Intramolecular base pairing between the nematode spliced leader and its 5′ splice site is not essential for trans-splicing in vitro.

Maroney, Patricia A.; Hannon, Gregory J.; Shambaugh, Joseph D.; Nilsen, Timothy W.

doi:10.1002/j.1460-2075.1991.tb04956.x

Cited by 36 publications

(25 citation statements)

References 24 publications

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“…Although this level of dissimilarity has not been shown in other species containing multiple spliced leaders, this finding is not necessarily unexpected. Mutagenesis studies have shown there is considerable flexibility in the sequence and structural requirements for splicing of the C. elegans SL-1 RNA (34,35). We now have multiple examples of genes whose mRNAs can accept either SL-A or -B.…”

Section: Discussionmentioning

confidence: 99%

Trans-spliced leader addition to mRNAs in a cnidarian

Stover

Steele

2001

Proc. Natl. Acad. Sci. U.S.A.

108

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Section: Discussionmentioning

confidence: 99%

Trans-spliced leader addition to mRNAs in a cnidarian

Stover

Steele

2001

Proc. Natl. Acad. Sci. U.S.A.

108

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“…Therefore, it was hypothesized that the base pairing of the trans-splice site with the SL mimicked the interaction of the 5Ј splice site with the U1 snRNA (5). However, the Ascaris SL RNA can function in vitro without such base pairing (34). Indeed, the entire SL exon is dispensable and can be replaced with sequences varying in length from two to 246 nt while maintaining the ability to trans splice (34).…”

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

“…In particular, different groups have reported very different results when they mutate or delete portions of the SL itself (15,19,32,34,47,56). In nematodes it is clear that the SL1 sequence can be replaced by unrelated sequences of various lengths without loss of in vitro trans splicing (34), but at least one SL mutation was found to prevent trans splicing in vivo (15). In contrast, mutational analysis of the intron portion of the molecule demonstrated that both stem-loop II and the Sm binding region are crucial for SL RNA function in both trypanosomes and nematodes (11,23,49).…”

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confidence: 99%

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trans Splicing of PolycistronicCaenorhabditis elegans Pre-mRNAs: Analysis of the SL2 RNA

Evans

Blumenthal

2000

Molecular and Cellular Biology

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Genes in Caenorhabditis elegans operons are transcribed as polycistronic pre-mRNAs in which downstream gene products are trans spliced to a specialized spliced leader, SL2. SL2 is donated by a 110-nucleotide RNA, SL2 RNA, present in the cell as an Sm-bound snRNP. SL2 RNA can be conceptually folded into a phylogenetically conserved three-stem-loop secondary structure. Here we report an in vivo mutational analysis of the SL2 RNA. Some sequences can be changed without consequence, while other changes result in a substantial loss of trans splicing. Interestingly, the spliced leader itself can be dramatically altered, such that the first stem-loop cannot form, with only a relatively small loss in trans-splicing efficiency. However, the primary sequence of stem II is crucial for SL2 trans splicing. Similarly, the conserved primary sequence of the third stem-loop plays a key role in trans splicing. While mutations in stem-loop III allow snRNP formation, a single nucleotide substitution in the loop prevents trans splicing. In contrast, the analogous region of SL1 RNA is not highly conserved, and its mutation does not abrogate function. Thus, stem-loop III appears to confer a specific function to SL2 RNA. Finally, an upstream sequence, previously predicted to be a proximal sequence element, is shown to be required for SL2 RNA expression.The mature 5Ј ends of mRNAs in many lower eucaryotes, including trypanosomes (41, 50), nematodes (28), trematodes (43), and Euglena (51), are generated through trans splicing. This process involves the donation of a common spliced leader (SL) exon and its associated modified cap structure to the 5Ј ends of mRNAs. The reaction proceeds via two transesterification reactions, analogous to cis splicing of introns, and in fact requires most of the same U snRNP cofactors, including U2, U4, U5, and U6 snRNAs (12,22,36,54). However, because the 5Ј exon is initially located on a separately transcribed RNA, the SL RNA, the intermediate of the trans-splicing reaction is a Y-branched molecule instead of the lariat molecule found in cis splicing. The SL RNA exists as an RNP particle (20,33,52,55) that includes the Sm core proteins that are also associated with most of the U snRNAs involved in cis splicing (30). Unlike the other U snRNPs, which are recycled after catalysis, the SL RNP is consumed during the trans-splicing reaction.In trypanosomes, pre-mRNAs are transcribed polycistronically (26, 40) and the mature mRNAs are generated by trans splicing and 3Ј end formation. Approximately 25% of Caenorhabditis elegans genes are also organized in polycistronic transcription units, or operons (57). However, unlike trypanosomes, C. elegans possesses a distinct SL RNA, SL2 RNA (25), that is specifically utilized to process the products of downstream genes in operons (46). Therefore, the discovery of SL2 on an mRNA is taken as an indication that the gene is in an operon (1, 2).Experiments designed to uncover the mechanism of transsplicing specificity in C. elegans have shown that SL1 trans splicing is usually...

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“…However, conservation of the spliced leader sequence, length, or structure is not required for trans-splicing in vitro (46). Similarly, it is possible that the SL2 sequence itself may not be required for the trans-splicing reaction, but for the transcription of the SL2 RNA gene.…”

Section: Discussionmentioning

confidence: 99%

Operons and SL2 trans-splicing exist in nematodes outside the genus Caenorhabditis

Evans

Zorio

MacMorris

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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The genomes of most eukaryotes are composed of genes arranged on the chromosomes without regard to function, with each gene transcribed from a promoter at its 5 end. However, the genome of the free-living nematode Caenorhabditis elegans contains numerous polycistronic clusters similar to bacterial operons in which the genes are transcribed sequentially from a single promoter at the 5 end of the cluster. The resulting polycistronic pre-mRNAs are processed into monocistronic mRNAs by conventional 3 end formation, cleavage, and polyadenylation, accompanied by trans-splicing with a specialized spliced leader (SL), SL2. To determine whether this mode of gene organization and expression, apparently unique among the animals, occurs in other species, we have investigated genes in a distantly related free-living rhabditid nematode in the genus Dolichorhabditis (strain CEW1). We have identified both SL1 and SL2 RNAs in this species. In addition, we have sequenced a Dolichorhabditis genomic region containing a gene cluster with all of the characteristics of the C. elegans operons. We show that the downstream gene is trans-spliced to SL2. We also present evidence that suggests that these two genes are also clustered in the C. elegans and Caenorhabditis briggsae genomes. Thus, it appears that the arrangement of genes in operons pre-dates the divergence of the genus Caenorhabditis from the other genera in the family Rhabditidae, and may be more widespread than is currently appreciated.In bacteria and archaea, the genomes are primarily organized in arrays of genes whose products have related functions. These gene clusters, called operons, are cotranscribed from an upstream promoter and the resulting polycistronic mRNA is translated by ribosomes initiating at or near the 5Ј end of the RNA. These operons serve to efficiently coregulate proteins that function together. In contrast, eukaryotes have genomes composed of genes arranged apparently at random, with each transcribed by a promoter at its 5Ј end. However, in a group of primitive eukaryotic protozoa, the trypanosomes, genes are transcribed polycistronically (1-3). In this case, the polycistronic pre-mRNA is processed by 3Ј end formation and trans-splicing to create conventional eukaryotic monocistronic mRNAs. The trans-splicing reaction that creates the 5Ј ends of the mRNAs is related to the cis-splicing of higher eukaryotes; it proceeds through a 2Ј-5Ј branched intermediate, the splice sites have the same consensus sequences, and it is catalyzed by some of the same small nuclear ribonucleoprotein particles (4, 5).Trans-splicing was first discovered in trypanosomatids (4, 6), and later shown to occur also in Caenorhabditis elegans and other nematodes (ref. 7; reviewed in refs. 8 and 9), in Euglena (10), and in flatworms (11,12). In contrast to trypanosomes, in which only trans-splicing is present, the genes in the other organisms also contain cis-spliced introns. It was presumed that these genes were monocistronic and arranged randomly on the chromosomes as in other...

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Intramolecular base pairing between the nematode spliced leader and its 5′ splice site is not essential for trans-splicing in vitro.

Cited by 36 publications

References 24 publications

Trans-spliced leader addition to mRNAs in a cnidarian

Trans-spliced leader addition to mRNAs in a cnidarian

trans Splicing of PolycistronicCaenorhabditis elegans Pre-mRNAs: Analysis of the SL2 RNA

Operons and SL2 trans-splicing exist in nematodes outside the genus Caenorhabditis

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