Efficient trans-Splicing of Mutated Spliced Leader Exons in Leishmania tarentolae

Abstract

Every kinetoplastid mRNA receives a common, conserved leader sequence via the process of trans-splicing. In Leishmania tarentolae the precursor spliced leader RNA is 96 nucleotides, with a 39-nucleotide exon that is 7meG-capped and methylated on the first 4 nucleotides. trans-Splicing was inferred from the presence of tagged leader in the high molecular weight RNA population and confirmed for accuracy by cDNA cloning. Linker scan substitutions within the exon between positions 10 and 39 did not affect trans-splicing. The trans-splicing efficiency for three of the scan exons was proportional to the tagged:wild type ratio in the spliced leader precursor population. Two scan leader RNAs that were efficiently spliced showed reduced methylation. Longer exons showed reduced splicing, whereas 10-or 20-base pair deletions abolished splicing. These results indicate that size, but not content, of the exon is a constraint on the splicing process. These results, in combination with previous data eliminating a role in transcription initiation, suggest that translation may be the selective pressure on the leader content.The 39-nt 1 spliced leader (SL) in the kinetoplastids is extensively conserved (1-3), such that PCR amplification of SL RNA genes from diverse kinetoplastids can be performed with a single set of oligonucleotide primers (4, 5). These conserved sequences are presumed to be important for one or more basic biological processes, which could include transcription, transsplicing, and translation. In the nematode Ascaris, part of the 22-nt SL sequence functions as a promoter (6); however, a major role for the SL in transcription initiation has been eliminated in Leishmania tarentolae and Trypanosoma brucei, which have upstream promoters (7,8). An Ascaris SL of two nucleotides is accurately trans-spliced in vitro (9), indicating that information for splicing is not contained within the SL. By contrast, the SL sequence is important for both transcription (10) and trans-splicing (11) in some kinetoplastids.Although the U5 small nuclear RNA may be cross-linked to exons near the splice junction (12), the roles of exons in cissplicing are generally constrained by their protein-coding capacities. There are no such constraints apparent in trans-splicing. The kinetoplastid SL functions as a cap 4 donor (13) and as a 5Ј-untranslated region, allowing the possibility for an active role in trans-splicing. Several inter-and intramolecular interactions have been predicted to occur within the SL. Two small RNAs, SL-associated 1 and U5, have been demonstrated through in vivo cross-linking to interact with the SL (14, 15). Intramolecular interactions of the SL include alternation between two structural forms involving SL-SL (Form I) and SLintron (Form II) base pairing in vitro (16) and in vivo (17) and proposed U1 and U5 functions (18).To determine the importance of the conserved kinetoplastid SL in trans-splicing we evaluated several SL mutants for transsplicing in vivo. We show that SLs containing 10-bp replacement mutations between...

On the Role of Exon and Intron Sequences intrans-Splicing Utilization and cap 4 Modification of the Trypanosomatid Leptomonas collosoma SL RNA

“…A summary of the results of our present (this study) and past mutational analysis of the L. collosoma SL RNA (17) is presented in Table I. In the following sections we discuss and contrast our results with those obtained in the L. seymouri and L. tarentolae systems (13,15,16). Fig.…”

“…In contrast, the importance of the stem I sequence and structure for trans-splicing, which is of special interest as it includes the 5Ј-splice site, has led to opposite results in the L. seymouri system (13) as compared with those obtained in the L. tarentolae system (15) and in this study. Mutations that substituted the 5Ј-portion of stem I, which is absolutely conserved in all known trypanosomatid protozoa, did not affect trans-splicing in L. tarentolae (linker scan mutant 20/29) (15) and in this study (mutants E-5 and E-4, Table I), whereas in L. seymouri mutations in the same domain (mutant SL-sub5) led to severe inhibition of transsplicing (13). However, whereas the SL-sub5 mutation in L. seymouri disrupted the integrity of most of the stem I structure creating in its place a bulge of 6 nt, the E-4 and E-5 mutations in L. collosoma and mutation 20/29 in L. tarentolae still had the potential to form stem structures of five or more adjacent nucleotides.…”

“…tion substituting nt 1-9 of the exon gave rise to SL RNA that was properly modified as determined by the primer extension assay (15). This was highly surprising in light of the fact that the linker substitution changed the first four nucleotides from AACU to CTCG, and thus it was not clear how the SL-specific methyltransferases could generate a cap 4 on such a sequence.…”

“…This was based on the observation that two linker scanning mutant substituting positions 10 -19 and 20 -29 of the exon showed about 5% of "properly" modified cap 4 but had wild-type activity when trans-splicing activity was determined by an RT-PCR assay (15). These studies also showed that a linker-scanning muta- 9.…”

“…tarentolae indicated that mutations encompassing positions 1-4 of the SL RNA, corresponding to the methylated nucleotides of the cap 4 structure, affect modification and transsplicing utilization of the SL RNA, but the function of the individual nucleotide was not investigated (13,15). To address this question positions 1-4 of the SL RNA were either substituted in block or individual nucleotides were changed one at the time (Fig.…”