Mapping divalent metal ion binding sites in a group II intron by Mn(2+)- and Zn(2+)-induced site-specific RNA cleavage.

The function of group II introns depends on positively charged divalent metal ions that stabilize the ribozyme structure and may be directly involved in catalysis. We investigated Mn2+- and Zn2+-induced site-specific RNA cleavage to identify metal ions that fit into binding pockets within the structurally conserved bI1 group II intron domains (DI-DVI), which might fulfill essential roles in intron function. Ten cleavage sites were identified in DI, two sites in DIII and two in DVI. All cleavage sites are located in the center or close to single-stranded and flexible RNA structures. Strand scissions mediated by Mn2+/Zn2+ are competed for by Mg2+, indicating the existence of Mg2+ binding pockets in physical proximity to the observed Mn2+-/Zn2+-induced cleavage positions. To distinguish between metal ions with a role in structure stabilization and those that play a more specific and critical role in the catalytic process of intron splicing, we combined structural and functional assays, comparing wild-type precursor and multiple splicing-deficient mutants. We identified six regions with binding pockets for Mg2+ ions presumably playing an important role in bI1 structure stabilization. Remarkably, assays with DI deletions and branch point mutants revealed the existence of one Mg2+ binding pocket near the branching A, which is involved in first-step catalysis. This pocket formation depends on precise interaction between the branching nucleotide and the 5' splice site, but does not require exon-binding site 1/intron binding site 1 interaction. This Mg2+ ion might support the correct placing of the branching A into the 'first-step active site'.     

Branch-point attack in group II introns is a highly reversible transesterification, providing a potential proofreading mechanism for 5'-splice site selection.

By examining the first step of group II intron splicing in the absence of the second step, we have found that there is an interplay of three distinct reactions at the 5'-splice site: branching, reverse branching, and hydrolytic cleavage. This approach has yielded the first kinetic parameters describing eukaryotic branching and establishes that group II intron catalysis can proceed on a rapid timescale. The efficient reversibility of the first step is due to increased conformational organization in the branched intermediate and it has several important mechanistic implications. Reversibility in the first step requires that the second step of splicing serve as a kinetic trap, thus driving splicing to completion and coordinating the first and second step of splicing. Facile reverse branching also provides the intron with a proofreading mechanism to control the fidelity of 5'-splice site selection and it provides a kinetic basis for the apparent mobility of group II introns.     

Relevance of the branch point adenosine, coordination loop, and 3' exon binding site for in vivo excision of the Sinorhizobium meliloti group II intron RmInt1

Excision of the bacterial group II intron RmInt1 has been demonstrated in vivo, resulting in the formation of both intron lariat and putative intron RNA circles. We show here that the bulged adenosine in domain VI of RmInt1 is required for splicing via the branching pathway, but branch site mutants produce small numbers of RNA molecules in which the first G residue of the intron is linked to the last C residue. Mutations in the coordination loop in domain I reduced splicing efficiency, but branched templates clearly predominated among splicing products. We also found that a single substitution at the EBS3 position (G329C), preventing EBS3-IBS3 pairing, resulted in the production of 50 to 100 times more RNA molecules in which the 5' and 3' extremities were joined. We provide evidence that these intron molecules may correspond to both, intron circles linked by a 2'-5' phosphodiester bond, and tandem, head-to-tail intron copies.     

RNA splicing: group I intron crystal structures reveal the basis of splice site selection and metal ion catalysis

The group I intron has served as a model for RNA catalysis since its discovery 25 years ago. Four recently determined high-resolution crystal structures complement extensive biochemical studies on this system. Structures of the Azoarcus, Tetrahymena and bacteriophage Twort group I introns mimic different states of the splicing or ribozyme reaction pathway and provide information on splice site selection and metal ion catalysis. The 5'-splice site is selected by formation of a conserved G.U wobble pair between the 5'-exon terminus and the intron. The 3'-splice site is identified through stacking of three base triples, in which the middle triple contains the conserved terminal nucleotide of the intron, OmegaG. The structures support a two-metal-ion mechanism for group I intron splicing that might have corollaries to group II intron and pre-mRNA splicing by the spliceosome.     

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.     

Circularization pathway of a bacterial group II intron

Group II introns are large RNA enzymes that can excise as lariats, circles or in a linear form through branching, circularization or hydrolysis, respectively. Branching is by far the main and most studied splicing pathway while circularization was mostly overlooked. We previously showed that removal of the branch point A residue from Ll.LtrB, the group II intron from Lactococcus lactis, exclusively leads to circularization. However, the majority of the released intron circles harbored an additional C residue of unknown origin at the splice junction. Here, we exploited the Ll.LtrB-ΔA mutant to study the circularization pathway of bacterial group II introns in vivo. We demonstrated that the non-encoded C residue, present at the intron circle splice junction, corresponds to the first nt of exon 2. Intron circularization intermediates, harboring the first 2 or 3 nts of exon 2, were found to accumulate showing that branch point removal leads to 3′ splice site misrecognition. Traces of properly ligated exons were also detected functionally confirming that a small proportion of Ll.LtrB-ΔA circularizes accurately. Overall, our data provide the first detailed molecular analysis of the group II intron circularization pathway and suggests that circularization is a conserved splicing pathway in bacteria.     

Unexpected metal ion requirements specific for catalysis of the branching reaction in a group II intron

The splicing process catalyzed by group II intron ribozymes follows the same two-step pathway as nuclear pre-mRNA splicing. In vivo, the first splicing step of wild-type introns is a transesterification reaction giving rise to a branched lariat intron-3'-exon intermediate characteristic of this splicing mode. In the wild-type introns, the ribozyme core and the substrate intron-exon junctions are carried by the same precursor molecule, making it difficult to distinguish between RNA folding and catalysis under normal splicing reactions. To characterize the catalytic step of the first transesterification reaction, we studied the reversal of this reaction, reverse branching. In this reverse reaction, the excised lariat intron and the substrate 5'-exon can be preincubated and folded separately, allowing the measure of the catalytic rate of the reaction. To measure the catalytic rate of the second splicing step, purified lariat intron-3'-exon intermediate molecules were preincubated and folded prior to the addition of 5'-exon. Conditions could be found where chemistry appeared rate limiting for both catalytic steps. Study of the metal ion requirements under these conditions resulted in the unexpected finding that, for the intron studied, substitution of magnesium ions by manganese ions enhanced the rate of the first transesterification reaction by two orders of magnitude but had virtually no effect on the second transesterification reaction or the 5' splice site cleavage by hydrolysis. Finally, the catalytic rates measured under optimal conditions for both splicing steps were faster by three orders of magnitude in the branching pathway than in the hydrolytic pathway.     

trans-splicing to spliceosomal U2 snRNA suggests disruption of branch site-U2 pairing during pre-mRNA splicing

Pairing between U2 snRNA and the branch site of spliceosomal introns is essential for spliceosome assembly and is thought to be required for the first catalytic step of splicing. We have identified an RNA comprising the 5' end of U2 snRNA and the 3' exon of the ACT1-CUP1 reporter gene, resulting from a trans-splicing reaction in which a 5' splice site-like sequence in the universally conserved branch site-binding region of U2 is used in trans as a 5' splice site for both steps of splicing in vivo. Formation of this product occurs in functional spliceosomes assembled on reporter genes whose 5' splice sites are predicted to bind poorly at the spliceosome catalytic center. Multiple spatially disparate splice sites in U2 can be used, calling into question both the fate of its pairing to the branch site and the details of its role in splicing catalysis.     

Splicing and spliceosome formation of the yeast MATa1 transcript require a minimum distance from the 5'' splice site to the internal branch acceptor site.

Small deletions of 6, 7, and 12 nucleotides introduced between the 5' splice site and the internal branch acceptor site of the first intron of the yeast MATa1 gene completely abolish accurate splicing in vitro in these constructs. Splicing only occurs at an alternative 5' splice site which was found in the first exon of the MATa1 gene and which is used both in vivo and in vitro. The splicing defect cannot be cured by expanding the distance from the branch point to the 3' splice site. If the alternative 5' splice site is deleted as well in these constructs, neither spliced products nor spliceosomes are formed. Our findings especially lead to the conclusion that a minimum distance between the 5' splice site and the internal branch acceptor site of the intron is required for the formation of splicing complexes and for accurate splicing.     

General and specific functions of exonic splicing silencers in splicing control

Correct splice site recognition is critical in pre-mRNA splicing. We find that almost all of a diverse panel of exonic splicing silencer (ESS) elements alter splice site choice when placed between competing sites, consistently inhibiting use of intron-proximal 5' and 3' splice sites. Supporting a general role for ESSs in splice site definition, we found that ESSs are both abundant and highly conserved between alternative splice site pairs and that mutation of ESSs located between natural alternative splice site pairs consistently shifted splicing toward the intron-proximal site. Some exonic splicing enhancers (ESEs) promoted use of intron-proximal 5' splice sites, and tethering of hnRNP A1 and SF2/ASF proteins between competing splice sites mimicked the effects of ESS and ESE elements, respectively. Further, we observed that specific subsets of ESSs had distinct effects on a multifunctional intron retention reporter and that one of these subsets is likely preferred for regulation of endogenous intron retention events. Together, our findings provide a comprehensive picture of the functions of ESSs in the control of diverse types of splicing decisions.     

Mutational analysis of the U12-dependent branch site consensus sequence

Highly conserved sequences at the 5′ splice site and branch site of U12-dependent introns are important determinants for splicing by U12-dependent spliceosomes. This study investigates the in vivo splicing phenotypes of mutations in the branch site consensus sequence of the U12-dependent intron F from a human NOL1 (P120) minigene. Intron F contains a fully consensus branch site sequence (UUCCUUAAC). Mutations at each position were analyzed for their effects on U12-dependent splicing in vivo. Mutations at most positions resulted in a significant reduction of correct U12-dependent splicing. Defects observed included increased unspliced RNA levels, the activation of cryptic U2-dependent 5′ and 3′ splice sites, and the activation of cryptic U12-dependent branch/3′ splice sites. A strong correlation was observed between the predicted thermodynamic stability of the branch site: U12 snRNA interaction and correct U12-dependent splicing. The lack of a polypyrimidine tract between the branch site and 3′ splice site of U12-dependent introns and the observed reliance on base-pairing interactions for correct U12-dependent splicing emphasize the importance of RNA/RNA interactions during U12-dependent intron recognition and proper splice site selection.     

Natural base-pairing interactions between 5' splice site and branch site sequences affect mammalian 5' splice site selection.

In the murine gene encoding the neuronal cell adhesion molecule (NCAM), the integrity of the 5' splice site of exon 18 (E18) is essential for regulation of alternative splicing. To further study the contribution of 5' splice site sequences, we used a simple NCAM pre-mRNA containing a portion of E18 fused to E19 and separated by a shortened intron. This RNA is spliced in vitro to produce five sets of lariat intermediates and products, the most abundant set displaying aberrant migration in acrylamide/urea gels. Base pairing interactions between positions +5 and +8 of the intron and positions -3 and -6 from the branch point were responsible for the faster migration of this set of lariat molecules. To test whether the duplex structure forms earlier and contributes to 5' splice site selection, we used NCAM substrates carrying the 5' splice sites of E17 and E18 in competition for the 3' splice site of E19. Mutations upstream of the major branch site improve E18/E19 splicing in NIH3T3 extracts, whereas compensatory mutations at positions +7 and +8 neutralize the effect of branch site mutations and curtail E18/E19 splicing. Our data suggest that duplex formation occurs early and interferes with the assembly of complexes initiated on the 5' splice site of NCAM E18. This novel type of intron interaction may exist in the introns of other mammalian pre-mRNAs.     

Efficient use of a 'dead-end' GA 5' splice site in the human fibroblast growth factor receptor genes

We have investigated use of a conserved non-canonical GA 5' splice site present in vertebrate fibroblast growth factor receptor (FGFR) genes. Despite previous studies suggesting that GA at the beginning of an intron is incompatible with splicing, we observe efficient utilization of this splice site for human FGFR1 gene constructs. We show that use of the GA splice site is dependent on both a conventional splice site six nucleotides upstream and sequence elements within the downstream intron. Furthermore, our results are consistent with competition between the tandem 5' splice sites being mediated by U6 snRNP, rather than U1 snRNP. Thus the GA 5' splice site represents an extension of the adjacent conventional 5' splice site, the first natural example of such a composite 5' splice site.     

The Ll.LtrB intron from Lactococcus lactis excises as circles in vivo: insights into the group II intron circularization pathway

Group II introns are large ribozymes that require the assistance of intron-encoded or free-standing maturases to splice from their pre-mRNAs in vivo. They mainly splice through the classical branching pathway, being released as RNA lariats. However, group II introns can also splice through secondary pathways like hydrolysis and circularization leading to the release of linear and circular introns, respectively. Here, we assessed in vivo splicing of various constructs of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis. The study of excised intron junctions revealed, in addition to branched intron lariats, the presence of perfect end-to-end intron circles and alternatively circularized introns. Removal of the branch point A residue prevented Ll.LtrB excision through the branching pathway but did not hinder intron circle formation. Complete intron RNA circles were found associated with the intron-encoded protein LtrA forming nevertheless inactive RNPs. Traces of double-stranded head-to-tail intron DNA junctions were also detected in L. lactis RNA and nucleic acid extracts. Some intron circles and alternatively circularized introns harbored variable number of non-encoded nucleotides at their splice junction. The presence of mRNA fragments at the splice junction of some intron RNA circles provides insights into the group II intron circularization pathway in bacteria.     

Metal ion binding sites in a group II intron core

Group II introns are catalytic RNA molecules that require divalent metal ions for folding, substrate binding, and chemical catalysis. Metal ion binding sites in the group II core have now been elucidated by monitoring the site-specific RNA hydrolysis patterns of bound ions such as Tb(3+) and Mg(2+). Major sites are localized near active site elements such as domain 5 and its surrounding tertiary interaction partners. Numerous sites are also observed at intron substructures that are involved in binding and potentially activating the splice sites. These results highlight the locations of specific metal ions that are likely to play a role in ribozyme catalysis.     

RS domain-splicing signal interactions in splicing of U12-type and U2-type introns

Serine-arginine (SR) proteins are general metazoan splicing factors that contain an essential arginine/serine-rich (RS) domain. On typical U2-type introns, RS domains contact the branchpoint and 5' splice site to promote base-pairing with U small nuclear RNAs (snRNAs). Here we analyze the role of SR proteins in splicing of U12-type introns and in the second step of U2-type intron splicing. We show that RS domains contact the branchpoint and 5' splice site of a U12-type intron. On a U2-type intron, we find that the RS domain contacts the site of the U6 snRNA-5' splice site interaction during the first step of splicing and shifts to contact the site of the U5 snRNA-exon 1 interaction during the second step. Our results reveal alternative interactions between the RS domain and 5' splice site region that coincide with remodeling of the spliceosome between the two catalytic steps.