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.     

The abundance of the spliceosomal snRNPs is not limiting the splicing of U12-type introns

The rate of excision of U12-type introns has been reported to be slower than that of U2-type introns, suggesting a rate-limiting bottleneck that could down-regulate genes containing U12-type introns. The mechanistic reasons for this slower rate of intron excision are not known, but lower abundance of the U12-type snRNPs and slower rate of assembly or catalytic activity have been suggested. To investigate snRNP abundance we concentrated on the U4atac snRNA, which is the least abundant of the U12-type snRNAs and is limiting the formation of U4atac/U6atac complex. We identified mouse NIH-3T3 cell line isolates in which the level of both U4atac snRNA and U4atac/U6atac complexes is reduced to 10%-20% of the normal level. We used these cell lines to investigate splicing efficiency by transient transfection of a reporter gene containing a U12-type intron and by quantitative PCR analysis of endogenous genes. The splicing of the reporter U12-type intron was very inefficient, but the activity could be restored by overexpression of U4atac snRNA. Using these U4atac-deficient NIH-3T3 cells, we confirmed the results of previous studies showing that U12-type introns of endogenous genes are, indeed, excised more slowly than U2-type introns, but we found that the rate did not differ from that measured in cells displaying normal levels of U4atac snRNA. Thus our results suggest that the cellular abundance of the snRNPs does not limit U12-type intron splicing under normal conditions.     

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.     

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.     

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.     

Gene expression profiling of U12-type spliceosome mutant Drosophila reveals widespread changes in metabolic pathways

Background

The U12-type spliceosome is responsible for the removal of a subset of introns from eukaryotic mRNAs. U12-type introns are spliced less efficiently than normal U2-type introns, which suggests a rate-limiting role in gene expression. The Drosophila genome contains about 20 U12-type introns, many of them in essential genes, and the U12-type spliceosome has previously been shown to be essential in the fly.

Methodology/Principal Findings

We have used a Drosophila line with a P-element insertion in U6atac snRNA, an essential component of the U12-type spliceosome, to investigate the impact of U12-type introns on gene expression at the organismal level during fly development. This line exhibits progressive accumulation of unspliced U12-type introns during larval development and the death of larvae at the third instar stage. Surprisingly, microarray and RT-PCR analyses revealed that most genes containing U12-type introns showed only mild perturbations in the splicing of U12-type introns. In contrast, we detected widespread downstream effects on genes that do not contain U12-type introns, with genes related to various metabolic pathways constituting the largest group.

Conclusions/Significance

U12-type intron-containing genes exhibited variable gene-specific responses to the splicing defect, with some genes showing up- or downregulation, while most did not change significantly. The observed residual U12-type splicing activity could be explained with the mutant U6atac allele having a low level of catalytic activity. Detailed analysis of all genes suggested that a defect in the splicing of the U12-type intron of the mitochondrial prohibitin gene may be the primary cause of the various downstream effects detected in the microarray analysis.     

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.     

Proximity of the U12 snRNA with both the 5' splice site and the branch point during early stages of spliceosome assembly

U12 snRNA is required for branch point recognition in the U12-dependent spliceosome. Using site-specific cross-linking, we have captured an unexpected interaction between the 5' end of the U12 snRNA and the -2 position upstream of the 5' splice site of P120 and SCN4a splicing substrates. The U12 snRNA nucleotides that contact the 5' exon are the same ones that form the catalytically important helix Ib with U6atac snRNA in the spliceosome catalytic core. However, the U12/5' exon interaction is transient, occurring prior to the entry of the U4atac/U6atac.U5 tri-snRNP to the spliceosome. This suggests that the helix Ib region of U12 snRNA is positioned near the 5' splice site early during spliceosome assembly and only later interacts with U6atac to form helix Ib. We also provide evidence that U12 snRNA can simultaneously interact with 5' exon sequences near 5' splice site and the branch point sequence, suggesting that the 5' splice site and branch point sequences are separated by <40 to 50 A in the complex A of the U12-dependent spliceosome. Thus, no major rearrangements are subsequently needed to position these sites for the first step of catalysis.     

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.     

A catalytically active group II intron domain 5 can function in the U12-dependent spliceosome

Both spliceosomal and self-splicing group II introns require the function of similar small, metal binding RNA stem-loop elements located in U6 or U6atac snRNAs of the spliceosome or domain 5 (D5) of group II introns. Here we report that two different D5 elements can functionally replace the U6atac snRNA stem-loop in an in vivo splicing assay. For efficient function in vivo, a single base pair from the upper helical section of the D5 sequence had to be removed. Introducing the equivalent base pair deletion into the D5 element of a group II intron reduced but did not eliminate self-splicing activity. Our results strengthen the case that these RNA elements play similar roles in the catalytic centers of both the spliceosome and a self-splicing ribozyme.     

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.     

Human U4/U6.U5 and U4atac/U6atac.U5 tri-snRNPs exhibit similar protein compositions

In the U12-dependent spliceosome, the U4atac/U6atac snRNP represents the functional analogue of the major U4/U6 snRNP. Little information is available presently regarding the protein composition of the former snRNP and its association with other snRNPs. In this report we show that human U4atac/U6atac di-snRNPs associate with U5 snRNPs to form a 25S U4atac/U6atac.U5 trimeric particle. Comparative analysis of minor and major tri-snRNPs by using immunoprecipitation experiments revealed that their protein compositions are very similar, if not identical. Not only U5-specific proteins but, surprisingly, all tested U4/U6- and major tri-snRNP-specific proteins were detected in the minor tri-snRNP complex. Significantly, the major tri-snRNP-specific proteins 65K and 110K, which are required for integration of the major tri-snRNP into the U2-dependent spliceosome, were among those proteins detected in the minor tri-snRNP, raising an interesting question as to how the specificity of addition of tri-snRNP to the corresponding spliceosome is maintained. Moreover, immunodepletion studies demonstrated that the U4/U6-specific 61K protein, which is involved in the formation of major tri-snRNPs, is essential for the association of the U4atac/U6atac di-snRNP with U5 to form the U4atac/U6atac.U5 tri-snRNP. Subsequent immunoprecipitation studies demonstrated that those proteins detected in the minor tri-snRNP complex are also incorporated into U12-dependent spliceosomes. This remarkable conservation of polypeptides between minor and major spliceosomes, coupled with the absence of significant sequence similarity between the functionally analogous snRNAs, supports an evolutionary model in which most major and minor spliceosomal proteins, but not snRNAs, are derived from a common ancestor.     

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.     

Minor-class splicing occurs in the nucleus of the Xenopus oocyte

A small fraction of premessenger RNA introns in certain eukaryotes is excised by the minor spliceosome, which contains low-abundance small nuclear ribonucleoproteins (snRNPs). Recently, it was suggested that minor-class snRNPs are localized to and function in the cytoplasm of vertebrate cells. To test whether U12-type splicing occurs in the cytoplasm of Xenopus oocytes, we performed microinjections of the well-characterized P120 minor-class splicing substrate into the nucleus or into the cytoplasm. Our results demonstrate that accurate splicing of this U12-dependent intron occurs exclusively in the nuclear compartment of the oocyte, where U12 and U6atac snRNPs are primarily localized. We further demonstrate that splicing of both a major-class and a minor-class intron is inhibited after nuclear envelope breakdown during meiosis.     

Whole-genome sequencing of a laboratory-evolved yeast strain

Background

Two categories of introns are known, a common U2 type and a rare U12 type. These two types of introns are removed by distinct spliceosomes. The phylogenetic distribution of spliceosomal RNAs that are characteristic of the U12 spliceosome, i.e. the U11, U12, U4atac and U6atac RNAs, suggest that U12 spliceosomes were lost in many phylogenetic groups. We have now examined the distribution of U2 and U12 introns in many of these groups.

Results

U2 and U12 introns were predicted by making use of available EST and genomic sequences. The results show that in species or branches where U12 spliceosomal components are missing, also U12 type of introns are lacking. Examples are the choanoflagellate Monosiga brevicollis, Entamoeba histolytica, green algae, diatoms, and the fungal lineage Basidiomycota. Furthermore, whereas U12 splicing does not occur in Caenorhabditis elegans, U12 introns as well as U12 snRNAs are present in Trichinella spiralis, which is deeply branching in the nematode tree. A comparison of homologous genes in T. spiralis and C. elegans revealed different mechanisms whereby U12 introns were lost.

Conclusions

The phylogenetic distribution of U12 introns and spliceosomal RNAs give further support to an early origin of U12 dependent splicing. In addition, this distribution identifies a large number of instances during eukaryotic evolution where such splicing was lost.     

U6atac snRNA,the highly divergent counterpart of U6 snRNA,is the specific target that mediates inhibition of AT-AC splicing by the influenza virus NS1 protein.

The influenza virus NS1 protein inhibits the splicing of the major class of mammalian pre-mRNAs (GU-AG Introns) by binding to a specific stem-bulge in U6 snRNA, thereby blocking the formation of U4/U6 and U2/U6 complexes. The splicing of the minor class of AT-AC introns takes place on spliceosomes that do not contain U6 snRNA, but rather U6atac snRNA-a highly divergent U6 snRNA counterpart. Nonetheless, we demonstrate that the NS1 protein inhibits AT-AC splicing in vitro, and specifically binds to only U6atac snRNA among the five minor class snRNAs. Chemical modification/interference assays show that the NS1 protein binds to the stem-bulge near the 3'' end of U6atac snRNA, encompassing nt 82-95 and nt 105-114. Although the sequence of this stem-bulge differs significantly from the sequence of the stem-bulge to which the NS1 protein binds in U6 snRNA, RNA competition experiments Indicate that U6 and U6atac snRNAs likely share the same binding site on the NS1 protein. Previously, the region of U6atac snRNA containing this 3'' stem-bulge had not been implicated in any interactions of this snRNA with either U4atac or U12 snRNA. However, as assayed by psoralen crosslinking, we show that the NS1 protein inhibits the formation of U12/U6atac complexes, but not the formation of U4atac/U6atac complexes. We can conclude that the inhibition of AT-AC splicing results largely from the inhibition of formation of U12/U6atac complexes caused by the binding of the NS1 protein to the 3'' stem-bulge of U6atac snRNA.     

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.     

Computational screen for spliceosomal RNA genes aids in defining the phylogenetic distribution of major and minor spliceosomal components

The RNA molecules of the spliceosome are critical for specificity and catalysis during splicing of eukaryotic pre-mRNA. In order to examine the evolution and phylogenetic distribution of these RNAs, we analyzed 149 eukaryotic genomes representing a broad range of phylogenetic groups. RNAs were predicted using high-sensitivity local alignment methods and profile HMMs in combination with covariance models. The results provide the most comprehensive view so far of the phylogenetic distribution of spliceosomal RNAs. RNAs were predicted in many phylogenetic groups where these RNA were not previously reported. Examples are RNAs of the major (U2-type) spliceosome in all fungal lineages, in lower metazoa and many protozoa. We also identified the minor (U12-type) spliceosomal U11 and U6atac RNAs in Acanthamoeba castellanii, where U12 spliceosomal RNA as well as minor introns were reported recently. In addition, minor-spliceosome-specific RNAs were identified in a number of phylogenetic groups where previously such RNAs were not observed, including the nematode Trichinella spiralis, the slime mold Physarum polycephalum and the fungal lineages Zygomycota and Chytridiomycota. The detailed map of the distribution of the U12-type RNA genes supports an early origin of the minor spliceosome and points to a number of occasions during evolution where it was lost.     

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.