(Received for publication, April 14, 1995; and in revised form, June 28, 1995)
From the
Characteristics of trans-splicing in Schistosoma mansoni were examined to explore the significance and determinants of spliced leader (SL) addition in flatworms. Only a small subset of mRNAs acquire the SL. Analysis of 30 trans-spliced mRNAs and four genes revealed no discernable patterns or common characteristics in the genes, mRNAs, or their encoded proteins that might explain the functional significance of SL addition. While the mRNA encoding the glycolytic enzyme enolase is trans-spliced, mRNAs encoding four other glycolytic enzymes are not, indicating trans-splicing is not prevalent throughout this metabolic pathway. Although the 3` end of flatworm SLs contribute an AUG to mRNAs, the SL AUG does not typically serve to provide a methionine for translation initiation of reading frames in recipient mRNAs. SL RNA expression exhibits no apparent sex, tissue, or cell specificity. Trans-spliced genes undergo both cis- and trans-splicing, and the sequence contexts for these respective acceptor sites are very similar. These results suggest trans-splicing in flatworms is most likely associated either with some property conferred on recipient mRNAs by SL addition or related to some characteristic of the primary transcripts or transcription of trans-spliced genes.
Trans-splicing is an RNA processing event that accurately joins
sequences derived from independently transcribed RNAs. In one form of
trans-splicing, a leader sequence (the spliced leader, SL) ()is donated from the 5` end of a small, non-polyadenylated
RNA (the spliced leader RNA, SL RNA) to pre-mRNAs to form the
5`-terminal exon of mature mRNAs (for recent reviews see (1, 2, 3, 4, 5, 6) ).
This form of RNA maturation was first described in trypanosomes (7, 8) and subsequently in other kinetoplastida and
the flagellated protozoan Euglena(9) . The
identification of trans-splicing in two divergent invertebrate phyla,
first in nematodes (10) and then in flatworms(11) ,
suggests that this particular form of RNA processing may be an
important form of gene expression common in early metazoa.
The
general distribution of trans-splicing and its origin in metazoa is
currently not known. Furthermore, both the origin of early metazoan
groups and the phylogenetic relationships between flatworms, nematodes,
and other early invertebrates have been difficult to
delineate(12, 13) . Trans-splicing may have arisen
independently in several invertebrate lineages (6) and, if
true, the characteristics and functional significance of spliced leader
addition might also be different in diverse metazoan groups.
Trans-splicing is of particular interest in flatworms (Phylum
Platyhelminthes) as these metazoa may represent the earliest bilateral
animals, and one possible evolutionary tree places a flatworm-like
ancestor as the progenitor of a number of other early invertebrate
groups(12, 13) . We have recently shown that
trans-splicing is present in diverse trematode flatworms and in a
predominantly free-living group generally considered to represent
primitive flatworms(14) . ()This suggests that
spliced leader addition may have been present in the flatworm
progenitor and in the ancestors of parasitic flatworms.
The primary function(s) of most trans-splicing in metazoa remains unknown. We have analyzed several characteristics of spliced leader addition in the flatworm Schistosoma mansoni to explore the biological significance of trans-splicing in flatworms and to provide a comparative metazoan perspective. We previously noted that not all mRNAs acquire the spliced leader in schistosomes(11) . In the present study, we identified and partially characterized 30 mRNAs and four genes that are trans-spliced in S. mansoni to increase our understanding of the molecular characteristics and general properties of trans-splicing in flatworms. The mRNAs were examined to determine 1) if there are any discernable patterns in the proteins they encode, 2) if mRNAs in a particular pathway are trans-spliced as a group, 3) if any other general characteristics of trans-spliced mRNAs were evident, and 4) if the AUG conserved at the 3` end of all flatworm SLs (11, 14) provides the methionine for translation initiation of recipient mRNAs. Genes coding for trans-spliced mRNAs were analyzed to investigate the general organization of these genes and for conserved elements associated with the trans-splice acceptor sites that might distinguish these sites from cis-splice acceptor sites or facilitate bringing the SL RNA and pre-mRNA substrates together for trans-splicing. Finally, the expression of the SL RNA and several trans-spliced mRNAs was also examined by in situ hybridization in adult worms to determine if there is any possible sex, tissue, or cell specificity in trans-splicing.
Our results described herein suggest that the functional significance of flatworm trans-splicing does not appear to be correlated with specific types of mRNAs or the proteins they encode nor with restricted expression of the SL RNA to specific cells, tissues, or sex. This suggests that the functional significance of trans-splicing in flatworms is more likely associated either with properties conferred on recipient mRNAs by addition of the spliced leader or related to the characteristics of transcription and the primary transcripts of trans-spliced genes.
Because only a relatively small subset of mRNAs appears to acquire the spliced leader in schistosomes, we identified and characterized trans-spliced mRNAs and several of their genes as one approach to determine if their type or organization could provide information on the potential function(s) and regulation of trans-splicing in flatworms. We used several approaches to construct cDNA libraries enriched for mRNAs with spliced leaders and isolated and characterized portions of 30 trans-spliced mRNAs (see ``Materials and Methods''). These cDNAs were analyzed to determine if there are any discernable patterns in the type of computer-predicted proteins encoded as wall as the general sequence or secondary structure characteristics of these mRNAs. cDNAs were also examined to determine if addition of the SL to mRNAs was required to provide the initiator methionine for open reading frames (ORFs) or contributed some other property to the 5` ends of the mRNAs. Representative cDNAs were selected from each of the libraries and analyzed either by primer extension analysis or direct sequencing of 5`-RACE products to provide independent confirmation that the cDNAs represented mRNAs with 5`-terminal SLs. From these analyses we estimated that at least 80% of the clones isolated from the SL-enriched libraries represent mRNAs with 5`-terminal spliced leaders.
Figure 1: Schistosoma mansoni trans-spliced gene organization. Schematics illustrate the exon (boxes) and intron organization for each gene, the location of the trans-splice acceptor site(s), and the location of the translation initiation site (AUG). The horizontal lines represent the extent of sequence generated for each locus. Note that the scales for each schematic vary. A, enolase gene (5,050 nucleotides). The discontinuity between exon 6 and 7 indicates that the entire sequence of the intron was not determined. B, L11 gene (1,020 nucleotides). C, 5` end of the HMG-CoA reductase gene (2,285 nucleotides). The discontinuity between exons 2 and 3 illustrates that the entire sequence of the intron was not determined. D, 5` end of the synaptobrevin gene (1060 nucleotides). The discontinuities (-//-) in the sequence are present to keep the figure to scale. The upstream region corresponds to 400 bases and the downstream region to 300 bases of nucleotide sequence.
Secondary structure and base pairing interactions have been implicated as phylogenetically conserved elements associated with self-splicing and snRNA-mediated cis- and trans-splicing. We examined the regions adjacent to the trans-splice acceptor sites in the four genes for homologous sequences or potential secondary structures that might be involved in facilitating the interaction of the two RNA substrates and/or the specificity of the trans-splicing reaction. Conserved elements were not observed in the trans-spliced genes.
Figure 2:
SL RNA expression in adult Schistosoma mansoni. In situ hybridization on
paraformaldehyde fixed paraffin sections of adult S. mansoni was performed with sense or antisense S-labeled SL
RNA probes. Control hybridization using a sense RNA corresponding to
the SL RNA sequence represents background (A and C).
SL RNA expression is shown using an antisense SL RNA probe (B and D-F). Grains associated with the antisense SL RNA
probe were absent when sections were pretreated prior to hybridization
with RNase A, but were not effected when pre-treated with DNase I (not
shown). The arrows in A and B denote one of
the five adjacent testes present in the males. F, represents
the grains over nuclei in the testes at higher magnification. The arrows in C-E denote nuclei. The nuclei marked in C (SL RNA probe) and E (anti-SL RNA probe) are from
adjacent sections. Exposure times for A and B were
five times longer than C-E. Magnification: A and B, =
20; C-F, =
200.
No apparent common motifs or patterns were observed in our sampling of 30 trans-spliced schistosome mRNAs and their encoded proteins. Similarly, analysis of the trans-spliced genes did not reveal any unique or inherent characteristics when compared with non-trans-spliced schistosome genes. Although the glycolytic enzyme enolase is derived from a trans-spliced mRNA, four other glycolytic enzymes are not, indicating that trans-splicing of mRNAs does not appear common to this particular metabolic pathway. Furthermore, in situ hybridization analysis of adult schistosomes indicates that the SL RNA exhibits no gross sex, tissue, or cell specificity. An AUG is absolutely conserved at the 3` terminus of all flatworm spliced leaders. We found, however, that addition of the spliced leader AUG is not typically required to initiate computer-predicted ORFs in trans-spliced schistosome mRNAs. Together, these observations suggest that the significance of trans-splicing in flatworms is more likely to be correlated either with other properties conferred by the SL on recipient mRNAs or related to some characteristic of the primary transcripts or transcription of trans-spliced genes.
Analysis of C. elegans and Ascaris mRNAs which acquire spliced leaders (3, 22, 23) and the current data base of trans-spliced nematode mRNAs indicates that it is also unlikely that trans-splicing is related to particular types of pathways, encoded proteins, or restricted to particular cells or tissues in nematodes(6, 34, 40) . Furthermore, there is no general conservation of particular genes that are trans-spliced in metazoa, since for example, glyceraldehyde 3-phosphate dehydrogenase is trans-spliced in Caenorhabditis spp., but not in schistosomes, and the homolog of the mitochondrial ATPase inhibitor in Caenorhabditis spp. is not trans-spliced, while the analogous mRNA in schistosomes acquires an SL(41) .
The 5` ends of both nematode (42, 43, 44) and flatworm (11, 14) SL RNAs have a trimethylguanosine (TMG) cap. This cap is transferred to nematode actin mRNAs during the trans-splicing reaction(45, 46) . Transfer of the TMG cap to mRNAs presumably also occurs in schistosomes. Capping of mRNAs by spliced leader addition appears essential for mRNA stability in trypanosomes(47, 48) , and the TMG cap or the SL sequence itself might also affect schistosome mRNA stability, translation, transport, cytoplasmic localization, cis-splicing, or other processing of precursor mRNAs.
Two spliced leaders are present in the nematode C. elegans, SL1 and SL2. Although SL1 trans-splicing constitutes the majority of trans-splicing in both C. elegans and Ascaris, its function remains largely unknown. In trypanosomes, trans-splicing plays a role in resolving polycistronic transcription units into individual mRNAs(49, 50, 51, 52) . These individual mRNAs are generated by 5` processing through trans-splicing of the SL and 3` processing via cleavage and polyadenylation. Recently, Blumenthal and colleagues (34, 41) have shown that the subset of trans-spliced C. elegans mRNAs acquiring SL2 are processed from internal genes within operons transcribed as polycistronic transcripts. SL2 appears specialized for processing of genes located within these operons in C. elegans(6) . Except for one unusual case(53) , SL1 is not known to be associated with the resolution of polycistronic transcripts in C. elegans. It will be of interest to determine if regions upstream or downstream from trans-spliced schistosome genes express detectable mature mRNAs (derived from the same DNA coding strand) to explore the possibility for polycistronic transcription across these loci.
Trans-splicing could be functionally associated with transcription initiation. Transcription initiation sites for these genes might be located significantly upstream of the trans-splice acceptor site or be unusually heterogeneous (1) producing long 5`-untranslated regions or ones of highly mixed lengths. Trans-splicing might then function to trim the mRNAs and generate shorter, uniform 5` ends. Although inherently difficult in trans-spliced genes, it will be of interest to attempt to identify and characterize transcription initiation sites in the genes described here to investigate this potential function for spliced leader addition in schistosomes.
All
four trans-spliced schistosome genes we characterized undergo
cis-splicing. Similarly, all nematode genes which undergo
trans-splicing almost invariably exhibit cis-splicing. The presence of
cis- and trans-splicing within the same primary transcript would
ostensibly require the splicing machinery to discriminate between these
sites for accurate RNA processing and generation of functionally mature
mRNAs. Our comparison of a small sampling of trans-splice and
cis-splice acceptor site sequences and their contexts indicates that
the two types of schistosome splice acceptor sites are similar. In
nematodes, significant differences between cis- and trans-splice
acceptor site sequences have also not been
observed(6, 40, 54) . The consensus for
cis-splicing in nematodes (UUUC/AGG) is similar to that which we
describe here in schistosomes (UYU
/AGR),
although the polypyrimidine tract upstream from the acceptor site in
schistosomes is more pronounced than in nematodes. Both nematodes and
flatworms have higher A/U content within introns than within exons. The
transition in A/U content between introns and exons is significantly
greater in nematodes (54) and is a determinant in splice site
recognition(55) .
Detailed studies using a hybrid gene in transgenic nematodes suggest that when the 5` most splice acceptor site within a primary transcript is not preceded by an upstream splice donor, that these elements are sufficient to identify a transcript as an appropriate SL1 trans-splice acceptor substrate(56) . Addition of a 5` splice site upstream of a trans-splice acceptor site in this paradigm alters the splicing exclusively to cis-splicing(57) . Thus, a 5` unpaired splice-acceptor site appears necessary and sufficient to direct SL1 trans-splicing to an appropriate site. Similar 5` unpaired splice-acceptor sites may direct trans-splicing to appropriate acceptor sites in schistosomes. In the S. mansoni HMG-CoA and the synaptobrevin genes, two distinct trans-spliced mRNAs are produced(11) . Whether the two distinct trans-spliced mRNAs from these two genes are derived by alternative trans-splicing within the same primary transcript, distinct transcription initiation sites for the mRNAs, or if inefficient cis-splicing is responsible for the generation of these different mRNAs is currently not known. Analysis of transcription initiation sites and the primary transcription units for schistosome genes will be necessary to provide a better understanding of the substrates, splice acceptor site choices, and processing of trans-spliced genes in schistosomes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U30175[GenBank]-U30183[GenBank], U30258[GenBank]-U30266[GenBank], and U30291[GenBank].