Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing

U2 small nuclear RNA is a highly conserved component of the eukaryotic cell nucleus involved in splicing messenger RNA precursors. In the yeast Saccharomyces cerevisiae, U2 RNA interacts with the intron by RNA-RNA pairing between the conserved branchpoint sequence UACUAAC and conserved nucleotides near the 5' end of U2 (ref. 4). Metazoan U2 RNA is less than 200 nucleotides in length, but yeast U2 RNA is 1,175 nucleotides long. The 5' 110 nucleotides of yeast U2 are homologous to the 5' 100 nucleotides of metazoan U2 (ref. 6), and the very 3' end of yeast U2 bears a weak structural resemblance to features near the 3' end of metazoan U2. Internal sequences of yeast U2 share primary sequence homology with metazoan U4, U5 and U6 small nuclear RNA (ref. 6), and have regions of complementarity with yeast U1 (ref. 7). We have investigated the importance of the internal U2 sequences by their deletion. Yeast cells carrying a U2 allele lacking 958 nucleotides of internal U2 sequence produce a U2 small nuclear RNA similar in size to that found in other organisms. Cells carrying only the U2 deletion grow normally, have normal levels of spliced mRNA and do not accumulate unspliced precursor mRNA. We conclude that the internal sequences of yeast U2 carry no essential function. The extra RNA may have a non-essential function in efficient ribonucleoprotein assembly or RNA stability. Variation in amount of RNA in homologous structural RNAs has precedence in ribosomal RNA and RNaseP.     

Human U2 snRNA can function in pre-mRNA splicing in yeast

The removal of introns from messenger RNA precursors requires five small nuclear RNAs (snRNAs), contained within ribonucleoprotein particles (snRNPs), which complex with the pre-mRNA and other associated factors to form the spliceosome. In both yeast and mammals, the U2 snRNA base pairs with sequences surrounding the site of lariat formation. Binding of U2 snRNP to the highly degenerate branchpoint sequence in mammalian introns is absolutely dependent on an auxiliary protein, U2AF, which recognizes a polypyrimidine stretch adjacent to the 3' splice site. The absence of this sequence motif in yeast introns has strengthened arguments that the two systems are fundamentally different. Deletion analyses of the yeast U2 gene have confirmed that the highly conserved 5' domain is essential, although the adjacent approximately 950 nucleotides can be deleted without any phenotypic consequence. A 3'-terminal domain of approximately 100 nucleotides is also required for wild-type growth rates; the highly conserved terminal loop within this domain (loop IV) may provide specific binding contacts for two U2-specific snRNP proteins. We have replaced the single copy yeast U2 (yU2) gene with human U2 (hU2), expecting that weak or no complementation would provide an assay for cloning additional splicing factors, such as U2AF. We report here that hU2 can complement the yeast deletion with surprising efficiency. The interactions governing spliceosome assembly and intron recognition are thus more conserved than previously suspected. Paradoxically, the conserved loop IV sequence is dispensable in yeast.     

Cleavage of 5' splice site and lariat formation are independent of 3' splice site in yeast mRNA splicing

Analysis of messenger RNA splicing in yeast and in metazoa has led to the identification of an RNA molecule in a lariat conformation. This structure has been found as an mRNA splicing intermediate in vitro and identical molecules have been identified in vivo. Lariat formation involves cleavage of the precursor at the 5' splice site (5' SS) and the formation of a 2'-5' phosphodiester bond between the guanosine residue at the 5' end of the intron and an adenosine within the intron. The yeast branchpoint is located within the absolutely conserved TACTAAC box (that is, the last A of the TACTAAC box is the site of formation of the 2'-5' phosphodiester bond with the 5' end of the intron)3,4. Moreover, efficient 5' SS cleavage and lariat formation require proper sequences at the 5' splice junction and within the TACTAAC box. Here we demonstrate that 5' SS cleavage and lariat formation take place in vitro in the absence of the 3' SS and much of the 3' junction. These results are discussed in light of possible differences between yeast and metazoan mRNA splicing.     

Mechanism of recognition of the 5' splice site in self-splicing group I introns

Group I introns include many mitochondrial ribosomal RNA and messenger RNA introns and the nuclear rRNA introns of Tetrahymena and Physarum. The splicing of precursor RNAs containing these introns is a two-step reaction. Cleavage at the 5' splice site precedes cleavage at the 3' splice site, the latter cleavage being coupled with exon ligation. Following the first cleavage, the 5' exon must somehow be held in place for ligation. We have now tested the reactivity of two self-splicing group I RNAs, the Tetrahymena pre-rRNA and the intron 1 portion of the Neurospora mitochondrial cytochrome b (cob) pre-mRNA, in the intermolecular exon ligation reaction (splicing in trans) described by Inoue et al. The different sequence specificity of the reactions supports the idea that the nucleotides immediately upstream from the 5' splice site are base-paired to an internal, 5' exon-binding site, in agreement with RNA structure models proposed by Davies and co-workers and others. The internal binding site is proposed to be involved in the formation of a structure that specifies the 5' splice site and, following the first step of splicing, to hold the 5' exon in place for exon ligation.     

Reverse self-splicing of group II intron RNAs in vitro.

Group II introns, which are classed together on the basis of a conserved secondary structure, are found in organellar genes of lower eukaryotes and plants. Like introns in nuclear pre-messenger RNA, they are excised by a two-step splicing reaction to generate branched circular RNAs, the so-called lariats. A remarkable feature of group II introns is their self-splicing activity in vitro. In the absence of a nucleotide cofactor, the intron RNAs catalyse two successive transesterification reactions which lead to autocatalytic excision of the lariat IVS from pre-mRNA and concomitantly to exon ligation. By virtue of its ability to specifically bind the 5' exon, the intron can also catalyse such reactions on exogenous RNA substrates. This sequence-specific attachment could enable group II introns to integrate into unrelated RNAs by reverse splicing, in a process similar to that described for the self-splicing Tetrahymena group I intron. Here we report that group II lariat IVS can indeed reintegrate itself into an RNA composed of the ligated exons in vitro. This occurs by a process of self-splicing that completely reverses both transesterification steps of the forward reaction: it involves a transition of the 2'-5' phosphodiester bond of the lariat RNA into the 3'-5' bond of the reconstituted 5' splice junction.     

Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans

Introns are defined by sequences that bind components of the splicing machinery. The branchpoint consensus, polypyrimidine (poly(Y)) tract, and AG at the splice boundary comprise the mammalian 3' splice site. Although the AG is crucial for the recognition of introns with relatively short poly(Y) tracts, which are termed 'AG-dependent introns', the molecule responsible for AG recognition has never been identified. A key player in 3' splice site definition is the essential heterodimeric splicing factor U2AF, which facilitates the interaction of the U2 small nuclear ribonucleoprotein particle with the branch point. The U2AF subunit with a relative molecular mass (Mr 65K) of 65,000 (U2AF65) binds to the poly(Y) tract, whereas the role of the 35K subunit (U2AF35) has not been clearly defined. It is not required for splicing in vitro but it plays a critical role in vivo. Caenorhabditis elegans introns have a highly conserved U4CAG/ R at their 3' splice sites instead of branch-point and poly(Y) consensus sequences. Nevertheless, C. elegans has U2AF, 12). Here we show that both U2AF subunits crosslink to the 3' splice site. Our results suggest that the U2AF65-U2AF35 complex identifies the U4CAG/R, with U2AF35 being responsible for recognition of the canonical AG.     

Are vertebrate exons scanned during splice-site selection?

Pairwise recognition of splice sites as a result of a scanning mechanism is an attractive model to explain the coordination of vertebrate splicing. Such a mechanism would predict a polarity-of-site recognition in the scanned unit, but no evidence for a polarity gradient across introns has been found. We have suggested that the exon rather than the intron is the unit of recognition in vertebrates and that polyadenylation and splicing factors interact during recognition of 3'-terminal exons. Interaction is reflected in maximal rates of in vitro polyadenylation. If scanning across the exon is operating during this interaction, then insertion of a 5' splice site should depress polyadenylation. Here we report recognition in vitro and in vivo of a 5' splice site situated within a 3'-terminal exon, and a concomitant depression of polyadenylation and ultraviolet crosslinking of a polyadenylation factor. Decreased crosslinking was only found when the 3' and 5' splice sites were within 300 nucleotides of each other. These results are consistent with an exon scanning mechanism for splice-site selection.     

A 3' splice site-binding sequence in the catalytic core of a group I intron

Ribozymes use specific RNA-RNA interactions for substrate binding and active-site formation. Self-splicing group I introns have approximately 70 nucleotides constituting the core, a region containing sequences and structures indispensable for catalytic function. The catalytic core must interact with the substrates used for the two steps of the self-splicing reaction, that is, guanosine, the 5'-splice-site helix (P1) and the 3' splice site. Mutational evidence suggests that core sequences near segment J6/7 that joins the base-paired stems P6 and P7, and the bulged base of P7(5'), participate in binding guanosine substrate, but nothing is known about the interactions between the core, the 5'-splice-site helix and the 3' splice site. On the basis of comparative sequence data, it has been suggested that two specific bases in the catalytic core of group I introns might form a binding sequence for the 3' splice site. Here we present genetic evidence that such a binding site exists in the core of the Tetrahymena large subunit ribosomal RNA intron. We demonstrate that this pairing, termed P9.0, is functionally important in the exon ligation step of self-splicing, but is not itself responsible for 3'-splice-site selection.     

The U1 snRNP protein U1C recognizes the 5' splice site in the absence of base pairing

Splicing of precursor messenger RNA takes place in the spliceosome, a large RNA/protein macromolecular machine. Spliceosome assembly occurs in an ordered pathway in vitro and is conserved between yeast and mammalian systems. The earliest step is commitment complex formation in yeast or E complex formation in mammals, which engages the pre-mRNA in the splicing pathway and involves interactions between U1 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA 5' splice site. Complex formation depends on highly conserved base pairing between the 5' splice site and the 5' end of U1 snRNA, both in vivo and in vitro. U1 snRNP proteins also contribute to U1 snRNP activity. Here we show that U1 snRNP lacking the 5' end of its snRNA retains 5'-splice-site sequence specificity. We also show that recombinant yeast U1C protein, a U1 snRNP protein, selects a 5'-splice-site-like sequence in which the first four nucleotides, GUAU, are identical to the first four nucleotides of the yeast 5'-splice-site consensus sequence. We propose that a U1C 5'-splice-site interaction precedes pre-mRNA/U1 snRNA base pairing and is the earliest step in the splicing pathway.     

Spliced leader RNAs from lower eukaryotes are trans-spliced in mammalian cells.

Exon sequences present on separate RNA molecules can be joined by trans-splicing in trypanosomatids, Euglena, and in the nematode and trematode worms. Trans-splicing involves an interaction between a 5' splice site present in a spliced leader RNA and a 3' splice site located near the 5' end of pre-messenger RNAs. In vitro trans-splicing of artificial mammalian pre-mRNAs has been reported, but the efficiency of splicing appears to depend on sequence complementarity between the two substrates. There has been speculation that some natural pre-mRNAs can be trans-spliced in mammalian cells in vivo, but alternative interpretations have not been ruled out. Here we show that spliced leader RNAs can be accurately trans-spliced in mammalian cells in vivo and in vitro. Both nematode and mammalian 3' splice sites can function as acceptors for trans-splicing in vivo. These results reveal functional conservation in the splicing machinery between lower eukaryotes and mammals, and they directly demonstrate the potential for trans-splicing in mammalian cells.     

Genetic evidence for base pairing between U2 and U6 snRNA in mammalian mRNA splicing.

Removal of introns from eukaryotic nuclear messenger RNA precursors is catalysed by a large ribonucleoprotein complex called the spliceosome, which consists of four small nuclear ribonucleoprotein particles (U1, U2, U5, and U4/U6 snRNPs) and auxiliary protein factors. We have begun a genetic analysis of mammalian U2 snRNA by making second-site mutations in a suppressor U2 snRNA. Here we find that several mutations in the 5' end of U2 (nucleotides 3-8) are deleterious and that one of these can be rescued by compensatory base changes in the 3' end of U6 (nucleotides 92-95). The results demonstrate genetically that the base-pairing interaction between U2 (nucleotides 3-11) and U6 snRNA (nucleotides 87-95), originally proposed on the basis of psoralen photocrosslinking experiments, can influence the efficiency of mRNA splicing in mammals. The U2/U6 interaction in yeast, however, is fairly tolerant to mutation (D.J. Field and J.D. Friesen, personal communication), emphasizing the potential for facultative RNA interactions within the spliceosome.     

Translational control of intron splicing in eukaryotes

Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs. Splicing is guided locally by short conserved sequences, but genes typically contain many potential splice sites, and the mechanisms specifying the correct sites remain poorly understood. In most organisms, short introns recognized by the intron definition mechanism cannot be efficiently predicted solely on the basis of sequence motifs. In multicellular eukaryotes, long introns are recognized through exon definition and most genes produce multiple mRNA variants through alternative splicing. The nonsense-mediated mRNA decay (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals. Here we show that the tiny introns of the ciliate Paramecium tetraurelia are under strong selective pressure to cause premature termination of mRNA translation in the event of intron retention, and that the same bias is observed among the short introns of plants, fungi and animals. By knocking down the two P. tetraurelia genes encoding UPF1, a protein that is crucial in NMD, we show that the intrinsic efficiency of splicing varies widely among introns and that NMD activity can significantly reduce the fraction of unspliced mRNAs. The results suggest that, independently of alternative splicing, species with large intron numbers universally rely on NMD to compensate for suboptimal splicing efficiency and accuracy.     

An intron with a constitutive transport element is retained in a Tap messenger RNA

Alternative splicing is a key factor contributing to genetic diversity and evolution. Intron retention, one form of alternative splicing, is common in plants but rare in higher eukaryotes, because messenger RNAs with retained introns are subject to cellular restriction at the level of cytoplasmic export and expression. Often, retention of internal introns restricts the export of these mRNAs and makes them the targets for degradation by the cellular nonsense-mediated decay machinery if they contain premature stop codons. In fact, many of the database entries for complementary DNAs with retained introns represent them as artefacts that would not affect the proteome. Retroviruses are important model systems in studies of regulation of RNAs with retained introns, because their genomic and mRNAs contain one or more unspliced introns. For example, Mason-Pfizer monkey virus overcomes cellular restrictions by using a cis-acting RNA element known as the constitutive transport element (CTE). The CTE interacts directly with the Tap protein (also known as nuclear RNA export factor 1, encoded by NXF1), which is thought to be a principal export receptor for cellular mRNA, leading to the hypothesis that cellular mRNAs with retained introns use cellular CTE equivalents to overcome restrictions to their expression. Here we show that the Tap gene contains a functional CTE in its alternatively spliced intron 10. Tap mRNA containing this intron is exported to the cytoplasm and is present in polyribosomes. A small Tap protein is encoded by this mRNA and can be detected in human and monkey cells. Our results indicate that Tap regulates expression of its own intron-containing RNA through a CTE-mediated mechanism. Thus, CTEs are likely to be important elements that facilitate efficient expression of mammalian mRNAs with retained introns.