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

Abstract

In trypanosomatid protozoa the biogenesis of mature mRNA involves addition of the spliced leader (SL) sequence from the SL RNA to polycistronic pre-mRNA via trans-splicing. Here we present a mutational analysis of the trypanosomatid Leptomonas collosoma SL RNA to further our understanding of its functional domains important for trans-splicing utilization. Mutant SL RNAs were analyzed for defects in modification of the hypermethylated cap structure (cap 4) characteristic of trypanosomatid SL RNAs, for defects in the first step of the reaction and overall utilization in trans-splicing. Single substitution of the cap 4 nucleotides led to undermethylation of the cap 4 structure, and these mutants were all impaired in their utilization in trans-splicing. Abrogation of the sequence of the Sm-like site and sequences downstream to it also showed cap modification and trans-splicing defects, thus providing further support for a functional linkage between cap modifications and trans-splicing. Further, we report that in L. collosoma both the exon and intron of the SL RNA contribute information for efficient function of the SL RNA in trans-splicing. This study, however, did not provide support for the putative SL RNA-U6 small nuclear RNA (snRNA) interaction at the Sm site like in the nematodes, suggesting differences in the bridging role of U6 in the two trans-splicing systems.In trypanosomatid protozoa all mRNAs carry at their 5Ј-end a common spliced leader sequence (SL) 1 that is acquired by trans-splicing, a bimolecular splicing reaction involving a small capped RNA, the SL RNA, and the pre-mRNA (1). Transsplicing occurs in a variety of eukaryotic organisms including trypanosomes (1), Euglena (2), nematodes (3), trematodes (4), and more recently in chordates (5). The SL RNA of all organisms has two domains, namely the SL sequence or exon, which in trypanosomatid protozoa is 39 -41 nt long, followed by the intron of variable length, and can be folded into a conserved secondary structure with three stem-loops (see Fig. 1). The first stem-loop encompasses part of the SL sequence just upstream from the 5Ј-splice site, whereas the intron has the potential to be folded into two stem-loops separated by a single-stranded region. This latter region, also known as the Sm-like site, contains the binding site for the core proteins of the SL ribonucleoprotein particle (SL RNP) and is analogous to the Sm site of snRNAs in higher eukaryotes and yeast. Despite the evolutionary conservation of the SL RNA secondary structure, the SL RNA sequence is not conserved among the different groups of organisms listed above. However, within trypanosomatid protozoa, as well within nematodes and trematodes, the exon sequence shows a considerable degree of conservation at the primary sequence level (6). One distinguishing feature of SL RNAs of trypanosomatid protozoa is the presence of a hypermethylated cap with the structure 7-methylguanosine-. By convention this cap structure is referred to as a cap 4, because four nucleotides after the m 7 G moiet...

A protein related to the vaccinia virus cap-specific methyltransferase VP39 is involved in cap 4 modification in Trypanosoma brucei

“…We think this result is compatible with existing evidence and, in particular, with the outcome of the study carried out in the L. collosoma system (Mandelboim et al 2002). Mutation of the +3 cytosine residue produced severe defects in the L. collosoma SL RNA cap structure and in the accumulation of Y-structure intermediates.…”

“…3A). As shown previously, at low deoxyribonucleotide triphosphate concentrations a series of characteristic primer extension stops at the 5¢ end of the SL are indicative of a fully methylated cap 4 structure (Mandelboim et al 2002). Specifically, the strong +5 primer extension stop, which is detected in the SL RNA from uninduced MT57-RNAi cells (lane 2), is diagnostic of the presence of N 3 ,2¢-O-dimethyluridine at position +4 of the SL RNA, because methylation at N 3 of uridine prevents hydrogen bonding with adenosine.…”

“…These studies also revealed that the cap 4 modifications are not required for the assembly of the SL RNA into a core ribonucleoprotein particle (RNP), for the stability of the SL RNA or for the proper folding of the SL RNA in vivo (Harris et al 1995). In addition, genetic analysis in Leptomonas collosoma revealed that each of the four nucleotides of the cap 4 plays a crucial role for SL RNA function in trans-splicing (Mandelboim et al 2002). However, the most dramatic phenotypes were obtained by replacement of nucleotides at positions +1, +2 and +3, whereas substitution of position +4 led only to partial inhibition of trans-splicing utilization of the SL RNA.…”

“…Since single nucleotide substitutions at positions +3 or +4 were severely detrimental to trans-splicing utilization of the L. collosoma SL RNA (Mandelboim et al 2002), we examined trans-splicing activity in mt57 À/À cells. To this end we carried out primer extension analysis with an oligonucleotide primer complementary to nucleotides 110-131 of the SL RNA intron (Fig.…”

Structure-Function Analysis of Trypanosoma brucei RNA Triphosphatase and Evidence for a Two-metal Mechanism

“…Kinetoplastid mRNAs acquire their 5Ј caps via trans-splicing of an RNA leader sequence containing the cap 4 structure. The hypermethylated cap is required for trans-splicing (7)(8)(9).…”