Alternative trans-splicing: a novel mode of pre-mRNA processing

Alternative splicing is an important process contributing to proteome diversity without involving an increase in the number of genes. In some cases, alternative splicing is carried out under 'trans-mode', called alternative trans-splicing, in which exons located on separate pre-mRNA molecules are selectively joined to produce mature mRNAs encoding proteins with distinct structures and functions. However, it is not known how widespread or how frequently trans-splicing occurs in vivo. Recently, trans-allelic trans-splicing has been unambiguously demonstrated in Drosophila using a SNP (single nucleotide polymorphism) as a marker. In this review, we provide an overview of alternative trans-splicing in Drosophila and mammals, and discuss its mechanisms.     

Bioinformatic analysis of exon repetition, exon scrambling and trans-splicing in humans

MOTIVATION: Using bioinformatic approaches we aimed to characterize poorly understood abnormalities in splicing known as exon scrambling, exon repetition and trans-splicing. RESULTS: We developed a software package that allows large-scale comparison of all human expressed sequence tags (EST) sequences to the entire set of human gene sequences. Among 5,992,495 EST sequences, 401 cases of exon repetition and 416 cases of exon scrambling were found. The vast majority of identified ESTs contain fragments rather than full-length repeated or scrambled exons. Their structures suggest that the scrambled or repeated exon fragments may have arisen in the process of cDNA cloning and not from splicing abnormalities. Nevertheless, we found 11 cases of full-length exon repetition showing that this phenomenon is real yet very rare. In searching for examples of trans-splicing, we looked only at reproducible events where at least two independent ESTs represent the same putative trans-splicing event. We found 15 ESTs representing five types of putative trans-splicing. However, all 15 cases were derived from human malignant tissues and could have resulted from genomic rearrangements. Our results provide support for a very rare but physiological occurrence of exon repetition, but suggest that apparent exon scrambling and trans-splicing result, respectively, from in vitro artifact and gene-level abnormalities. AVAILABILITY: Exon-Intron Database (EID) is available at http://www.meduohio.edu/bioinfo/eid. Programs are available at http://www.meduohio.edu/bioinfo/software.html. The Laboratory website is available at http://www.meduohio.edu/medicine/fedorov Supplementary information: Supplementary file is available at http://www.meduohio.edu/bioinfo/software.html.     

利用反式剪接核酶修复突变绿色荧光蛋白mRNA

为构建修复突变绿色荧七蛋白（GFP）基因的反式剪接核酶,分别构建包含突变的GFP基因的XYQ5／10-pGEM重组质粒、XYQ5／10—pEGFP—C2重组质粒及用于修复该突变基因的反式剪接核酶载体trans—rib—CMV2。通过对体外共转录XYQ5／10—pGEM和trans—rib—CMV2重组质粒的RNA产物进行RT—PCR检测核酶细胞外剪接效果;通过XYQ5／10-pEGFP-C2和trans—rib—CMV2重组质粒共转染HeLa细胞检测核酶细胞内的剪接效果。结果显示,XYQ5／10—pGEM、XYQ5／10-pEGFP-C2及trans—rib—CMV2重组质粒构建成功,反式剪接核酶在细胞外及细胞内都可以修复突变基因。虽然效率不高,但为今后更大规模地研究设计反式剪接核酶打下了基础。     

Alternative mRNA processing increases the complexity of microRNA-based gene regulation in Arabidopsis

During trans-splicing of discontinuous organellar introns, independently transcribed coding sequences are joined together to generate a continuous mRNA. The chloroplast psaA gene from Chlamydomonas reinhardtii encoding the P(700) core protein of photosystem I (PSI) is split into three exons and two group IIB introns, which are both spliced in trans. Using forward genetics, we isolated a novel PSI mutant, raa4, with a defect in trans-splicing of the first intron. Complementation analysis identified the affected gene encoding the 112.4 kDa Raa4 protein, which shares no strong sequence identity with other known proteins. The chloroplast localization of the protein was confirmed by confocal fluorescence microscopy, using a GFP-tagged Raa4 fusion protein. RNA-binding studies showed that Raa4 binds specifically to domains D2 and D3, but not to other conserved domains of the tripartite group II intron. Raa4 may play a role in stabilizing folding intermediates or functionally active structures of the split intron RNA.     

Novel and essential subunits in the 300-kilodalton nuclear cap binding complex of Trypanosoma brucei

One of the unique aspects of RNA processing in trypanosomatid protozoa is the presence of a cap 4 structure (m7Gpppm2(6)AmpAmpCmpm3Um) at the 5' end of all mRNAs. The cap 4 becomes part of the mRNA through trans-splicing of a 39-nucleotide-long sequence donated by the spliced leader RNA. Although the cap 4 modifications are required for trans-splicing to occur, the underlying mechanism remains to be determined. We now describe an unconventional nuclear cap binding complex (CBC) in Trypanosoma brucei with an apparent molecular mass of 300 kDa and consisting of five protein components: the known CBC subunits CBP20 and importin-alpha and three novel proteins that are only present in organisms featuring a cap 4 structure and trans-splicing. Competitive binding studies are consistent with a specific interaction between the CBC and the cap 4 structure. Downregulation of several individual components of the T. brucei CBC by RNA interference demonstrated an essential function at an early step in trans-splicing. Thus, our studies are consistent with the CBC providing a mechanistic link between cap 4 modifications and trans-splicing.     

The spliceosomal snRNAs of Caenorhabditis elegans.

Nematodes are the only group of organisms in which both cis- and trans-splicing of nuclear mRNAs are known to occur. Most Caenorhabditis elegans introns are exceptionally short, often only 50 bases long. The consensus donor and acceptor splice site sequences found in other animals are used for both cis- and trans-splicing. In order to identify the machinery required for these splicing events, we have characterized the C. elegans snRNAs. They are similar in sequence and structure to those characterized in other organisms, and several sequence variations discovered in the nematode snRNAs provide support for previously proposed structure models. The C. elegans snRNAs are encoded by gene families. We report here the sequences of many of these genes. We find a highly conserved sequence, the proximal sequence element (PSE), about 65 bp upstream of all 21 snRNA genes thus far sequenced, including the SL RNA genes, which specify the snRNAs that provide the 5' exons in trans-splicing. The sequence of the C. elegans PSE is distinct from PSE's from other organisms.     

U small nuclear ribonucleoprotein requirements for nematode cis- and trans-splicing in vitro.

In nematodes, a fraction of mRNAs acquires a common 22-nucleotide 5'-terminal spliced leader sequence via a trans-splicing reaction. The same premessenger RNAs which receive the spliced leader are also processed by conventional cis-splicing. Whole cell extracts prepared from synchronous embryos of the parasitic nematode Ascaris lumbricoides catalyze both cis- and trans-splicing. We have used this cell-free system and oligodeoxynucleotide directed RNase H digestion to assess the U small nuclear RNA requirements for nematode cis- and trans-splicing. These experiments indicated that both cis- and trans-splicing require intact U2 and U4/U6 small nuclear ribonucleoproteins (snRNPs). However, whereas cis-splicing displays the expected requirement for an intact U1 snRNP, trans-splicing is unaffected when approximately 90% of U1 snRNP is degraded. These results suggest that 5' splice site identification differs in nematode cis- and trans-splicing.     

On the role of exon and intron sequences in trans-splicing utilization and cap 4 modification of the trypanosomatid Leptomonas collosoma SL RNA

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.     

Functional selection of splicing enhancers that stimulate trans-splicing in vitro.

The role of exonic sequences in naturally occurring trans-splicing has not been explored in detail. Here, we have identified trans-splicing enhancers through the use of an iterative selection scheme. Several classes of enhancer sequences were identified that led to dramatic increases in trans-splicing efficiency. Two sequence families were investigated in detail. These include motifs containing the element (G/C)GAC(G/C) and also 5' splice site-like sequences. Distinct elements were tested for their ability to function as splicing enhancers and in competition experiments. In addition, discrete trans-acting factors were identified. This work demonstrates that splicing enhancers are able to effect a large increase in trans-splicing efficiency and that the process of exon definition is able to positively enhance trans-splicing even though the reaction itself is independent of the need for the 5' end of U1 snRNA. Due to the presence of internal introns in messages that are trans-spliced, the natural arrangement of 5' splice sites downstream of trans-splicing acceptors may lead to a general promotion of this unusual reaction.     

Direct analysis of nematode cis- and trans-spliceosomes: a functional role for U5 snRNA in spliced leader addition trans-splicing and the identification of novel Sm snRNPs.

Most nuclear pre-mRNAs in nematodes are processed by both cis- and trans-splicing. In trans-splicing, the 5' terminal exon, the spliced leader sequence (SL), is derived from a trans-splicing specific Sm snRNP, the SL RNP. Because U snRNPs are required cofactors for trans-splicing, and because this processing reaction proceeds via a two-step reaction pathway identical to that of cis-splicing, it has long been assumed that trans-splicing is catalyzed in a complex analogous to the cis-spliceosome. However, similarities or differences between cis- and trans-spliceosomes have not been established. In particular, the role of U5 snRNP in trans-splicing has been unclear. Here, we have used affinity selection to analyze the U snRNA constituents of nematode cis- and trans-spliceosomes. We find that U5 snRNP is an integral component of the trans-spliceosome and, using site-specific crosslinking, we show that U5 snRNP establishes specific Interactions with the SL RNA exon. We also identify two novel Sm snRNPs that are enriched in both cis- and trans-spliceosomes. Finally, we provide evidence that a SL RNP-containing multi-snRNP (SL, U4, U5, and U6 RNPs) may be a functional precursor in trans-spliceosome assembly.     

Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides.

Synthesis of mRNA in kinetoplastid protozoa involves the process of trans-splicing, in which an identical 39-41-nucleotide (depending on the species) mini-exon is placed at the 5' end of mature mRNAs. The mini-exon sequence is highly conserved among all members of the Kinetoplastida, nucleotides 1-6 being identical in the four genera so far examined. Prior to trans-splicing, the mini-exon donor RNA is capped by the addition of a (5'-5') triphosphate-linked 7-methylguanosine, followed by modification of the first four transcribed nucleotides. Partial structures have been previously deduced for this cap 4 moiety from Trypanosoma brucei and Leptomonas collosoma. We have purified enough cap 4 from T. brucei and Crithidia fasciculata to allow definitive structural analysis by combined liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. The results, together with the known mini-exon sequence, show that cap 4 in both species has the structure m7G(5')ppp(5')m6(2)AmpAmpCmpm3Ump. The presence of N6,N6,2'-O-trimethyladenosine and 3,2'-O-dimethyluridine, nucleosides previously unknown in nature, were confirmed by rigorous comparison with synthetic standards. The conservation of cap 4 between these divergent genera suggests that this structure may be common to most if not all Kinetoplastida.     

Operon structure and trans-splicing in the nematode Pristionchus pacificus

In the nematode Caenorhabditis elegans, up to 15% of the genes are organized in operons. Polycistronic precursor RNAs are processed by trans-splicing at the 5' ends of genes by adding a specific trans-spliced leader. Ten different spliced leaders are known in C. elegans that differ in sequence and abundance. The SL1 leader is most abundant and is spliced to the 5' ends of monocistronic genes and to upstream genes in operons. Trans-splicing is common among nematodes and was observed in the genera Panagrellus, Ascaris, Haemonchus, Anisakis, and Brugia. However, little is known about operons in nonrhabditid nematodes. Dolichorhabditis CEW1, another rhabditid nematode that is now called Oscheius CEW1, contains operons and SL2 trans-splicing. We have studied the presence of operons and trans-splicing in Pristionchus pacificus, a species of the Diplogastridae that has recently been developed as a satellite organism in evolutionary developmental biology. We provide evidence that P. pacificus contains operons and that downstream genes are trans-spliced to SL2. Surprisingly, the one operon analyzed so far in P. pacificus is not conserved in C. elegans, suggesting unexpected genomic plasticity.