Sde2 is an intron‐specific pre‐mRNA splicing regulator activated by ubiquitin‐like processing

The expression of intron‐containing genes in eukaryotes requires generation of protein‐coding messenger RNAs (mRNAs) via RNA splicing, whereby the spliceosome removes non‐coding introns from pre‐mRNAs and joins exons. Spliceosomes must ensure accurate removal of highly diverse introns. We show that Sde2 is a ubiquitin‐fold‐containing splicing regulator that supports splicing of selected pre‐mRNAs in an intron‐specific manner in Schizosaccharomyces pombe. Both fission yeast and human Sde2 are translated as inactive precursor proteins harbouring the ubiquitin‐fold domain linked through an invariant GGKGG motif to a C‐terminal domain (referred to as Sde2‐C). Precursor processing after the first di‐glycine motif by the ubiquitin‐specific proteases Ubp5 and Ubp15 generates a short‐lived activated Sde2‐C fragment with an N‐terminal lysine residue, which subsequently gets incorporated into spliceosomes. Absence of Sde2 or defects in Sde2 activation both result in inefficient excision of selected introns from a subset of pre‐mRNAs. Sde2 facilitates spliceosomal association of Cactin/Cay1, with a functional link between Sde2 and Cactin further supported by genetic interactions and pre‐mRNA splicing assays. These findings suggest that ubiquitin‐like processing of Sde2 into a short‐lived activated form may function as a checkpoint to ensure proper splicing of certain pre‐mRNAs in fission yeast.     

Tra2-Mediated Recognition of HIV-1 5′ Splice Site D3 as a Key Factor in the Processing of vpr mRNA

Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3′ splice site (3′ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5′ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE_vpr) localized between exonic splicing silencer ESSV and 5′ss D3. The ESE_vpr sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE_vpr. Further analyses revealed that ESE_vpr supports the binding of U1 snRNA at 5′ss D3, allowing bridging interactions across the upstream exon with 3′ss A2. In line with this, an increase or decrease in the complementarity of 5′ss D3 to the 5′ end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3′ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5′ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3′ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.     

Biased exon/intron distribution of cryptic and de novo 3' splice sites

We compiled sequences of previously published aberrant 3′ splice sites (3′ss) that were generated by mutations in human disease genes. Cryptic 3′ss, defined here as those resulting from a mutation of the 3′YAG consensus, were more frequent in exons than in introns. They clustered in ~20 nt region adjacent to authentic 3′ss, suggesting that their under-representation in introns is due to a depletion of AG dinucleotides in the polypyrimidine tract (PPT). In contrast, most aberrant 3′ss that were induced by mutations outside the 3′YAG consensus (designated ‘de novo’) were in introns. The activation of intronic de novo 3′ss was largely due to AG-creating mutations in the PPT. In contrast, exonic de novo 3′ss were more often induced by mutations improving the PPT, branchpoint sequence (BPS) or distant auxiliary signals, rather than by direct AG creation. The Shapiro–Senapathy matrix scores had a good prognostic value for cryptic, but not de novo 3′ss. Finally, AG-creating mutations in the PPT that produced aberrant 3′ss upstream of the predicted BPS in vivo shared a similar ‘BPS-new AG’ distance. Reduction of this distance and/or the strength of the new AG PPT in splicing reporter pre-mRNAs improved utilization of authentic 3′ss, suggesting that AG-creating mutations that are located closer to the BPS and are preceded by weaker PPT may result in less severe splicing defects.     

Increased efficiency of evolved group I intron spliceozymes by decreased side product formation

The group I intron ribozyme from Tetrahymena was recently reengineered into a trans-splicing variant that is able to remove 100-nt introns from pre-mRNA, analogous to the spliceosome. These spliceozymes were improved in this study by 10 rounds of evolution in Escherichia coli cells. One clone with increased activity in E. coli cells was analyzed in detail. Three of its 10 necessary mutations extended the substrate binding duplexes, which led to increased product formation and reduced cleavage at the 5′-splice site. One mutation in the conserved core of the spliceozyme led to a further reduction of cleavage at the 5′-splice site but an increase in cleavage side products at the 3′-splice site. The latter was partially reduced by six additional mutations. Together, the mutations increased product formation while reducing activity at the 5′-splice site and increasing activity at the 3′-splice site. These results show the adaptation of a ribozyme that evolved in nature for cis-splicing to trans-splicing, and they highlight the interdependent function of nucleotides within group I intron ribozymes. Implications for the possible use of spliceozymes as tools in research and therapy, and as a model for the evolution of the spliceosome, are discussed.     

Multiple physical forms of excised group II intron RNAs in wheat mitochondria

Plant mitochondrial group II introns do not all possess hallmark ribozymic features such as the bulged adenosine involved in lariat formation. To gain insight into their splicing pathways, we have examined the physical form of excised introns in germinating wheat embryos. Using RT–PCR and cRT–PCR, we observed conventional lariats consistent with a two-step transesterification pathway for introns such as nad2 intron 4, but this was not the case for the cox2 intron or nad1 intron 2. For cox2, we detected full-length linear introns, which possess non-encoded 3′terminaladenosines, as well as heterogeneous circular introns, which lack 3′ nucleotide stretches. These observations are consistent with hydrolytic splicing followed by polyadenylation as well as an in vivo circularization pathway, respectively. The presence of both linear and circular species in vivo is supported by RNase H analysis. Furthermore, the nad1 intron 2, which lacks a bulged nucleotide at the branchpoint position, comprised a mixed population of precisely full-length molecules and circular ones which also include a short, discrete block of non-encoded nucleotides. The presence of these various linear and circular forms of excised intron molecules in plant mitochondria points to multiple novel group II splicing mechanisms in vivo.     

Aberrant 3' splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization

The frequency distribution of mutation-induced aberrant 3′ splice sites (3′ss) in exons and introns is more complex than for 5′ splice sites, largely owing to sequence constraints upstream of intron/exon boundaries. As a result, prediction of their localization remains a challenging task. Here, nucleotide sequences of previously reported 218 aberrant 3′ss activated by disease-causing mutations in 131 human genes were compared with their authentic counterparts using currently available splice site prediction tools. Each tested algorithm distinguished authentic 3′ss from cryptic sites more effectively than from de novo sites. The best discrimination between aberrant and authentic 3′ss was achieved by the maximum entropy model. Almost one half of aberrant 3′ss was activated by AG-creating mutations and ~95% of the newly created AGs were selected in vivo. The overall nucleotide structure upstream of aberrant 3′ss was characterized by higher purine content than for authentic sites, particularly in position −3, that may be compensated by more stringent requirements for positive and negative nucleotide signatures centred around position −11. A newly developed online database of aberrant 3′ss will facilitate identification of splicing mutations in a gene or phenotype of interest and future optimization of splice site prediction tools.     

Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5′ splice sites (5′ ss) and three 3′ splice sites (3′ ss) normally used in HPV16⁺ cervical cancer and its derived cell lines. The choice of two novel alternative 5′ ss (nt 221 5′ ss and nt 191 5′ ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5′ ss and nt 409 3′ ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3′ ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3′ ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of ⁹¹QYNK⁹⁴ to ⁹¹PSFW⁹⁴ displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.     

In vitro characterization of the splicing efficiency and fidelity of the RmInt1 group II intron as a means of controlling the dispersion of its host mobile element

Group II introns are catalytic RNAs that are excised from their precursors in a protein-dependent manner in vivo. Certain group II introns can also react in a protein-independent manner under nonphysiological conditions in vitro. The efficiency and fidelity of the splicing reaction is crucial, to guarantee the correct formation and expression of the protein-coding mRNA. RmInt1 is an efficient mobile intron found within the ISRm2011-2 insertion sequence in the symbiotic bacterium Sinorhizobium meliloti. The RmInt1 intron self-splices in vitro, but this reaction generates side products due to a predicted cryptic IBS1* sequence within the 3′ exon. We engineered an RmInt1 intron lacking the cryptic IBS1* sequence, which improved the fidelity of the splicing reaction. However, atypical circular forms of similar electrophoretic mobility to the lariat intron were nevertheless observed. We analyzed a run of four cytidine residues at the 3′ splice site potentially responsible for a lack of fidelity at this site leading to the formation of circular intron forms. We showed that mutations of residues base-pairing in the tertiary EBS3–IBS3 interaction increased the efficiency and fidelity of the splicing reaction. Our results indicate that RmInt1 has developed strategies for decreasing its splicing efficiency and fidelity. RmInt1 makes use of unproductive splicing reactions to limit the transposition of the insertion sequence into which it inserts itself in its natural context, thereby preventing potentially harmful dispersion of ISRm2011-2 throughout the genome of its host.     

SL1 trans Splicing and 3′-End Formation in a Novel Class of Caenorhabditis elegans Operon

Many Caenorhabditis elegans genes exist in operons in which polycistronic precursors are processed by cleavage at the 3′ ends of upstream genes and trans splicing 100 to 400 nucleotides away, at the 5′ ends of downstream genes, to generate monocistronic messages. Of the two spliced leaders, SL1 is trans spliced to the 5′ ends of upstream genes, whereas SL2 is reserved for downstream genes in operons. However, there are isolated examples of what appears to be a different sort of operon, in which trans splicing is exclusively to SL1 and there is no intercistronic region; the polyadenylation signal is only a few base pairs upstream of the trans-splice site. We have analyzed the processing of an operon of this type by inserting the central part of mes-6/cks-1 into an SL2-type operon. In this novel context, cks-1 is trans spliced only to SL1, and mes-6 3′-end formation occurs normally, demonstrating that this unique mode of processing is indeed intrinsic to this kind of operon, which we herein designate “SL1-type.” An exceptionally long polypyrimidine tract found in the 3′ untranslated regions of the three known SL1-type operons is shown to be required for the accumulation of both upstream and downstream mRNAs. Mutations of the trans-splice and poly(A) signals indicate that the two processes are independent and in competition, presumably due to their close proximity, raising the possibility that production of upstream and downstream mRNAs is mutually exclusive.     

Prp4 Kinase Grants the License to Splice: Control of Weak Splice Sites during Spliceosome Activation

The genome of the fission yeast Schizosaccharomyces pombe encodes 17 kinases that are essential for cell growth. These include the cell-cycle regulator Cdc2, as well as several kinases that coordinate cell growth, polarity, and morphogenesis during the cell cycle. In this study, we further characterized another of these essential kinases, Prp4, and showed that the splicing of many introns is dependent on Prp4 kinase activity. For detailed characterization, we chose the genes res1 and ppk8, each of which contains one intron of typical size and position. Splicing of the res1 intron was dependent on Prp4 kinase activity, whereas splicing of the ppk8 intron was not. Extensive mutational analyses of the 5’ splice site of both genes revealed that proper transient interaction with the 5’ end of snRNA U1 governs the dependence of splicing on Prp4 kinase activity. Proper transient interaction between the branch sequence and snRNA U2 was also important. Therefore, the Prp4 kinase is required for recognition and efficient splicing of introns displaying weak exon1/5’ splice sites and weak branch sequences.     

Group II intron in Bacillus cereus has an unusual 3' extension and splices 56 nucleotides downstream of the predicted site

All group II introns known to date fold into six functional domains. However, we recently identified an intron in Bacillus cereus ATCC 10987, B.c.I4, that splices 56nt downstream of the expected 3′ splice site in vivo (Tourasse et al. 2005, J. Bacteriol., 187, 5437–5451). In this study, we confirmed by ribonuclease protection assay that the 56-bp segment is part of the intron RNA molecule, and computational prediction suggests that it might form a stable stem-loop structure downstream of domain VI. The splicing of B.c.I4 was further investigated both in vivo and in vitro. Lariat formation proceeded primarily by branching at the ordinary bulged adenosine in domain VI without affecting the fidelity of splicing. In addition, the splicing efficiency of the wild-type intron was better than that of a mutant construct deleted of the 56-bp 3′ extension. These results indicate that the intron has apparently adapted to the extra segment, possibly through conformational adjustments. The extraordinary group II intron B.c.I4 harboring an unprecedented extra 3′ segment constitutes a dramatic example of the flexibility and adaptability of group II introns.     

A Role for SRp54 during Intron Bridging of Small Introns with Pyrimidine Tracts Upstream of the Branch Point

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3′ splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3′ splice site. Many small introns, especially those in nonvertebrates, lack a classical 3′ pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5′ splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5′ and 3′ splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3′ pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.Pre-mRNA splicing is a conserved process occurring in a wide variety of eucaryotes with differing exon/intron architectures (reviewed in references 4, 6, 9, 15, 20, and 26). Vertebrates typically have small exons and large introns. Nonmetazoans frequently have the opposite genetic organization, with introns smaller than the minimum permissible for splicing of a vertebrate intron. Drosophila melanogaster possesses a mixture of these two classes of intron sizes (16, 23). In addition, more than half of the small introns in Drosophila are missing a prominent vertebrate splicing signal, the 3′ polypyrimidine tract (23). For these reasons, Drosophila provides a model system in which to study potential mechanistic variations operating during recognition of splicing signals.In the general model of early vertebrate spliceosome complex assembly, U1 snRNP binds to the 5′ splice site and U2 snRNP auxiliary factor (U2AF) binds to the 3′ polypyrimidine tract, thereby facilitating U2 snRNP interaction with the branch point. Various members of the serine/arginine (SR) family of proteins may participate by promoting or stabilizing these interactions (reviewed in references 13, 22, and 31). This family of proteins may also act as exon or intron bridging factors via their SR-mediated interaction with SR domains on the small subunit of U2AF (U2AF³⁵) and the U1 70K protein (32, 33, 38). SF1, originally discovered as an essential splicing factor in reconstitution assays (19), has also been observed to bind to the branch point (7, 8). In yeast, BBP (branch point bridging protein), the ortholog to SF1, functions as an intron bridging factor via interactions with U1 snRNP-associated proteins and the large subunit of U2AF (U2AF⁶⁵) (1, 2). It is assumed that vertebrate SF1 can play a similar role, although the mammalian equivalents to the yeast U1 snRNP proteins that interact with BBP have not yet been identified. Furthermore, the relationship between bridging by SR proteins and that afforded by SF1 is unclear.We have previously examined the cis-acting sequences required for efficient splicing of a constitutively spliced small (59-nucleotide [nt]) intron from the D. melanogaster mle gene that lacks a well-defined pyrimidine tract between the branch point and 3′ splice site (18, 29). Assembly of initial ATP-dependent spliceosomes (complex A) on the mle intron requires both the 5′ and 3′ splice sites, suggesting concerted recognition of the entire intron (29). Instead of a classic pyrimidine tract, the mle intron contains two C-rich tracts located between the 5′ splice site and branch point that are necessary for efficient splicing of this intron (18). In addition to a requirement for maximal splicing efficiency, the pyrimidine stretches are also necessary for binding of U2AF, interaction of factors with the 5′ splice site, and proper assembly of the active spliceosome, suggesting that these sequences affect early assembly events at both ends of this small intron. Interestingly, the upstream C-rich tracts are inhibitory if a classical 3′ pyrimidine tract is introduced between the branch point and 3′ splice site (18). This observation suggests competing pathways of factor binding to this substrate and also raises the possibility of alternative gene-specific modes of association of constitutive factors with introns.Here we demonstrate that both U2AF and an SR protein, SRp54, interact with the C-rich tracts in the mle intron. The central location of the pyrimidine tracts, their importance for maximal splicing, and the ability of human SRp54 to interact with U2AF⁶⁵ instead of U2AF³⁵ (37) suggested that the binding of SRp54 to the tracts could replace SF1 in bridging this intron. Immunoprecipitation studies using an antibody specific for SF1 indicated that SF1 did not contact mle precursor RNA unless a pyrimidine tract was introduced downstream of the branch point. Furthermore, antibodies against either SRp54 or U2AF immunoprecipitated both halves of a precleaved mle splicing substrate, suggesting that these factors either directly or indirectly interact with both the 5′ and 3′ splice sites. We suggest that SRp54 participates in bridging the small mle intron via its ability to bind both the C-rich tracts and the large subunit of U2AF.