Linking the branchpoint helix to a newly found receptor allows lariat formation by a group II intron

Like spliceosomal introns, the ribozyme-containing group II introns are excised as branched, lariat structures: a 2'-5' bond is created between the first nucleotide of the intron and an adenosine in domain VI, a component which is missing from available crystal structures of the ribozyme. Comparative sequence analysis, modelling and nucleotide substitutions point to the existence, and probable location, of a specific RNA receptor for the section of domain VI that lies just distal to the branchpoint adenosine. By designing oligonucleotides that tether domain VI to this novel binding site, we have been able to specifically activate lariat formation in an engineered, defective group II ribozyme. The location of the newly identified receptor implies that prior to exon ligation, the distal part of domain VI undergoes a major translocation, which can now be brought under control by the system of anchoring oligonucleotides we have developed. Interestingly, these oligonucleotides, which link the branchpoint helix and the binding site for intron nucleotides 3-4, may be viewed as counterparts of U2-U6 helix III in the spliceosome.     

The different intron 2 species excised in vivo from the E2A premRNA of adenovirus-2: an approach to analyse alternative splicing.

In the early period of cellular infection by adenovirus 2, the E2A region gives rise to 2 major mRNA species of 2.0 and 2.3 kilobases, formed by alternative excisions of intron 2 (Gattoni et al., 1986, J. Mol. Biol. 187, 379-307). We have analysed the excision pathways of this intron. Two major intron species of 626 and 337 nucleotides, generated by the use of 2 consensus 3' splicing sites and a minor intron species of 520 nucleotides, generated by the use of another weaker 3' splicing site, are identified, the 3 species sharing a common 5' splicing site. They are detected predominantly in the lariat form. For the 2 major species we analyzed, the branched nucleotides are localized at consensus branching sequences, 26 or 25 nucleotides upstream from the 3' terminal AG. Our results confirm that the first reactions of cleavage at the 5' end of introns and branching occur in vivo as described in in vitro systems. The second predominant form of intron 2 is the linear segment, whereas the nicked lariat form which is very minor, might not be a genuine product of in vivo splicing. All intron 2 molecules show practically intact 5' and 3' terminal sequences, indicating that they are well protected against nuclease attack throughout their life. Therefore, these results indicate that the primary reaction following the excision of the lariat intron is debranching. In addition, the existence of a potential 5' splicing site contiguous to the major internal 3' splicing site raised the possibility of an elimination of the major 626 nucleotide intron in 2 cycles of excision. However, we demonstrate that intron 2 is systematically excised by a one cycle process, which is likely to represent the general rule for the production of correctly spliced mRNA.     

U5 snRNA interacts with exon sequences at 5' and 3' splice sites.

U5 snRNA is an essential pre-mRNA splicing factor whose function remains enigmatic. Specific mutations in a conserved single-stranded loop sequence in yeast U5 snRNA can activate cleavage of G1----A mutant pre-mRNAs at aberrant 5' splice sites and facilitate processing of dead-end lariat intermediates to mRNA. Activation of aberrant 5' cleavage sites involves base pairing between U5 snRNA and nucleotides upstream of the cleavage site. Processing of dead-end lariat intermediates to mRNA correlates with base pairing between U5 and the first two bases in exon 2. The loop sequence in U5 snRNA may therefore by intimately involved in the transesterification reactions at 5' and 3' splice sites. This pattern of interactions is strikingly reminiscent of exon recognition events in group II self-splicing introns and is consistent with the notion that U5 snRNA may be related to a specific functional domain from a group II-like self-splicing ancestral intron.     

An alternative intron-exon pairing scheme implied by unexpected in vitro activities of group II intron RmInt1 from Sinorhizobium meliloti

RmInt1 is a mobile group II intron which interrupts ISRm2011-2, another mobile element from the bacterium Sinorhizobium meliloti. Ribozyme constructs derived from intron RmInt1 self-splice in vitro when incubated under permissive conditions, but the excised intron and ligated exons are largely replaced by unconventional products. These include a slightly shorter, 5'-end truncated 3' exon, truncated variants of the linear and lariat forms of the intron-3' exon reaction intermediate, as well as presumably circular molecules derived from the latter. Two factors explain the abundance of these products: (i) nucleotides 5-11 of the 3' exon (IBS1*) provide a better match to the EBS1 5'-exon-binding site than the authentic IBS1 sequence in the 5' exon; (ii) exon ligation is unusually inefficient, and especially so when the 5' exon is truncated close to the second (IBS2) intron-binding site. We propose that reactions at the IBS1* site play a part in the regulation of the intron ISRm2011-2 host in vivo.     

Exon definition may facilitate splice site selection in RNAs with multiple exons.

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.     

Conserved putative signals in 3' intron junctions in rodents

Several 3' splice signals are known todate. At the 3' splice site an AG doublet is frequently found. Just upstream of the splice site there is a string of 6-11 pyrimidines. More recently it has been found that one of the stages in the splicing process involves formation of a lariat, in which the 5' end of the intron forms a 2'-5' branch with an A residue located 18-37 nucleotides upstream of the 3' splice site. The branching-point consensus is weakly defined and consists of the sequence YNYTRAY, where Y is a pyrimidine, R a purine and N any base. The A in the sixth position is the one with which branching occurs. Here we present the results of extensive searches for additional putative signals around the branching-point consensus and the 3' splice site in rodent nuclear precursor mRNAs. The signals obtained for the over 370 rodent introns are compared with those found in a larger eukaryotic sample containing over 900 nuclear pre-mRNA introns. Of particular interest are GGGA and CCCA. In both analyses GGGA occurs about 60 nucleotides upstream and CCCA is found 3-40 nucleotides downstream from the 3' splice site. A model explaining some of the putative signals discussed here is also proposed. This model involves formation of alternate stem-loop structures around the branching point and 3' splice site. Such signals and structures can possibly aid in protein or nucleoprotein branching point and splice site recognition.     

Antisense-targeted immuno-EM localization of the pre-mRNA path in the spliceosomal C complex

A first step in understanding the architecture of the spliceosome is elucidating the positions of individual spliceosomal components and functional centers. Catalysis of the first step of pre-mRNA splicing leads to the formation of the spliceosomal C complex, which contains the pre-mRNA intermediates--the cleaved 5' exon and the intron-3' exon lariat. To topographically locate the catalytic center of the human C complex, we first determined, by DNA oligonucleotide-directed RNAse H digestions, accessible pre-mRNA regions closest to nucleotides of the cleaved 5' splice site (i.e., the 3' end of exon 1 and the 5' end of the intron) and the intron lariat branch point, which are expected to be at/near the catalytic center in complex C. For electron microscopy (EM) localization studies, C complexes were allowed to form, and biotinylated 2'-OMe RNA oligonucleotides were annealed to these accessible regions. To allow localization by EM of the bound oligonucleotide, first antibiotin antibodies and then protein A-coated colloidal gold were additionally bound. EM analyses allowed us to map the position of exon and intron nucleotides near the cleaved 5' splice site, as well as close to the anchoring site just upstream of the branch adenosine. The identified positions in the C complex EM map give first hints as to the path of the pre-mRNA splicing intermediates in an active spliceosomal C complex and further define a possible location for its catalytic center.     

Self-splicing of a group II intron in yeast mitochondria: dependence on 5'' exon sequences.

It has been previously suggested that self-splicing of group II introns starts with a nucleophilic attack of the 2' OH group from the branchpoint adenosine on the 5' splice junction. To investigate the sequences governing the specificity of this attack, a series of Bal31 nuclease deletion mutants was constructed in which progressively larger amounts of 5' exon have been removed starting from its 5' end. The ability of mutant RNAs to carry out self-splicing in vitro was studied. Involvement of 5' exon sequences in self-splicing activity is indicated by the fact that a mutant in which as many as 18 nucleotides of 5' exon remain is seriously disturbed in splicing, while larger deletions eliminate splicing entirely. Mutants containing a truncated 5' exon form aberrant RNAs. One of these is a 425-nucleotide RNA containing the 5' exon as well as sequences of the 5' part of the intron. Its 3' end maps at position 374 of the 887-nucleotide intron. The other is a less abundant lariat RNA probably originating from the remainder of the intron linked to the 3' exon. We interpret this large dependence of reactivity of the intron on 5' exon and adjoining intron sequences as evidence for base-pairing interactions between the exon and parts of the intron, leading to an RNA folding necessary for splicing. Possible folding models are discussed.     

Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency

Intron lariat formation between the 5' end of an intron and a branchpoint adenosine is a fundamental aspect of the first step in animal and yeast nuclear pre-mRNA splicing. Despite similarities in intron sequence requirements and the components of splicing, differences exist between the splicing of plant and vertebrate introns. The identification of AU-rich sequences as major functional elements in plant introns and the demonstration that a branchpoint consensus sequence was not required for splicing have led to the suggestion that the transition from AU-rich intron to GC-rich exon is a major potential signal by which plant pre-mRNA splice sites are recognized. The role of putative branchpoint sequences as an internal signal in plant intron recognition/definition has been re-examined. Single nucleotide mutations in putative branchpoint adenosines contained within CUNAN sequences in four different plant introns all significantly reduced splicing efficiency. These results provide the most direct evidence to date for preferred branchpoint sequences being required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences in dicot intron recognition. The observed patterns of 3' splice site selection in the introns studied are consistent with the scanning model described for animal intron 3' splice site selection. It is suggested that, despite the clear importance of AU sequences for plant intron splicing, the fundamental processes of splice site selection and splicing in plants are similar to those in animals.     

A novel H-2K splice form: predictions for other alternative H-2 splicing events

A large number of H-2K and H-2D cDNA clones from a C3HfB/HeN spleen cDNA library were extensively characterized. All H-2D^k cDNAs were shown to exhibit the short form of exon 8, consistent with the presence of a single lariat branchpoint site within intron 7. Twenty-five H-2K^km2 cDNAs were found to bear a short exon 8, whereas only two clones were shown to carry the longer form of this exon. In one of the H-2K^km2 cDNAs, a novel pattern of H-2 splicing was identified, in which an extra 15 nucleotides, derived from the 3′ end of intron 5, were inserted between the intact and unaltered exon 5 and exon 6 sequences. Resulting from the apparent use of a cryptic splice acceptor site in place of the canonical intron 5 site, this insertion is predicted to generate an in-frame insertion of five nonpolar amino acid residues within a highly polar region of the intracytoplasmic domain of the H-2K polypeptide. The features of this novel splice form served as the basis for predicting additional rare, alternative H-2 pre-mRNA splicing events that might produce functionally relevant microheterogeneity in the encoded H-2 gene products.     

Self-splicing of group II introns in vitro: lariat formation and 3' splice site selection in mutant RNAs

Deletion or substitution of the branch A residue in group II intron bl1 significantly reduces splicing activity; yet, residual exon ligation is correct, and lariats have their branch points at the normal distance from the 3' end of the intron. Mutations in the sequence facing the branch point also allow residual lariat formation; however, free 3' exons are generated with false 5' termini, all of which are within a UCACA consensus sequence located upstream or downstream of the normal 3' splice site. These results indicate that both the conserved 3' splice site APy and the spatial arrangements in stem 6 are crucial for correct 3' splice site selection.     

Intron splicing: a conserved internal signal in introns of Drosophila pre-mRNAs.

The introns of Drosophila pre-mRNAs have been analysed for conserved internal sequence elements near the 3' intron boundary similar to the T-A-C-T-A-A-C in yeast introns and the C/T-T-A/G-A-C/T in introns of other organisms. Such conserved internal elements are the 3' splice signals recognized in intron splicing. In the lariat splicing mechanism, the G at the 5' end of an intron joins covalently to the last A of a 3' splice signal to form a branch point in a splicing intermediate. Analysis of 39 published sequences of Drosophila introns reveals that potential 3' splice signals with the consensus C/T-T-A/G-A-C/T are present in 18 cases. In 17 of the remaining cases signals are present which vary from this consensus just in the middle or last position. In Drosophila introns the 3' splice signal is usually located in a discrete region between 18 and 35 nucleotides upstream from the 3' splice point. We note that the Drosophila small nuclear U2-RNA has sequences complementary to C-T-G-A-T, one variant of the signal, and to C-A-G, one variant of the 3' terminus of an intron. We also note that the absence of any A-G between -3 and -19 from the 3' splice point may be an essential feature of a strong 3' boundary.     

Yeast mRNA splicing in vitro

Synthetic actin and CYH2 pre-mRNAs containing a single intron are accurately spliced in a soluble whole cell extract of yeast. Splicing in vitro requires ATP. The excised intron is released as a lariat in which an RNA branch connects the 5' end of the molecule to the last A in the "intron conserved sequence" UACUAAC. Two other discrete RNA species produced during splicing in vitro may represent reaction intermediates: free, linear exon 1 and a form of the intron lariat extending beyond the 3' splice site to include exon 2. Both lariat forms correspond to molecules previously shown to be produced during yeast pre-mRNA splicing in vivo.     

Self-splicing of group II introns in vitro: mapping of the branch point and mutational inhibition of lariat formation

Group II intron bl1 from yeast mitochondria can undergo self-splicing in vitro. Exons become correctly ligated, and the excised intron has a lariat structure similar to that of introns from nuclear mRNA. The branch point of the bl1 lariat is located eight or nine nucleotides upstream of the 3' end of the intron and is part of a hairpin structure that is well conserved among group II introns. Several mutations next to the branch point and in other parts of the core structure of group II introns are shown to affect lariat formation. One of them, carried by strain M4873, abolishes splicing in vivo and in vitro, apparently by changing the architecture of the hairpin structure containing the branch point. Similarities between group II introns and nuclear pre-mRNA introns are discussed in terms of evolutionary relatedness.     

Group II introns in wheat mitochondria have degenerate structural features and varied splicing pathways

Mitochondrial introns in flowering plant genes are virtually all classified as members of the group II ribozyme family although certain structural features have degenerated to varying degrees over evolutionary time. We are interested in the impact that unconventional intron architecture might have on splicing biochemistry in vivo and we have focused in particular on intronic domains V and VI, which for self-splicing introns provide a key component of the catalytic core and the bulged branchpoint adenosine, respectively. Notably, the two transesterification steps in classical group II splicing are the same as for nuclear spliceosomal introns and release the intron as a lariat. Using RT-PCR and circularized RT-PCR, we had previously demonstrated that several wheat mitochondrial introns which lack a branchpoint adenosine have atypical splicing pathways, and we have now extended this analysis to the full set of wheat introns, namely six trans-splicing and sixteen cis-splicing ones. A number of introns are excised using non-lariat pathways and interestingly, we find that several introns which do have a conventional domain VI also use pathways that appear to exploit other internal or external nucleophiles, with the lariat form being relatively minor. Somewhat surprisingly, several introns with weakly-structured domain V/VI helices still exhibit classical lariat splicing, suggesting that accessory factors aid in restoring a splicing-competent conformation. Our observations illustrate that the loss of conventional group II features during evolution is correlated with altered splicing biochemistry in an intron-distinctive manner.