Alternate trans splicing in Trypanosoma equiperdum: implications for splice site selection.

Abstract

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 ...

Coupling of poly(A) site selection and trans-splicing in Leishmania.

“…In many eukaryotes the splice acceptor is characterized by well-defined motifs, including a pyrimidine-rich tract and an AG dinucleotide, and data in both trypanosomes and Leishmania on abundant mRNAs are consistent with this (Layden and Eisen 1988;Huang and Van der Ploeg 1991a;de Lafaille et al 1992). However, our data on nonabundant RNAs, such as DHFR-TS and DST, suggest that the situation may be more complex.…”

“…These two cleavages result in the formation of monocistronic mRNAs bearing both 5' caps and 3' poly(A) sequences. Consensus motifs for the trans-splicing acceptor site of Leishmania and trypanosomes resemble those of mammals in that they possess an AG acceptor site and an adjacent upstream pyrimidine-rich region that are essential for correct splicing of the procyclic acidic repetitive protein (PARP) and cx-tubulin genes in trypanosomes (Mount 1982;Layden and Eisen 1988;Huang and Van der Ploeg 1991a;de Lafaille et al 1992). In contrast, no conserved motifs participating in polyadenylation have been identified in trypanosomatid genes (Borst 1986;Kapler et al 1990a); and heterologous polyadenylation signals, such as the herpes simplex virus thymidine kinase (HSV tk) poly(A) site, do not function in Leishmania (Kapler et al 1990b).…”

Structural isoforms of a membrane transport protein from Leishmania enriettii.

“…This result is typical for comparison of Si nuclease-protected and primer-extended fragments derived from the 5' termini of Leishmania mRNAs (11) and is due to the presence of the 39-nucleotide (16) spliced leader or miniexon sequence (13) that is transspliced onto the 5' ends of all known mRNAs from kinetoplastid protozoa (2). Furthermore, two potential splice acceptor sites (12) occur within the p2.75 sequence (underlined AGs in Fig. 3) at the position where spliced leader addition should occur, as determined by these mapping experiments.…”

“…The two AG sequences underlined (nucleotides 1211 to 1212 and 1221 to 1222) are potential splice acceptor sequences that are near the 5' end of the p2.75 exon, as determined by S1 mapping (Fig. 5), and could serve as acceptor sites for the 39-nucleotide spliced leader (12). The amino acids in italics are from the previously determined (3) sequence of the Pro-1 polypeptide; hence, these represent the amino-terminal domain of isoform 2 (cross-hatched boxes in Fig.…”

Mapping of branch sites in trans-spliced pre-mRNAs of Trypanosoma brucei.

“…One hypothesis is that in mammalian cells, U2 RNA (as part of the U2 small nuclear ribonucleoprotein particle) might first interact with the pre-mRNA via the polypyrimidine regions and subsequently select a suitable branch point sequence via base pairing. It is currently not possible to carry out similar experiments with trypanosomes because of the lack of an in vitro trans-splicing system; however, data obtained by Layden and Eisen (13) suggest that pyrimidine-rich sequences act as splicing signals in trypanosomes also. They note that for the VSG-178 and VSG-78 genes of Trypanosoma equiperdum, which have multiple SL addition sites, those sites that are used most frequently contain the highest pyrimidine content in the adjacent upstream sequences.…”