- Email: [email protected]
Trans-Splicing
2040
Tra ns - S p li c i n g
Parinov S and Sundaresan V (2000) Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project. Current Opinion in Biotechnology 11: 157±161.
See also: Insertion Sequence; P Elements; Phage Mu; Transposable Elements; Transposable Elements in Plants; Transposase
Trans-Splicing T Blumenthal Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1334
Most instances of RNA splicing involve removal of internal sequences of a single molecule and splicing together of the two surrounding sequences. In contrast, trans-splicing results in the splicing of two originally separate RNA molecules. Trans-splicing can take several different forms: (1) the splicing of a so-called spliced leader (SL) onto the 50 ends of mRNAs, which occurs in trypanosomes, euglena, roundworms, flatworms, and primitive chordates (2) group II splicing of separate RNAs in certain organellar systems, and (3) situations in which separate RNAs undergo trans-splicing by group-I-dependent, groupII-dependent, or spliceosome-dependent mechanisms, either because they have been engineered to do so or because of a rare, poorly understood, low-frequency event. Because these three kinds of trans-splicing are unrelated processes and are grouped here only because they are each classified as `trans-splicing,' they will be considered separately.
Spliced Leader Addition In this kind of trans-splicing, a short donor RNA contributes its 50 end to form the 50 end of an mRNA. The spliced leader (SL) replaces the 50 sequences of the premRNA, and the reaction is catalyzed by most of the same machinery that catalyzes nuclear intron removal. That is, these are spliceosome-catalyzed reactions. The donor in trans-splicing is itself a small nuclear ribonucleoprotein particle. It is comprised of a short RNA, the SL RNA, which is 100±135 nucleotides in length, and several bound proteins. The first 21 to 51 nucleotides of the SL RNA are transferred to a recipient RNA by trans-splicing. The SL RNA is folded into a three-stem/loop structure with a conventional Sm protein-binding site located between the second and third stem. The SL snRNP contains the Sm proteins also found on U1, U2, U4, and U5 snRNPs, as well as some unique proteins that have not been found
on other snRNPs. The spliced leader itself is immediately followed by a conventional 50 splice site that acts as the donor in trans-splicing. The recipient in transsplicing is a standard pre-mRNA in most respects. However it differs from most pre-mRNAs by beginning with an intron-like sequence, sometimes called an outron, instead of the usual exon at the 50 end. The outron ends with a conventional 30 splice site that acts as the trans-splice acceptor. The 50 splice site on the SL RNA interacts with a branch point in the outron to form a Y-branched intermediate that is subsequently resolved by splicing of the short SL to the 30 splice site at the end of the outron. The resulting products are (1) the SL spliced to the first exon of the acceptor RNA, and (2) the outron branched to the downstream portion of the SL RNA. The latter is presumably debranched and the nucleotides recycled as with the lariat byproducts of cis-splicing or intron removal, the more familiar nuclear splicing event. Transsplicing is catalyzed by most of the same snRNPs as catalyze cis-splicing. One exception though is the U1 snRNP responsible for recognition and choice of the 50 splice site. In trans-splicing U1 plays no role since the 50 splice site is present on a snRNP already. In fact it is base paired in all known SL snRNPs to the SL itself in a short helix reminiscent of the U1 RNA/50 splice site helix. However, this base pairing is not required for trans-splicing in vitro or in vivo. In trypanosomes and at least some nematodes (and possibly some flatworms) many or all of the acceptor molecules are synthesized as polycistronic precursors. Each pre-mRNA contains RNA copies of several genes, and in these cases trans-splicing is used to resolve the polycistronic precursor into mature monocistronic mRNAs. In addition, 30 end formation occurs just upstream (generally about 100±400 nt upstream) which results in a polyadenylated upstream mRNA and an SL-containing downstream mRNA. In these cases, the trans-splicing reaction follows the same course as described above except the branching occurs at a branch point between genes rather than near the 50 end of the mRNA. In trypanosomes, there is only a single SL RNA, which is used for trans-splicing both at the 50 ends and at internal sites in polycistronic mRNAs. In the nematode Caenorhabditis elegans, about 25% of genes are transcribed as parts of polycistronic precursors containing two to more than five genes. There is a special SL RNA, called SL2, which is used for trans-splicing at trans-splice sites between genes in these polycistronic pre-mRNAs. SL2 RNA has a secondary structure similar to the SL RNAs described above, but its sequence is different. In C. elegans, many polycistronic precursors must undergo SL1-trans-splicing at their 50 ends, intron removal throughout, and SL2-trans-splicing at
Tran sve ct i on 2041 internal trans-splice sites between genes. How are these different processes accomplished with specificity? Not all the players in the reaction are known yet, but it is clear that an intron or synthetic intronlike RNA can serve as an outron if placed at the 50 end of a pre-mRNA. Furthermore an outron can be excised as an intron if a 50 splice site is placed within it. Thus the context of a 30 splice site, rather than any particular sequence, determines whether it is subjected to trans- or cis-splicing. In general, a 30 splice site near the 50 end of a pre-mRNA, with no upstream 50 splice site, will be trans-spliced. This can be most easily understood by envisioning a spliceosome beginning to form around a 30 splice site; if an upstream U1 snRNP bound to a 50 splice site pairs with it, then cissplicing occurs, whereas if no upstream site is found, then the SL snRNP provides a 50 splice site in trans. The rules for SL2 trans-splicing at internal sites in polycistronic pre-mRNAs are less clear. In trypanosomes the downstream trans-splicing event determines the location of upstream 30 end formation. However, in worms the events are largely independent, although interference with 30 end formation does affect the SL2 specificity of trans-splicing. In the only operon studied so far, a 22-nucleotide U-rich sequence about 30 nt downstream of the 30 end formation site has been shown to be required for utilization of SL2. It is not yet known what trans-acting factors interact with this sequence. The sequence of the remainder of the intercistronic region is not required for transsplicing.
Group II Trans-Splicing Group II introns occur in plant mitochondria and chloroplasts. Most exist between adjacent exons and their removal by cis-splicing is dependent on a complex secondary structure containing six stem/loop domains. However, in some instances, especially in the nad1, nad2, and nad5 genes of higher plant mitochondria, the exons have become rearranged. In these cases the individual pieces of the genes are transcribed separately. The transcripts can then form the analogous stems by intermolecular base pairing and the splicing occurs in trans just as if it were occurring within a single transcript. In some cases one of the transcripts contains no exon sequences; apparently its only purpose is to bring the correct exons together in a trimolecular stem±loop structure to allow the correct trans-splicing to occur.
II intron splicing that normally occurs in cis. Furthermore the eukaryotic nuclear mRNA splicing machinery can splice together two separate RNAs by conventional mechanisms in vitro. This reaction is relatively efficient when spliceosomes are offered two substrates, one of which contains only a 30 splice site while the other has only a 50 splice site. In these cases, splice sites normally used for cis-splicing are used in trans. A low level of trans-splicing occurs in vivo as well. Trans-splicing has been detected in a variety of cells, always cases of splicing between two different mRNAs at cis-splice sites. These have been detected by RT-PCR, which greatly amplifies rare products, and by isolation of single rare cDNA clones. Nevertheless, there have now been numerous reports that can be explained only by trans-splicing having occurred. Most of these examples have occurred in mammalian cells. So far, it is not clear what has brought the two exons from separate mRNAs together. One possibility would be formation of an RNA double helix or other tertiary structure involving the two molecules which could artificially bring the two splice sites into proximity. It has been possible to force trans-splicing to occur in mammalian systems by engineering two molecules in which portions of the `intron' sequences can anneal. In these cases it appears as if the splicing machinery is `fooled' into believing the 50 and 30 splice sites are on the same molecule and so it splices them together, creating a hybrid molecule. This is splicing in trans, but it is presumably mechanistically identical to normal splicing, since conventional 50 and 30 splice sites are used. The significance of these rare events is unclear, since there are no cases in which the products of these trans-spliced chimeric RNAs have been shown to function. Presumably this sort of trans-splicing is just an unintended consequence of normal nuclear pre-mRNA processing events. See also: Pre-mRNA Splicing
Transvection Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.2057
Other Instances of Trans-Splicing
Transvection is the ability of a locus to influence activity of an allele on the other homolog only when the two chromosomes are synapsed.
It is well established that autocatalytic Group I splicing can be engineered to occur in trans, as can Group
See also: Synapsis in DNA Transactions
Tra ns - S p li c i n g
Parinov S and Sundaresan V (2000) Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project. Current Opinion in Biotechnology 11: 157±161.
See also: Insertion Sequence; P Elements; Phage Mu; Transposable Elements; Transposable Elements in Plants; Transposase
Trans-Splicing T Blumenthal Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1334
Most instances of RNA splicing involve removal of internal sequences of a single molecule and splicing together of the two surrounding sequences. In contrast, trans-splicing results in the splicing of two originally separate RNA molecules. Trans-splicing can take several different forms: (1) the splicing of a so-called spliced leader (SL) onto the 50 ends of mRNAs, which occurs in trypanosomes, euglena, roundworms, flatworms, and primitive chordates (2) group II splicing of separate RNAs in certain organellar systems, and (3) situations in which separate RNAs undergo trans-splicing by group-I-dependent, groupII-dependent, or spliceosome-dependent mechanisms, either because they have been engineered to do so or because of a rare, poorly understood, low-frequency event. Because these three kinds of trans-splicing are unrelated processes and are grouped here only because they are each classified as `trans-splicing,' they will be considered separately.
Spliced Leader Addition In this kind of trans-splicing, a short donor RNA contributes its 50 end to form the 50 end of an mRNA. The spliced leader (SL) replaces the 50 sequences of the premRNA, and the reaction is catalyzed by most of the same machinery that catalyzes nuclear intron removal. That is, these are spliceosome-catalyzed reactions. The donor in trans-splicing is itself a small nuclear ribonucleoprotein particle. It is comprised of a short RNA, the SL RNA, which is 100±135 nucleotides in length, and several bound proteins. The first 21 to 51 nucleotides of the SL RNA are transferred to a recipient RNA by trans-splicing. The SL RNA is folded into a three-stem/loop structure with a conventional Sm protein-binding site located between the second and third stem. The SL snRNP contains the Sm proteins also found on U1, U2, U4, and U5 snRNPs, as well as some unique proteins that have not been found
on other snRNPs. The spliced leader itself is immediately followed by a conventional 50 splice site that acts as the donor in trans-splicing. The recipient in transsplicing is a standard pre-mRNA in most respects. However it differs from most pre-mRNAs by beginning with an intron-like sequence, sometimes called an outron, instead of the usual exon at the 50 end. The outron ends with a conventional 30 splice site that acts as the trans-splice acceptor. The 50 splice site on the SL RNA interacts with a branch point in the outron to form a Y-branched intermediate that is subsequently resolved by splicing of the short SL to the 30 splice site at the end of the outron. The resulting products are (1) the SL spliced to the first exon of the acceptor RNA, and (2) the outron branched to the downstream portion of the SL RNA. The latter is presumably debranched and the nucleotides recycled as with the lariat byproducts of cis-splicing or intron removal, the more familiar nuclear splicing event. Transsplicing is catalyzed by most of the same snRNPs as catalyze cis-splicing. One exception though is the U1 snRNP responsible for recognition and choice of the 50 splice site. In trans-splicing U1 plays no role since the 50 splice site is present on a snRNP already. In fact it is base paired in all known SL snRNPs to the SL itself in a short helix reminiscent of the U1 RNA/50 splice site helix. However, this base pairing is not required for trans-splicing in vitro or in vivo. In trypanosomes and at least some nematodes (and possibly some flatworms) many or all of the acceptor molecules are synthesized as polycistronic precursors. Each pre-mRNA contains RNA copies of several genes, and in these cases trans-splicing is used to resolve the polycistronic precursor into mature monocistronic mRNAs. In addition, 30 end formation occurs just upstream (generally about 100±400 nt upstream) which results in a polyadenylated upstream mRNA and an SL-containing downstream mRNA. In these cases, the trans-splicing reaction follows the same course as described above except the branching occurs at a branch point between genes rather than near the 50 end of the mRNA. In trypanosomes, there is only a single SL RNA, which is used for trans-splicing both at the 50 ends and at internal sites in polycistronic mRNAs. In the nematode Caenorhabditis elegans, about 25% of genes are transcribed as parts of polycistronic precursors containing two to more than five genes. There is a special SL RNA, called SL2, which is used for trans-splicing at trans-splice sites between genes in these polycistronic pre-mRNAs. SL2 RNA has a secondary structure similar to the SL RNAs described above, but its sequence is different. In C. elegans, many polycistronic precursors must undergo SL1-trans-splicing at their 50 ends, intron removal throughout, and SL2-trans-splicing at
Tran sve ct i on 2041 internal trans-splice sites between genes. How are these different processes accomplished with specificity? Not all the players in the reaction are known yet, but it is clear that an intron or synthetic intronlike RNA can serve as an outron if placed at the 50 end of a pre-mRNA. Furthermore an outron can be excised as an intron if a 50 splice site is placed within it. Thus the context of a 30 splice site, rather than any particular sequence, determines whether it is subjected to trans- or cis-splicing. In general, a 30 splice site near the 50 end of a pre-mRNA, with no upstream 50 splice site, will be trans-spliced. This can be most easily understood by envisioning a spliceosome beginning to form around a 30 splice site; if an upstream U1 snRNP bound to a 50 splice site pairs with it, then cissplicing occurs, whereas if no upstream site is found, then the SL snRNP provides a 50 splice site in trans. The rules for SL2 trans-splicing at internal sites in polycistronic pre-mRNAs are less clear. In trypanosomes the downstream trans-splicing event determines the location of upstream 30 end formation. However, in worms the events are largely independent, although interference with 30 end formation does affect the SL2 specificity of trans-splicing. In the only operon studied so far, a 22-nucleotide U-rich sequence about 30 nt downstream of the 30 end formation site has been shown to be required for utilization of SL2. It is not yet known what trans-acting factors interact with this sequence. The sequence of the remainder of the intercistronic region is not required for transsplicing.
Group II Trans-Splicing Group II introns occur in plant mitochondria and chloroplasts. Most exist between adjacent exons and their removal by cis-splicing is dependent on a complex secondary structure containing six stem/loop domains. However, in some instances, especially in the nad1, nad2, and nad5 genes of higher plant mitochondria, the exons have become rearranged. In these cases the individual pieces of the genes are transcribed separately. The transcripts can then form the analogous stems by intermolecular base pairing and the splicing occurs in trans just as if it were occurring within a single transcript. In some cases one of the transcripts contains no exon sequences; apparently its only purpose is to bring the correct exons together in a trimolecular stem±loop structure to allow the correct trans-splicing to occur.
II intron splicing that normally occurs in cis. Furthermore the eukaryotic nuclear mRNA splicing machinery can splice together two separate RNAs by conventional mechanisms in vitro. This reaction is relatively efficient when spliceosomes are offered two substrates, one of which contains only a 30 splice site while the other has only a 50 splice site. In these cases, splice sites normally used for cis-splicing are used in trans. A low level of trans-splicing occurs in vivo as well. Trans-splicing has been detected in a variety of cells, always cases of splicing between two different mRNAs at cis-splice sites. These have been detected by RT-PCR, which greatly amplifies rare products, and by isolation of single rare cDNA clones. Nevertheless, there have now been numerous reports that can be explained only by trans-splicing having occurred. Most of these examples have occurred in mammalian cells. So far, it is not clear what has brought the two exons from separate mRNAs together. One possibility would be formation of an RNA double helix or other tertiary structure involving the two molecules which could artificially bring the two splice sites into proximity. It has been possible to force trans-splicing to occur in mammalian systems by engineering two molecules in which portions of the `intron' sequences can anneal. In these cases it appears as if the splicing machinery is `fooled' into believing the 50 and 30 splice sites are on the same molecule and so it splices them together, creating a hybrid molecule. This is splicing in trans, but it is presumably mechanistically identical to normal splicing, since conventional 50 and 30 splice sites are used. The significance of these rare events is unclear, since there are no cases in which the products of these trans-spliced chimeric RNAs have been shown to function. Presumably this sort of trans-splicing is just an unintended consequence of normal nuclear pre-mRNA processing events. See also: Pre-mRNA Splicing
Transvection Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.2057
Other Instances of Trans-Splicing
Transvection is the ability of a locus to influence activity of an allele on the other homolog only when the two chromosomes are synapsed.
It is well established that autocatalytic Group I splicing can be engineered to occur in trans, as can Group
See also: Synapsis in DNA Transactions