRNA, only intron 2 was correctly and efficiently processed in HeLa cells. The 5' splice sites of the remaining two introns were faithfully recognized, but correct processing of the 3' sites took place only rarely (intron 1) or not at all (intron 3); cryptic 3' splice sites were used instead. While the first intron in human ,-globin pre-mRNA was not spliced in transfected plant protoplasts, intron 2 processing occurred at a low level, indicating that some mammalian introns can be recognized by the plant intron-splicing machinery. However, excision of intron 2 proved to be incorrect, involving the authentic 5' splice site and a cryptic 3' splice site. Our results indicate that the mechanism of 3'-splice-site selection during intron excision differs between plants and animals. This conclusion is supported by analysis of the 3'-splice-site consensus sequences in animal and plant introns which revealed that polypyrimidine tracts, characteristic of animal introns, are not present in plant pre-mRNAs. It is proposed that an elevated AU content of plant introns is important for their processing.The splicing of nuclear pre-mRNAs in eucaryotes is a complex multistep process (for reviews, see references 16 and 33). The pre-mRNA is first assembled into a large ribonucleoprotein complex called the spliceosome. In mammalian systems its assembly requires, in addition to the appropriate signals in the RNA substrate, the heterogeneous nuclear ribonucleoprotein polypeptides, small nuclear ribonucleoprotein particles Ul, U2, U4 to U6, and US, and several other less-defined soluble proteins. Excision of an intron (IVS) then occurs in two stages. First, the pre-mRNA is cleaved at the intron 5' border to release exon 1 (Exl). An intron-Ex2 branched intermediate (a lariat) is formed simultaneously. In the second step, cleavage at the 3' splice site and ligation of the two exons occur, accompanied by the release of the lariat intron.Although much information is now available about spliceosome assembly and the importance of the splice junction and branch point consensus sequences in splicing (for reviews, see references 16 and 33), little is known about the structural features which distinguish introns from exons and which establish introns as the sequences to be removed from pre-mRNA. It is evident that the consensus nucleotides are in most instances not sufficient for this purpose; similar sequences are frequently present elsewhere in RNA but are not recognized. It has been proposed that the spatial structure of pre-mRNA may be an important factor in determining the choice of splice sites for processing (...