PRPF8 defects cause missplicing in myeloid malignancies.

Mutations of spliceosome components are common in myeloid neoplasms. One of the affected genes, PRPF8, encodes the most evolutionarily conserved spliceosomal protein. We identified either recurrent somatic PRPF8 mutations or hemizygous deletions in 15/447 and 24/450 cases, respectively. Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis. PRPF8 defects correlated with increased myeloblasts and ring sideroblasts in cases without SF3B1 mutations. Knockdown of PRPF8 in K562 and CD34+ primary bone marrow cells increased proliferative capacity. Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects. In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions. In sum, the exploration of clinical and functional consequences suggests that PRPF8 is a novel leukemogenic gene in myeloid neoplasms with a distinct phenotype likely manifested through aberrant splicing.

Alternative splicing of U12-dependent introns in vivo responds to purine-rich enhancers.

Alternative splicing increases the coding capacity of genes through the production of multiple protein isoforms by the conditional use of splice sites and exons. Many alternative splice sites are regulated by the presence of purine-rich splicing enhancer elements (ESEs) located in the downstream exon. Although the role of ESEs in alternative splicing of the major class U2-dependent introns is well established, no alternatively spliced minor class U12-dependent introns have so far been described. Although in vitro studies have shown that ESEs can stimulate splicing of individual U12-dependent introns, there is no direct evidence that the U12-dependent splicing system can respond to ESEs in vivo. To investigate the ability of U12-dependent introns to use alternative splice sites and to respond to ESEs in an in vivo context, we have constructed two sets of artificial minigenes with alternative splicing pathways and evaluated the effects of ESEs on their alternative splicing patterns. In minigenes with alternative U12-dependent 3' splice sites, a purine-rich ESE promotes splicing to the immediately upstream 3' splice site. As a control, a mutant ESE has no stimulatory effect. In minigene constructs with two adjacent U12-dependent introns, the predominant in vivo splicing pattern results in the skipping of the internal exon. Insertion of a purine-rich ESE into the internal exon promotes the inclusion of the internal exon. These results show that U12-dependent introns can participate in alternative splicing pathways and that U12-dependent splice sites can respond to enhancer elements in vivo.

Role of the 3' splice site in U12-dependent intron splicing.

U12-dependent introns containing alterations of the 3' splice site AC dinucleotide or alterations in the spacing between the branch site and the 3' splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5' splice site AU dinucleotide, any nucleotide could serve as the 3'-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3' splice site function. A branch site-to-3' splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3' splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3' splice site but that formation of the prespliceosomal complex A requires an active 3' splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3' splice site.

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.

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.

The two steps of group II intron self-splicing are mechanistically distinguishable.

The two transesterification reactions catalyzed by self-splicing group II introns take place in either two active sites or two conformations of a single active site involving rearrangements of the positions of the reacting groups. We have investigated the effects on the rates of the chemical steps of the two reactions due to sulfur substitution of nonbridging oxygens at both the 5' and 3' splice sites as well as the deoxyribose substitution of the ribose 2' hydroxyl group at the 5' splice site. The data suggest that the two active sites differ in their interactions with several of these groups. Specifically, sulfur substitution of the pro-Sp nonbridging oxygen at the 5' splice site reduces the chemical rate of the step one branching reaction by at least 250-fold, whereas substitution of the pro-Sp oxygen at the 3' splice site has only a 4.5-fold effect on the chemical rate of step two. Previous work demonstrated that the Rp phosphorothioate substitutions at both the 5' and 3' splice sites reduced the rate of both steps of splicing to an undetectable level. These results suggest that either two distinct active sites catalyze the two steps or that more significant alterations must be made in a single bifunctional active site to accommodate the two different reactions.

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.

Evolutionary fates and origins of U12-type introns.

U2-type and U12-type introns are spliced by distinct spliceosomes in eukaryotic nuclei. A classification method was devised to distinguish these two types of introns based on splice site sequence properties and was used to identify 56 different genes containing U12-type introns in available genomic sequences. U12-type introns occur with consistently low frequency in diverse eukaryotic taxa but have almost certainly been lost from C. elegans. Comparisons with available homologous sequences demonstrate subtype switching of U12 introns between termini of AT-AC and GT-AG as well as conversion of introns from U12-type to U2-type and provide evidence for a fission/fusion model in which the two splicing systems evolved in separate lineages that were fused in a eukaryotic progenitor.

U11 snRNA interacts in vivo with the 5' splice site of U12-dependent (AU-AC) pre-mRNA introns.

A notable feature of the newly described U12 snRNA-dependent class of eukaryotic nuclear pre-mRNA introns is the highly conserved 8-nt 5' splice site sequence. This sequence is virtually invariant in all known members of this class from plants to mammals. Based on sequence complementarity between this sequence and the 5' end of the U11 snRNA, we proposed that U11 snRNP may play a role in identifying and/or activating the 5' splice site for splicing. Here we show that mutations of the conserved 5' splice site sequence of a U12-dependent intron severely reduce correct splicing in vivo and that compensatory mutations in U11 snRNA can suppress the effects of the 5' splice site mutations to varying extents. This provides evidence for a required interaction between U11 snRNA and the 5' splice site sequence involving Watson-Crick base pairing. This data, in addition to a report that U11 snRNP is bound transiently to the U12-dependent spliceosome, suggests that U11 snRNP is the analogue of U1 snRNP in splicing this rare class of introns.

Terminal intron dinucleotide sequences do not distinguish between U2- and U12-dependent introns.

Two types of eukaryotic nuclear introns are known: the common U2-dependent class with /GU and AG/ terminal intron dinucleotides, and the rare U12-dependent class with /AU and AC/ termini. Here we show that the U12-dependent splicing system can splice introns with /GU and AG/ termini and that such introns occur naturally. Further, U2-dependent introns with /AU and AC/termini also occur naturally and are evolutionarily conserved. Thus, the sequence of the terminal dinucleotides does not determine which spliceosomal system removes an intron. Rather, the four classes of introns described here can be sorted into two mechanistic classes (U2- or U12-dependent) by inspection of the complete set of conserved splice site sequences.

Requirement of U12 snRNA for in vivo splicing of a minor class of eukaryotic nuclear pre-mRNA introns.

A conserved sequence element in a minor class of eukaryotic pre-messenger RNA (pre-mRNA) introns was previously proposed to base pair with a complementary sequence in the U12 small nuclear RNA (snRNA) in a manner analogous to the pairing of US snRNA with the branch site sequence of the major class of introns. Here, mutations generated in this conserved sequence element block the splicing of a member of this minor intron class in vivo. The block was relieved by coexpression of a U12 snRNA containing compensatory mutations that restore the proposed base pairing interaction. These results show that this minor class of pre-mRNA introns is a distinct class existing alongside the major class of introns in animal genomes, and these results also establish an in vivo function for U12 snRNA.

Stereochemical selectivity of group II intron splicing, reverse splicing, and hydrolysis reactions.

We have previously shown, using phosphorothioate substitutions at splice site, that both transesterification steps of group II intron self-splicing proceed, by stereochemical inversion, with an Sp but not an Rp phosphorothioate. Under alternative reaction conditions or with various intron fragments, group II introns can splice following hydrolysis at the 5' splice site and can also hydrolyze the bond between spliced exons (the spliced-exon reopening reaction). In this study, we have determined the stereochemical specificities of all of the major model hydrolytic reactions carried out by the aI5 gamma intron from Saccharomyces cerevisiae mitochondria. For all substrates containing exon 1 and most of the intron, the stereospecificity of hydrolysis is the same as for the step 1 transesterification reaction. In contrast, the spliced-exon reopening reaction proceeds with an Rp but not an Sp phosphorothioate at the scissile bond, as does true reverse splicing. Thus, by stereochemistry, this reaction appears to be related to the reverse of step 2 of self-splicing. Finally, a substrate RNA that contains the first exon and nine nucleotides of the intron, when reacted with the intron ribozyme, releases the first exon regardless of the configuration of the phosphorothioate at the 5' splice site, suggesting that this substrate can be cleaved by either the step 1 or the step 2 reaction site. Our findings clarify the relationships of these model reactions to the transesterification reactions of the intact self-splicing system and permit new studies to be interpreted more rigorously.

The stereochemical course of group II intron self-splicing.

The stereochemical specificities and reaction courses for both self-splicing steps of a group II intron have been determined by phosphorothioate substitution at the 5' and 3' splice site phosphodiester bonds. Both steps of the splicing reaction proceeded with a phosphorothioate in the Sp configuration but were blocked by the Rp diastereomer. Both steps also proceeded with inversion of stereochemical configuration around phosphorus, consistent with a concerted transesterification reaction. These results are identical to those found for nuclear precursor mRNA (pre-mRNA) splicing and provide support for the hypothesis that group II introns and nuclear pre-mRNA introns share a common evolutionary history.

Conserved sequences in a class of rare eukaryotic nuclear introns with non-consensus splice sites.

Eukaryotic nuclear genomes contain a rare class of pre-mRNA introns with consensus sequence features that differ markedly from most pre-mRNA introns. Four genes have so far been identified that contain one copy each of this rare intron class in addition to several standard introns. These introns and homologous introns from several species were compared to identify conserved sequence elements and to establish consensus sequences for these elements. The only well-conserved elements are found at the 5' and 3' ends of the introns. The 5' splice site sequence is ATATCCTT beginning with the first nucleotide of the intron and is invariant in the introns examined to date. The 3' splice site consensus sequence is YCCAC ending at the last nucleotide of the intron. An almost invariant sequence of TCCTTAAC is also found near the 3' end of the intron (the 3' upstream element). The length of the introns varies between 95 and 2940 nucleotides. The sequence organization of these introns suggests that they represent a variant class of pre-mRNA introns that might be spliced via a spliceosome mechanism employing factors distinct from those used by other pre-mRNA introns. A search of small nuclear RNA (snRNA) sequences for regions complementary to the conserved elements of this rare class of introns found a strong match between U12 snRNA and the 3' upstream element and a weaker match between U11 snRNA and the 5' splice site sequence.

The stereochemical course of the first step of pre-mRNA splicing.

We have determined the effects on splicing of sulfur substitution of the non-bridging oxygens in the phosphodiester bond at the 5' splice site of a pre-mRNA intron. Pre-mRNAs containing stereochemically pure Rp and Sp phosphorothioate isomers were produced by ligation of a chemically synthesized modified RNA oligonucleotide to enzymatically synthesized RAs. When these modified pre-mRNA substrates were tested for in vitro splicing activity in a HeLa cell nuclear extract system, the RNA with the Rp diastereomeric phosphorothioate was not spliced while the Sp diastereomeric RNA spliced readily. The sulfur-containing branched trinucleotide was purified from the splicing reaction of the Sp RNA and analyzed by cleavage with a stereospecific nuclease. The results showed that the Sp phosphorothioate was inverted during the splicing reaction to the Rp configuration; a finding previously obtained for a Group I self-splicing RNA. This inversion of configuration is consistent with a transesterification mechanism for pre-mRNA splicing. The lack of splicing of the Rp modified RNA also suggests that the pro-Rp oxygen at the 5' splice site is involved in a critical chemical contact in the splicing mechanism. Additionally, we have found that the HeLa cell RNA debranching enzyme is inactive on branches containing an Rp phosphorothioate.

Phosphorothioate substitution identifies phosphate groups important for pre-mRNA splicing.

Substitution of pre-mRNA in vitro splicing substrates with alpha-phosphorothioate ribonucleotide analogs has multiple effects on the processes of spliceosome formation and splicing. A major effect of substitution is on the splicing cleavage/ligation reactions. Substitution at the 5' splice junction blocks the first cleavage/ligation reaction while substitution at the 3' splice junction blocks the second cleavage/ligation reaction. A second effect of phosphorothioate substitution is the inhibition of spliceosome formation. A substitution/interference assay was used to determine positions where substitution inhibits spliceosome formation or splicing. Substitution in the 3' splice site polypyrimidine tract was found to inhibit spliceosome formation and splicing. This effect was enhanced with multiple substitutions in the region. No sites of substitution within the exons were found which affected spliceosome formation or splicing.

Hydroxyl radical "footprinting" of RNA: application to pre-mRNA splicing complexes.

We present an adaptation of the hydroxyl radical DNA "footprinting" technique that permits high-resolution mapping of protected regions of RNA. Hydroxyl radical cleaves RNA independently of base sequence and secondary structure of the RNAs examined and allows resolution of protected regions at the single nucleotide level. By using this technique, we show that several regions of the 3' splice site of mRNA precursors are protected during the formation of splicing-specific ribonucleoprotein complexes in an in vitro splicing system. These regions include the 3' intron/exon junction and a portion of the adjacent exon, the polypyrimidine tract, and the site of branch formation. These protections appear to be due to splicing specific complexes since their formation is sensitive to point mutations at crucial residues and requires ATP and incubation. The formation of these protected regions is independent of the presence of a 5' splice site.

Sequence requirements for splicing of higher eukaryotic nuclear pre-mRNA.

We determined the effect on splicing of 24 point mutations in the 5' and 3' splice region of the large rabbit beta-globin intron. In vitro, 3' AG mutations drastically reduce 5' cleavage and abolish splicing. In vivo, the same mutations elicit efficient splicing at a cryptic, rather than the correct, 3' splice site. In vitro, mutations at all but 2 positions of the consensus 5' splice region impair correct splicing and promote joining of exon 1 to exon 3. In vivo, the same mutations show no effect, except for those converting 5' GT to AT or GA, which cause accumulation of lariat intermediate in vitro and in vivo. We conclude that the 5' GT need not be conserved for 5' cleavage and that it plays an important role in cleavage and exon joining at the 3' splice site.