The role of branchpoint-3'' splice site spacing and interaction between intron terminal nucleotides in 3'' splice site selection in Saccharomyces cerevisiae.

A conserved 3' splice site YAG is essential for the second step of pre-mRNA splicing but no trans-acting factor recognizing this sequence has been found. A direct, non-Watson-Crick interaction between the intron terminal nucleotides was suggested to affect YAG selection. The mechanism of YAG recognition was proposed to involve 5' to 3' scanning originating from the branchpoint or the polypyrimidine tract. We have constructed a yeast intron harbouring two closely spaced 3' splice sites. Preferential selection of a wild-type site over mutant ones indicated that the two sites are competing. For two identical sequences, the proximal site is selected. As previously observed, an A at the first intron nucleotide spliced most efficiently with a 3' splice site UAC. In this context, UAA or UAU were also more efficient 3' splice sites than UAG and competed more efficiently than the wild-type sequence with a 3' splice site UAC. We observed that a U at the first intron nucleotide is used for splicing in combination with 3' splice sites UAG, UAA or UAU. Our data indicate that the 3' splice site is not primarily selected through an interaction with the first intron nucleotide. Selection of the 3' splice site depends critically on its distance from the branchpoint but does not occur by a simple leaky scanning mechanism.     

RNA sequences upstream of the 3' splice site repress splicing of mutantyeast ACT1 introns.

A yeast ACT1 intron in which both the first and last intron nucleotides are mutated, the /a-c/ intron, splices 10% as well as wild type. We selected for additional cis-acting mutations that improve the splicing of /a-c/ introns and recovered small deletions upstream of the 3' splice site. For example, deletion of nucleotides -9 and -10 upstream of the 3' splice site increased the splicing activity of the /a-c/ intron to 30% that of the wild-type ACT1 intron. To determine if the increased /a-c/ splicing was due to changes in intron spacing or sequence, we made mutations that mimicked the local sequence of the delta-9, -10 deletion without deleting any nucleotides. These mutants also increased /a-c/ splicing, indicating that the increased splicing activity was due to changes in intron sequence. The delta-9, -10 deletion was not allele specific to the /a-c/ intron, and improved the splicing efficiency of many mutant introns with step II splicing defects. To further define the sequences required for improved splicing of mutant introns, we randomized the region upstream of the ACT1 3' splice site. We found that almost all sequence alterations improved the splicing of the /a-c/ intron. We postulate that this sequence near the 3' end of the intron represses the splicing of mutant introns, perhaps by serving as the binding site for a negative splicing factor.     

Activation of cryptic splice sites in murine sarcoma virus-124 mutants.

We have examined splice site activation in relation to intron structure in murine sarcoma virus (MuSV)-124 RNA. MuSV-124 contains inactive murine leukemia virus env gene splice sites (termed 5' env and 3' env) as well as cryptic sites in the gag and v-mos genes (termed 5' gag and 3' mos) which are activated for thermosensitive splicing by a 1,487-base intronic deletion in the MuSV-124 derived MuSVts110 retrovirus. To determine conditions permissive for splice site activation, we examined MuSV-124 mutants deleted in the 1,919-base intron bounded by the 5' gag and 3' mos sites. Several of these deletions activated thermosensitive splicing either at the same sites used in MuSVts110 or in a previously unreported temperature-sensitive splice event between the 5' gag and 3' env sites. These data suggested that the thermosensitive splicing phenotype characteristic of MuSVts110 required neither a specialized intron nor selection of a particular 3' splice site. The 3' env and 3' mos sites were found to compete for splicing to the 5' gag site; the more upstream 3' env site was exclusively used in MuSV-124 mutants containing both sites, whereas selection of the 3' mos site required removal of the 3' env site. Branchpoint sequences were found to have a potential regulatory role in thermosensitive splicing. Insertion of a beta-globin branchpoint sequence in a splicing-inactive MuSV-124 mutant activated efficient nonthermosensitive splicing at the 3' mos site, whereas a mutated branchpoint activated less efficient but thermosensitive splicing.     

Deletion mutants that alter differential RNA processing in the E3 complex transcription unit of adenovirus

Region E3 of the adenovirus encodes about ten overlapping mRNAs (a to j) with different splicing patterns and with two RNA 3' end sites termed E3A and E3B. We have examined how deletions in 12 viable virus mutants affect differential RNA processing in E3. We assayed E3 mRNAs by the nuclease-gel and RNA blot procedures. Some deletions had no effect whereas others (e.g. deletion of a 3' splice or the E3A 3' end signal) had the anticipated effects on RNA processing. However, deletions in two regions had surprising effects. Deletions in one region (nucleotides 1691 to 2044) enhanced splicing at the upstream 951 5' splice site and the downstream 2157 and/or 2880 3' splice sites. Some of these deletions prevented RNA 3' end formation at the downstream E3A site. Deletion in the other region (nucleotides 2173 to 2237) enhanced an upstream splice site (951 to 2157) such that almost all pre-mRNA was processed into mRNA f. We suggest that these two regions contain cis-acting signals that regulate differential RNA processing. We discuss the results in terms of RNA folding and scanning models for splicing, as well as models for differential RNA 3' end formation at the E3A versus the E3B site.     

Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency

Intron lariat formation between the 5' end of an intron and a branchpoint adenosine is a fundamental aspect of the first step in animal and yeast nuclear pre-mRNA splicing. Despite similarities in intron sequence requirements and the components of splicing, differences exist between the splicing of plant and vertebrate introns. The identification of AU-rich sequences as major functional elements in plant introns and the demonstration that a branchpoint consensus sequence was not required for splicing have led to the suggestion that the transition from AU-rich intron to GC-rich exon is a major potential signal by which plant pre-mRNA splice sites are recognized. The role of putative branchpoint sequences as an internal signal in plant intron recognition/definition has been re-examined. Single nucleotide mutations in putative branchpoint adenosines contained within CUNAN sequences in four different plant introns all significantly reduced splicing efficiency. These results provide the most direct evidence to date for preferred branchpoint sequences being required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences in dicot intron recognition. The observed patterns of 3' splice site selection in the introns studied are consistent with the scanning model described for animal intron 3' splice site selection. It is suggested that, despite the clear importance of AU sequences for plant intron splicing, the fundamental processes of splice site selection and splicing in plants are similar to those in animals.     

Self-splicing of a group II intron in yeast mitochondria: dependence on 5'' exon sequences.

It has been previously suggested that self-splicing of group II introns starts with a nucleophilic attack of the 2' OH group from the branchpoint adenosine on the 5' splice junction. To investigate the sequences governing the specificity of this attack, a series of Bal31 nuclease deletion mutants was constructed in which progressively larger amounts of 5' exon have been removed starting from its 5' end. The ability of mutant RNAs to carry out self-splicing in vitro was studied. Involvement of 5' exon sequences in self-splicing activity is indicated by the fact that a mutant in which as many as 18 nucleotides of 5' exon remain is seriously disturbed in splicing, while larger deletions eliminate splicing entirely. Mutants containing a truncated 5' exon form aberrant RNAs. One of these is a 425-nucleotide RNA containing the 5' exon as well as sequences of the 5' part of the intron. Its 3' end maps at position 374 of the 887-nucleotide intron. The other is a less abundant lariat RNA probably originating from the remainder of the intron linked to the 3' exon. We interpret this large dependence of reactivity of the intron on 5' exon and adjoining intron sequences as evidence for base-pairing interactions between the exon and parts of the intron, leading to an RNA folding necessary for splicing. Possible folding models are discussed.     

Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants

Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U-rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon.     

Intron definition in splicing of small Drosophila introns.

Approximately half of the introns in Drosophila melanogaster are too small to function in a vertebrate and often lack the pyrimidine tract associated with vertebrate 3' splice sites. Here, we report the splicing and spliceosome assembly properties of two such introns: one with a pyrimidine-poor 3' splice site and one with a pyrimidine-rich 3' splice site. The pyrimidine-poor intron was absolutely dependent on its small size for in vivo and in vitro splicing and assembly. As such, it had properties reminiscent of those of yeast introns. The pyrimidine-rich intron had properties intermediate between those of yeasts and vertebrates. This 3' splice site directed assembly of ATP-dependent complexes when present as either an intron or exon and supported low levels of in vivo splicing of a moderate-length intron. We propose that splice sites can be recognized as pairs across either exons or introns, depending on which distance is shorter, and that a pyrimidine-rich region upstream of the 3' splice site facilitates the exon mode.     

Splicing and spliceosome formation of the yeast MATa1 transcript require a minimum distance from the 5'' splice site to the internal branch acceptor site.

Small deletions of 6, 7, and 12 nucleotides introduced between the 5' splice site and the internal branch acceptor site of the first intron of the yeast MATa1 gene completely abolish accurate splicing in vitro in these constructs. Splicing only occurs at an alternative 5' splice site which was found in the first exon of the MATa1 gene and which is used both in vivo and in vitro. The splicing defect cannot be cured by expanding the distance from the branch point to the 3' splice site. If the alternative 5' splice site is deleted as well in these constructs, neither spliced products nor spliceosomes are formed. Our findings especially lead to the conclusion that a minimum distance between the 5' splice site and the internal branch acceptor site of the intron is required for the formation of splicing complexes and for accurate splicing.     

RNA splicing in Neurospora mitochondria: nuclear mutants defective in both splicing and 3' end synthesis of the large rRNA

We have identified nuclear mutants of Neurospora that are defective in splicing the mitochondrial large rRNA and that accumulate unspliced pre-rRNA (35S RNA). In cyt-4 mutants, the unspliced pre-rRNA contains short 3' end extensions (110 nucleotides) that are not present in pre-rRNAs from the other mutants. This and other characteristics suggest that the cyt-4 mutants may be primarily defective in 3' end synthesis and the RNA splicing defect occurs secondarily as a result of impaired RNA folding. The cyt-4 mutants also accumulate a "short" intron RNA and small exon RNAs that may reflect aberrant RNA cleavages. The 5' end of the short intron is about 285 nucleotides downstream from the 5' splice site at or near the base of the "central hairpin", a putative intermediate in folding of the pre-rRNA. Furthermore, the aberrant cleavage sites are immediately after a six nucleotide sequence (GAUAAU) homologous to the final splice junction (GAU/AAC).     

The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro.

We have shown previously that truncation of the human beta-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. We now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence. Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit beta-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a "poison" sequence that can inhibit the splicing of an upstream intron.     

An exonic splicing enhancer offsets the atypical GU-rich 3' splice site of human apolipoprotein A-II exon 3

Human apolipoprotein A-II (apoA-II) intron 2/exon 3 junction shows a peculiar tract of alternating pyrimidines and purines (GU tract) that makes the acceptor site deviate significantly from the consensus. However, apoA-II exon 3 is constitutively included in mRNA. We have studied this unusual exon definition by creating a construct with the genomic fragment encompassing the whole gene from apoA-II and its regulatory regions. Transient transfections in Hep3B cells have shown that deletion or replacement of the GU repeats at the 3' splice site resulted in a decrease of apoA-II exon 3 inclusion, indicating a possible role of the GU tract in splicing. However, a 3' splice site composed of the GU tract in heterologous context, such as the extra domain A of human fibronectin or cystic fibrosis transmembrane conductance regulator exon 9, resulted in total skipping of the exons. Next, we identified the exonic cis-acting elements that may affect the splicing efficiency of apoA-II exon 3 and found that the region spanning from nucleotide 87 to 113 of human apoA-II exon 3 is essential for its inclusion in the mRNA. Overlapping deletions and point mutations (between nucleotides 91 and 102) precisely defined an exonic splicing enhancer (ESEwt). UV cross-linking assays followed by immunoprecipitation with anti-SR protein monoclonal antibodies showed that ESEwt, but not mutated ESE RNA, was able to bind both alternative splicing factor/splicing factor 2 and SC35. Furthermore, overexpression of both splicing factors enhanced exon 3 inclusion. These results show that this protein-ESE interaction is able to promote the incorporation of exon 3 in mRNA and suggest that they can rescue the splicing despite the noncanonical 3' splice site.     

Species-specific signals for the splicing of a short Drosophila intron in vitro.

The effects of branchpoint sequence, the pyrimidine stretch, and intron size on the splicing efficiency of the Drosophila white gene second intron were examined in nuclear extracts from Drosophila and human cells. This 74-nucleotide intron is typical of many Drosophila introns in that it lacks a significant pyrimidine stretch and is below the minimum size required for splicing in human nuclear extracts. Alteration of sequences of adjacent to the 3' splice site to create a pyrimidine stretch was necessary for splicing in human, but not Drosophila, extracts. Increasing the size of this intron with insertions between the 5' splice site and the branchpoint greatly reduced the efficiency of splicing of introns longer than 79 nucleotides in Drosophila extracts but had an opposite effect in human extracts, in which introns longer than 78 nucleotides were spliced with much greater efficiency. The white-apricot copia insertion is immediately adjacent to the branchpoint normally used in the splicing of this intron, and a copia long terminal repeat insertion prevents splicing in Drosophila, but not human, extracts. However, a consensus branchpoint does not restore the splicing of introns containing the copia long terminal repeat, and alteration of the wild-type branchpoint sequence alone does not eliminate splicing. These results demonstrate species specificity of splicing signals, particularly pyrimidine stretch and size requirements, and raise the possibility that variant mechanisms not found in mammals may operate in the splicing of small introns in Drosophila and possibly other species.     

Intron sequences involved in lariat formation during pre-mRNA splicing

We have shown that lariat formation during in vitro splicing of several RNA precursors, from Drosophila to man, occurs at a unique and identifiable but weakly conserved site, 18 to 37 nucleotides proximal to the 3' splice site. Lariat formation within an artificial intron lacking a normal branch-point sequence occurs at a cryptic site a conserved distance (approximately 23 nucleotides) from the 3' splice site. Analysis of beta-thalassemia splicing mutations revealed that lariat formation in the first intron of the human beta-globin gene occurs at the same site in normal and mutant precursors, even though alternate 5' and 3' splice sites are utilized in the mutants. Remarkably, cleavage at the 5' splice site and lariat formation do not occur when the precursor contains a beta-thalassemia deletion removing the polypyrimidine stretch and AG dinucleotide at the 3' splice site. In contrast, a single base substitution in the AG dinucleotide blocks cleavage at the 3' splice site but not at the 5' site.     

tau Exon 10 expression involves a bipartite intron 10 regulatory sequence and weak 5' and 3' splice sites

tau mutations that deregulate alternative exon 10 (E10) splicing cause frontotemporal dementia with parkinsonism chromosome 17-type by several mechanisms. Previously we showed that E10 splicing involved exon splicing enhancer sequences at the 5' and 3' ends of E10, an exon splicing silencer, a weak 5' splice site, and an intron splicing silencer (ISS) within intron 10 (I10). Here, we identify additional regulatory sequences in I10 using both non-neuronal and neuronal cells. The ISS sequence extends from I10 nucleotides 11-18, which is sufficient to inhibit use of a weakened 5' splice site of a heterologous exon. Furthermore, ISS function is location-independent but requires proximity to a weak 5' splice site. Thus, the ISS functions as a linear sequence. A new cis-acting element, the intron splicing modulator (ISM), was identified immediately downstream of the ISS at I10 positions 19-26. The ISM and ISS form a bipartite regulatory element, within which the ISM functions when the ISS is present, mitigating E10 repression by the ISS. Additionally, the 3' splice site of E10 is weak and requires exon splicing enhancer elements for efficient E10 inclusion. Thus far, tau FTDP-17 splicing mutations affect six predicted cis-regulatory sequences.     

Identification of cis-acting intron and exon regions in influenza virus NS1 mRNA that inhibit splicing and cause the formation of aberrantly sedimenting presplicing complexes.

In in vitro splicing reactions, influenza virus NS1 mRNA was not detectably spliced, but nonetheless very efficiently formed ATP-dependent 55S complexes containing the U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) (C. H. Agris, M. E. Nemeroff, and R. M. Krug, Mol. Cell. Biol. 9:259-267, 1989). We demonstrate that the block in splicing was caused by two regions in NS1 mRNA: (i) a large intron region (not including the branchpoint sequence) and (ii) an 85-nucleotide 3' exon region near the 3' end of the exon. After removal of both of these regions, the 5' and 3' splice sites and branchpoint of NS1 mRNA functioned efficiently in splicing, indicating that they were not defective. The two inhibitory regions shared one property: splicing inhibition was independent of the identity of the nucleotide sequence in either region. In other respects, however, the two inhibitory regions differed. The inhibitory activity of the intron region was proportional to its length, indicating that the inhibition was probably due to size only. In contrast, the 3' exon, which was of small size, was a context element; i.e., it functioned only when it was located at a specific position in the 3' exon of NS1 mRNA. To determine how these intron and exon regions inhibited splicing, we compared the types of splicing complexes formed by intact NS1 mRNA with those formed by spliceable NS1 mRNA lacking the intron and exon regions. Splicing complexes were formed by using purified splicing factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deletion analysis of a unique 3'' splice site indicates that alternating guanine and thymine residues represent an efficient splicing signal.

The 3' splice site of the second intron (I2) of the human apolipoprotein-AII gene, (GT)16GGGCAG, is unique in that, although fully functional, a stretch of alternating guanine and thymine residues replaces the polypyrimidine tract usually associated with 3' splice junctions. The transient expression of successive 5' deletion mutants has defined the minimum number of nucleotides at the 3' end of apo-AII I2 that are required to direct efficient splicing. Processing in two cell-types, representing apo-AII producing and non-producing tissue was identical; in both, only by removing all the GT repeats did the 3' splice site of apo-AII I2 become completely non-functional. Similar deletion analyses of "classic" 3' splice sites, which conform to the consensus sequence (Y)nNYAG, have indicated that a minimum of 14 nucleotides of the polypyrimidine tract are required for detectable levels of processing to take place. Here we report that the six nucleotides (GT)2GG, which directly replace this tract in a deletion mutant of the 3' splice site of apo-AII I2 are sufficient to direct the splicing process efficiently and correctly.