We examined the structures of the 5' ends of mRNAs encoding variant surface glycoprotein 78 (VSG-78) and VSG-178 in Trypanosoma equiperdum. Several mRNA species were found for each gene, and all contained the 35-base miniexon (or spliced leader) sequence attached at different positions on their 5' ends. Thus, the generation of multiple messages for each VSG occurred by attachment of the miniexon at one of several 3' splice acceptor sites. The frequency with which individual splice sites were used varied from less than 1 to 95% of the RNA produced from a particular gene. We propose that the miniexon RNA and RNA from the VSG genes may interact via base pairing and that this in part specifies the use of particular acceptor sites. Sequences complementary to the miniexon primary transcript, termed the "med-comp site," were found in both genes and in several published sequences. Splice sites were most often used if they were the first site 3' of the med-comp site and contained a high pyrimidine content in the bases preceeding the AG acceptor signal.In higher eucaryotic cells, mRNA maturation involves several well-known alterations. These include terminal modifications at the 5' and 3' ends and the removal of intervening sequences. The maturation of mRNA in protozoan trypanosomatids involves some common features such as 3' poly(A) addition and 5' capping, but the latter similarity is somewhat complicated. The modified cap structure and a 35-base sequence called the miniexon are donated to all mRNAs by a small (approximately 135 nucleotides) short-lived (half-life of less than 6 min) RNA termed the medRNA or miniexon primary transcript (4,11,17).Several models for miniexon addition have been proposed. These include the medRNA acting as a primer for transcription, the medRNA ligating to nascent RNAs followed by cis splicing to produce the final mRNA, and trans splicing involving the formation of a branched intermediate between the non-miniexon portion of the medRNA (minRNA) and high-molecular-weight poly(A)+ RNAs (19,30). Models for trans splicing have been proposed which may or may not require base pairing (27,29).The sequence cleaved in the medRNA (releasing the miniexon) resembles the consensus 5' splice donor site seen in vertebrates and Saccharomyces cerevisiae (i.e., AGGU) (20). Also, the attachment of the miniexon always occurs at sites resembling a 3' splice acceptor site (i.e., after an AG dinucleotide) (20). Recently, two groups of investigators studying Trypanosoma brucei have found branched RNA which consists of high-molecular-weight poly(A)+ RNA with the minRNA attached (19, 30). The minRNA can be removed from this structure with (HeLa cell nucleus) debranching enzyme. Polyadenylated nuclear RNA (in yeast and higher eucaryotic cells) has been shown to contain branched RNA intermediates which are formed during cis splicing (21,24,25,34).We studied miniexon addition in variant surface glycoprotein (VSG) genes from Trypanosoma equiperdum. Our results show that several sites can be used for miniexon addition to ...