The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop

The U6 spliceosomal snRNA forms an intramolecular stem-loop structure during spliceosome assembly that is required for splicing and is proposed to be at or near the catalytic center of the spliceosome. U6atac snRNA, the analog of U6 snRNA used in the U12-dependent splicing of the minor class of spliceosomal introns, contains a similar stem-loop whose structure but not sequence is conserved between humans and plants. To determine if the U6 and U6atac stem-loops are functionally analogous, the stem-loops from human and budding yeast U6 snRNAs were substituted for the U6atac snRNA structure and tested in an in vivo genetic suppression assay. Both chimeric U6/U6atac snRNA constructs were active for splicing in vivo. In contrast, several mutations of the native U6atac stem-loop that either delete putatively unpaired residues or disrupt the putative stem regions were inactive for splicing. Compensatory mutations that are expected to restore base pairing within the stem regions restored splicing activity. However, other mutants that retained base pairing potential were inactive, suggesting that functional groups within the stem regions may contribute to function. These results show that the U6atac snRNA stem-loop structure is required for in vivo splicing within the U12-dependent spliceosome and that its role is likely to be similar to that of the U6 snRNA intramolecular stem-loop.     

Dynamics in the U6 RNA intramolecular stem-loop: a base flipping conformational change

The U6 RNA intramolecular stem-loop (ISL) structure is an essential component of the spliceosome and binds a metal ion required for pre-messenger RNA splicing. The metal binding internal loop region of the stem contains a partially protonated C67-(+)A79 base pair (pK(a) = 6.5) and an unpaired U80 nucleotide that is stacked within the helix at pH 7.0. Here, we determine that protonation occurs with an exchange lifetime of approximately 20 micros and report the solution structures of the U6 ISL at pH 5.7. The differences between pH 5.7 and 7.0 structures reveal that the pH change significantly alters the RNA conformation. At lower pH, U80 is flipped out into the major groove. Base flipping involves a purine stacking interaction of flanking nucleotides, inversion of the sugar pucker 5' to the flipped base, and phosphodiester backbone rearrangement. Analysis of residual dipolar couplings as a function of pH indicates that base flipping is not restricted to a local conformational change. Rather, base flipping alters the alignment of the upper and lower helices. The alternative conformations of the U6 ISL reveal striking structural similarities with both the NMR and crystal structures of domain 5 of self-splicing group II introns. These structures suggest that base flipping at an essential metal binding site is a conserved feature of the splicing machinery for both the spliceosome and group II self-splicing introns.     

Conservation of functional features of U6atac and U12 snRNAs between vertebrates and higher plants

Splicing of U12-dependent introns requires the function of U11, U12, U6atac, U4atac, and U5 snRNAs. Recent studies have suggested that U6atac and U12 snRNAs interact extensively with each other, as well as with the pre-mRNA by Watson-Crick base pairing. The overall structure and many of the sequences are very similar to the highly conserved analogous regions of U6 and U2 snRNAs. We have identified the homologs of U6atac and U12 snRNAs in the plant Arabidopsis thaliana. These snRNAs are significantly diverged from human, showing overall identities of 65% for U6atac and 55% for U12 snRNA. However, there is almost complete conservation of the sequences and structures that are implicated in splicing. The sequence of plant U6atac snRNA shows complete conservation of the nucleotides that base pair to the 5' splice site sequences of U12-dependent introns in human. The immediately adjacent AGAGA sequence, which is found in human U6atac and all U6 snRNAs, is also conserved. High conservation is also observed in the sequences of U6atac and U12 that are believed to base pair with each other. The intramolecular U6atac stem-loop structure immediately adjacent to the U12 interaction region differs from the human sequence in 9 out of 21 positions. Most of these differences are in base pairing regions with compensatory changes occurring across the stem. To show that this stem-loop was functional, it was transplanted into a human suppressor U6atac snRNA expression construct. This chimeric snRNA was inactive in vivo but could be rescued by coexpression of a U4atac snRNA expression construct containing compensatory mutations that restored base pairing to the chimeric U6atac snRNA. These data show that base pairing of U4atac snRNA to U6atac snRNA has a required role in vivo and that the plant U6atac intramolecular stem-loop is the functional analog of the human sequence.     

RNA structural requirements for the association of the spliceosomal hPrp31 protein with the U4 and U4atac small nuclear ribonucleoproteins

The kink-turn, a stem I-internal loop-stem II structure of the 5 ' stem-loop of U4 and U4atac small nuclear (sn) RNAs bound by 15.5K protein is required for binding of human Prp31 protein (hPrp31) during U4 and U4atac snRNP assembly. In box C/D snoRNPs a similar kink-turn with bound 15.5K protein is required for selective binding of proteins NOP56 and NOP58. Here we analyzed RNA structural requirements for association of hPrp31 with U4 snRNP in vitro by hydroxyl radical footprinting. hPrp31 induced protection of the terminal penta-loop, as well as of stems I and II flanking the kink-turn. Similar protection was found with U4/U6 snRNA duplex prebound with 15.5K protein. A detailed mutational analysis of the U4 snRNA elements by electrophoretic mobility shift analysis revealed that stem I could not be shortened, although it tolerated sequence alterations. However, introduction of a third Watson-Crick base pair into stem II significantly reduced hPrp31 binding. While stem I of U4atac snRNA showed relaxed binding requirements, its stem II requirements were likewise restricted to two base pairs. In contrast, as shown previously, stem II of the kink-turn motif in box C/D snoRNAs is comprised of three base pairs, and NOP56 and NOP58 require a G-C pair at the central position. This indicates that hPrp31 binding specificity is achieved by the recognition of the two base pair long stem II of the U4 and U4atac snRNAs and suggests how discrimination is achieved by RNA structural elements during assembly of U4/U6 and U4atac/U6atac snRNPs and box C/D snoRNPs.     

A dynamic bulge in the U6 RNA internal stem-loop functions in spliceosome assembly and activation

The highly conserved internal stem-loop (ISL) of U6 spliceosomal RNA is unwound for U4/U6 complex formation during spliceosome assembly and reformed upon U4 release during spliceosome activation. The U6 ISL is structurally similar to Domain 5 of group II self-splicing introns, and contains a dynamic bulge that coordinates a Mg++ ion essential for the first catalytic step of splicing. We have analyzed the causes of growth defects resulting from mutations in the Saccharomyces cerevisiae U6 ISL-bulged nucleotide U80 and the adjacent C67-A79 base pair. Intragenic suppressors and enhancers of the cold-sensitive A79G mutation, which replaces the C-A pair with a C-G pair, suggest that it stabilizes the ISL, inhibits U4/U6 assembly, and may also disrupt spliceosome activation. The lethality of mutations C67A and C67G results from disruption of base-pairing potential between U4 and U6, as these mutations are fully suppressed by compensatory mutations in U4 RNA. Strikingly, suppressor analysis shows that the lethality of the U80G mutation is due not only to formation of a stable base pair with C67, as previously proposed, but also another defect. A U6-U80G strain in which mispairing with position 67 is prevented grows poorly and assembles aberrant spliceosomes that retain U1 snRNP and fail to fully unwind the U4/U6 complex at elevated temperatures. Our data suggest that the U6 ISL bulge is important for coupling U1 snRNP release with U4/U6 unwinding during spliceosome activation.     

Domains of human U4atac snRNA required for U12-dependent splicing in vivo

U4atac snRNA forms a base-paired complex with U6atac snRNA. Both snRNAs are required for the splicing of the minor U12-dependent class of eukaryotic nuclear introns. We have developed a new genetic suppression assay to investigate the in vivo roles of several regions of U4atac snRNA in U12-dependent splicing. We show that both the stem I and stem II regions, which have been proposed to pair with U6atac snRNA, are required for in vivo splicing. Splicing activity also requires U4atac sequences in the 5' stem-loop element that bind a 15.5 kDa protein that also binds to a similar region of U4 snRNA. In contrast, mutations in the region immediately following the stem I interaction region, as well as a deletion of the distal portion of the 3' stem-loop element, were active for splicing. Complete deletion of the 3' stem-loop element abolished in vivo splicing function as did a mutation of the Sm protein binding site. These results show that the in vivo sequence requirements of U4atac snRNA are similar to those described previously for U4 snRNA using in vitro assays and provide experimental support for models of the U4atac/U6atac snRNA interaction.     

Base pairing with U6atac snRNA is required for 5' splice site activation of U12-dependent introns in vivo.

The minor U12-dependent class of eukaryotic nuclear pre-mRNA introns is spliced by a distinct spliceosomal mechanism that requires the function of U11, U12, U5, U4atac, and U6atac snRNAs. Previous work has shown that U11 snRNA plays a role similar to U1 snRNA in the major class spliceosome by base pairing to the conserved 5'' splice site sequence. Here we show that U6atac snRNA also base pairs to the 5'' splice site in a manner analogous to that of U6 snRNA in the major class spliceosome. We show that splicing defective mutants of the 5'' splice site can be activated for splicing in vivo by the coexpression of compensatory U6atac snRNA mutants. In some cases, maximal restoration of splicing required the coexpression of compensatory U11 snRNA mutants. The allelic specificity of mutant phenotype suppression is consistent with Watson-Crick base pairing between the pre-mRNA and the snRNAs. These results provide support for a model of the RNA-RNA interactions at the core of the U12-dependent spliceosome that is strikingly similar to that of the major class U2-dependent spliceosome.     

Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA.

Activation of the spliceosome for splicing catalysis requires the dissociation of U4 snRNA from the U4/U6 snRNA duplex prior to the first step of splicing. We characterize an evolutionarily conserved 15.5 kDa protein of the HeLa [U4/U6.U5] tri-snRNP that binds directly to the 5' stem-loop of U4 snRNA. This protein shares a novel RNA recognition motif with several RNP-associated proteins, which is essential, but not sufficient for RNA binding. The 15.5kD protein binding site on the U4 snRNA consists of an internal purine-rich loop flanked by the stem of the 5' stem-loop and a stem comprising two base pairs. Addition of an RNA oligonucleotide comprising the 5' stem-loop of U4 snRNA (U4SL) to an in vitro splicing reaction blocked the first step of pre-mRNA splicing. Interestingly, spliceosomal C complex formation was inhibited while B complexes accumulated. This indicates that the 15.5kD protein, and/or additional U4 snRNP proteins associated with it, play an important role in the late stage of spliceosome assembly, prior to step I of splicing catalysis. Our finding that the 15.5kD protein also efficiently binds to the 5' stem-loop of U4atac snRNA indicates that it may be shared by the [U4atac/U6atac.U5] tri-snRNP of the minor U12-type spliceosome.     

Dynamic exchanges of RNA interactions leading to catalytic core formation in the U12-dependent spliceosome

Important general insights into the mechanism of pre-mRNA splicing have emerged from studies of the U12-dependent spliceosome. Here, photochemical cross-linking analyses during U12-dependent spliceosome assembly have surprisingly revealed that an upstream 5' exon region is required for establishing two essential catalytic core interactions, U12/U6atac helix Ib and U6atac/5' splice site contacts, but not for U5/5' exon interactions or partial unwinding of U4atac/U6atac. A novel intermediate, representing an alternative pathway for catalytic core formation, is a ternary snRNA complex containing U4atac/U6atac stem II and U12/U6atac helix Ia that forms even without U6atac replacing U11 at the 5' splice site. A powerful oligonucleotide displacement method suggests that the blocked complexes analyzed to deduce the interdependence of these multiple RNA exchanges are authentic intermediates in U12-dependent spliceosome assembly.     

Structure of a self-splicing group II intron catalytic effector domain 5: parallels with spliceosomal U6 RNA

Domain 5 (D5) is absolutely required for all catalytic functions of group II introns. Here we describe the solution NMR structure, electrostatic calculations, and detailed magnesium ion-binding surface of D5 RNA from the Pylaiella littoralis large ribosomal RNA intron (D5-PL). The overall structure consists of a hairpin capped by a GNRA tetraloop. The stem is divided into lower and upper helices of 8 and 5 bp, respectively, separated by an internal bulge. The D5-PL internal bulge nucleotides stack into the helical junction, resulting in a coupling between the bulge A25 and the closing base pair (G8-C27) of the lower helix. Comparison of the D5-PL structure to previously reported related structures indicates that our structure is most similar, in the helical regions, to the crystal structure of D5 from yeast Ai5gamma (D5-Ai5gamma) and the NMR structure of the U6 snRNA stem-loop region. Our structure differs in many respects from both the NMR and X-ray structures of D5-Ai5gamma in the bulge region. Electrostatic calculations and NMR chemical shift perturbation analyses reveal magnesium ion-binding sites in the tetraloop, internal bulge, and the AGC triad in the lower stem. Our results suggest that the structure, electrostatic environment, and the magnesium ion-binding sites within the tetraloop, bulge, and triad regions are conserved features of the splicing machinery of both the group II introns and the spliceosome that are likely key for catalytic function.     

Both U2 snRNA and U12 snRNA are required for accurate splicing of exon 5 of the rat calcitonin/CGRP gene

Two classes of spliceosome are present in eukaryotic cells. Most introns in nuclear pre-mRNAs are removed by a spliceosome that requires U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles (snRNPs). A minor class of introns are removed by a spliceosome containing U11, U12, U5, U4atac, and U6 atac snRNPs. We describe experiments that demonstrate that splicing of exon 5 of the rat calcitonin/CGRP gene requires both U2 snRNA and U12 snRNA. In vitro, splicing to calcitonin/ CGRP exon 5 RNA was dependent on U2 snRNA, as preincubation of nuclear extract with an oligonucleotide complementary to U2 snRNA abolished exon 5 splicing. Addition of an oligonucleotide complementary to U12 snRNA increased splicing at a cryptic splice site in exon 5 from <5% to 50% of total spliced RNA. Point mutations in a candidate U12 branch sequence in calcitonin/CGRP intron 4, predicted to decrease U12-pre-mRNA base-pairing, also significantly increased cryptic splicing in vitro. Calcitonin/CGRP genes containing base changes disrupting the U12 branch sequence expressed significantly decreased CGRP mRNA levels when expressed in cultured cells. Coexpression of U12 snRNAs containing base changes predicted to restore U12-pre-mRNA base pairing increased CGRP mRNA synthesis to the level of the wild-type gene. These observations indicate that accurate, efficient splicing of calcitonin/CGRP exon 5 is dependent upon both U2 and U12 snRNAs.     

Evolutionary conservation of minor U12-type spliceosome between plants and humans

Splicing of rare, U12-type or AT-AC introns is mediated by a distinct spliceosome that assembles from U11, U12, U4atac, U6atac, and U5 snRNPs. Although in human cells the protein composition of minor and major snRNPs is similar, differences, particularly in U11 and U12 snRNPs, have been recently described. We have identified an Arabidopsis U11 snRNP-specific 35K protein as an interacting partner of an RS-domain-containing cyclophilin. By using a transient expression system in Arabidopsis protoplasts, we show that the 35K protein incorporates into snRNP. Oligo affinity selection and glycerol gradient centrifugation revealed that the Arabidopsis 35K protein is present in monomeric U11 snRNP and in U11/U12-di snRNP. The interaction of the 35K protein with Arabidopsis SR proteins together with its strong sequence similarity to U1-70K suggests that its function in splicing of minor introns is analogous to that of U1-70K. Analysis of Arabidopsis and Oryza sativa genome sequences revealed that all U11/U12-di-snRNP-specific proteins are conserved in dicot and monocot plants. In addition, we have identified an Arabidopsis gene encoding the homolog of U4atac snRNA and a second Arabidopsis gene encoding U6atac snRNA. Secondary structure predictions indicate that the Arabidopsis U4atac is able to form dimeric complexes with both Arabidopsis U6atac snRNAs. As revealed by RNaseA/T1 protection assay, the U4atac snRNA gene is expressed as an ~160-nt RNA, whereas the second U6atac snRNA gene seems to be a pseudogene. Taken together, our data indicate that recognition and splicing of minor, AT-AC introns in plants is highly similar to that in humans.     

The conserved 3′ end domain of U6atac snRNA can direct U6 snRNA to the minor spliceosome

U6 and U6atac snRNAs play analogous critical roles in the major U2-dependent and minor U12-dependent spliceosomes, respectively. Previous results have shown that most of the functional cores of these two snRNAs are either highly similar in sequence or functionally interchangeable. Thus, a mechanism must exist to restrict each snRNA to its own spliceosome. Here we show that a chimeric U6 snRNA containing the unique and highly conserved 3′ end domain of U6atac snRNA is able to function in vivo in U12-dependent spliceosomal splicing. Function of this chimera required the coexpression of a modified U4atac snRNA; U4 snRNA could not substitute. Partial deletions of this element in vivo, as well as in vitro antisense experiments, showed that the 3′ end domain of U6atac snRNA is necessary to direct the U4atac/U6atac.U5 tri-snRNP to the forming U12-dependent spliceosome. In vitro experiments also uncovered a role for U4atac snRNA in this targeting.     

U2-U6 RNA folding reveals a group II intron-like domain and a four-helix junction

Intron removal in nuclear precursor mRNA is catalyzed through two transesterification reactions by a multi-megaDalton ribonucleoprotein machine called the spliceosome. A complex between U2 and U6 small nuclear RNAs is a core component of the spliceosome. Here we present an NMR structural analysis of a protein-free U2-U6 complex from Saccharomyces cerevisiae. The observed folding of the U2-U6 complex is a four-helix junction, in which the catalytically important AGC triad base-pairs only within U6 and not with U2. The base-pairing of the AGC triad extends the U6 intramolecular stem-loop (U6 ISL), and the NMR structure of this extended U6 ISL reveals structural similarities with domain 5 of group II self-splicing introns. The observed conformation of the four-helix junction could be relevant to the first, but not the second, step of splicing and may help to position the U6 ISL adjacent to the 5' splice site.     

Conserved domains of human U4 snRNA required for snRNP and spliceosome assembly.

U4 snRNA is phylogenetically highly conserved and organized in several domains. To determine the function of each of the domains of human U4 snRNA in the multi-step process of snRNP and spliceosome assembly, we used reconstitution procedures in combination with snRNA mutagenesis. The highly conserved 5' terminal domain of U4 snRNA consists of the stem I and stem II regions that have been proposed to base pair with U6 snRNA, and the 5' stem-loop structure. We found that each of these structural elements is essential for spliceosome assembly. However, only the stem II region is required for U4-U6 interaction, and none of these elements for Sm protein binding. In contrast, the 3' terminal domain of U4 snRNA containing the Sm binding site is dispensable for both U4-U6 interaction and spliceosome assembly. Our results support an organization of the U4 snRNP into multiple functional domains, each of which acts at distinct stages of snRNP and spliceosome assembly.     

Studies of point mutants define three essential paired nucleotides in the domain 5 substructure of a group II intron.

Domain 5 (D5) is a highly conserved, largely helical substructure of group II introns that is essential for self-splicing. Only three of the 14 base pairs present in most D5 structures (A2.U33, G3.U32, and C4.G31) are nearly invariant. We have studied effects of point mutations of those six nucleotides on self-splicing and in vivo splicing of aI5 gamma, an intron of the COXI gene of Saccharomyces cerevisiae mitochondria. Though none of the point mutations blocked self-splicing under one commonly used in vitro reaction condition, the most debilitating mutations were at G3 and G4. Following mitochondrial Biolistic transformation, it was found that mutations at A2, G3, and C4 blocked respiratory growth and splicing while mutations at the other sites had little effect on either phenotype. Intra-D5 second-site suppressors showed that pairing between nucleotides at positions 2 and 33 and 4 and 31 is especially important for D5 function. At the G3.U32 wobble pair, the mutant A.U pair blocks splicing, but a revertant of that mutant that can form an A+.C base pair regains some splicing. A dominant nuclear suppressor restores some splicing to the G3A mutant but not the G3U mutant, suggesting that a purine is required at position 3. These findings are discussed in terms of the hypothesis of Madhani and Guthrie (H. D. Madhani and C. Guthrie, Cell 71:803-817, 1992) that helix 1 formed between yeast U2 and U6 small nuclear RNAs may be the spliceosomal cognate of D5.     

A structural analysis of the group II intron active site and implications for the spliceosome

Group II introns are self-splicing, mobile genetic elements that have fundamentally influenced the organization of terrestrial genomes. These large ribozymes remain important for gene expression in almost all forms of bacteria and eukaryotes and they are believed to share a common ancestry with the eukaryotic spliceosome that is required for processing all nuclear pre-mRNAs. The three-dimensional structure of a group IIC intron was recently determined by X-ray crystallography, making it possible to visualize the active site and the elaborate network of tertiary interactions that stabilize the molecule. Here we describe the molecular features of the active site in detail and evaluate their correspondence with prior biochemical, genetic, and phylogenetic analyses on group II introns. In addition, we evaluate the structural significance of RNA motifs within the intron core, such as the major-groove triple helix and the domain 5 bulge. Having combined what is known about the group II intron core, we then compare it with known structural features of U6 snRNA in the eukaryotic spliceosome. This analysis leads to a set of predictions for the molecular structure of the spliceosomal active site.     

Cwc2 and its human homologue RBM22 promote an active conformation of the spliceosome catalytic centre

RNA-structural elements play key roles in pre-mRNA splicing catalysis; yet, the formation of catalytically competent RNA structures requires the assistance of spliceosomal proteins. We show that the S. cerevisiae Cwc2 protein functions prior to step 1 of splicing, and it is not required for the Prp2-mediated spliceosome remodelling that generates the catalytically active B complex, suggesting that Cwc2 plays a more sophisticated role in the generation of a functional catalytic centre. In active spliceosomes, Cwc2 contacts catalytically important RNA elements, including the U6 internal stem-loop (ISL), and regions of U6 and the pre-mRNA intron near the 5' splice site, placing Cwc2 at/near the spliceosome's catalytic centre. These interactions are evolutionarily conserved, as shown by studies with Cwc2's human counterpart RBM22, indicating that Cwc2/RBM22-RNA contacts are functionally important. We propose that Cwc2 induces an active conformation of the spliceosome's catalytic RNA elements. Thus, the function of RNA-RNA tertiary interactions within group II introns, namely to induce an active conformation of domain V, may be fulfilled by proteins that contact the functionally analogous U6-ISL, within the spliceosome.     

Recycling of the U12-type spliceosome requires p110, a component of the U6atac snRNP

U12-dependent introns are spliced by the so-called minor spliceosome, requiring the U11, U12, and U4atac/U6atac snRNPs in addition to the U5 snRNP. We have recently identified U6-p110 (SART3) as a novel human recycling factor that is related to the yeast splicing factor Prp24. U6-p110 transiently associates with the U6 and U4/U6 snRNPs during the spliceosome cycle, regenerating functional U4/U6 snRNPs from singular U4 and U6 snRNPs. Here we investigated the involvement of U6-p110 in recycling of the U4atac/U6atac snRNP. In contrast to the major U6 and U4/U6 snRNPs, p110 is primarily associated with the U6atac snRNP but is almost undetectable in the U4atac/U6atac snRNP. Since p110 does not occur in U5 snRNA-containing complexes, it appears to be transiently associated with U6atac during the cycle of the minor spliceosome. The p110 binding site was mapped to U6 nucleotides 38 to 57 and U6atac nucleotides 10 to 30, which are highly conserved between these two functionally related snRNAs. With a U12-dependent in vitro splicing system, we demonstrate that p110 is required for recycling of the U4atac/U6atac snRNP.