Determinants of the 3' splice site for self-splicing of the Tetrahymena pre-rRNA

Tetrahymena preribosomal RNA undergoes self-splicing in vitro. The structural components involved in recognition of the 5' splice site have been identified, but the mechanism by which the 3' splice site is recognized is not established. To identify some components of 3'splice site recognition, we have generated mutations near the 3' splice site and determined their effects on self-splicing. Alteration of the 3'-terminal guanosine of the intervening sequence (IVS), a conserved nucleotide in group I IVSs, almost eliminated 3' splice site activity; the IVS-3' exon splicing intermediate accumulated, and exon ligation was extremely slow. These mutations do not result in recruitment of cryptic 3' splice sites, in contrast to mutations that affect the 5' splice site. Alteration of the cytidine preceding the 3'-terminal guanosine or of the first two nucleotides of the 3' exon had similar but less severe effects on exon ligation. Most of the mutants showed some reduction (less than threefold) in GTP addition at the 5' splice site. A mutation that placed a new guanosine residue just upstream from the 3'-terminal guanosine misspliced to produce ligated exons with one extra nucleotide between the 5' and 3' exons. We conclude that multiple nucleotides, located both at the 3' end of the IVS and in the 3' exon, are required for 3' splice site recognition.     

Identification of two distinct intron elements involved in alternative splicing of beta-tropomyosin pre-mRNA

The rat beta-tropomyosin gene encodes two isoforms, termed skeletal muscle beta-tropomyosin and fibroblast last tropomyosim 1 (TM-1), via an alternative RNA processing mechanism. The gene contains 11 exons. Exons 1-5 and exons 8 and 9 are common to all mRNAs expressed from the gene. Exons 6 and 11 are used in fibroblasts, as well as smooth muscle, whereas exons 7 and 10 are used only in skeletal muscle. In the present studies we focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). We have identified two distinct elements in the intron, upstream of exon 7, involved in splice site selection. The first element is comprised of a polypyrimidine tract located 89-143 nucleotides upstream of the 3' splice site, which specifies the location of the lariat branchpoints used, 144-153 nucleotides upstream of exon 7. The 3' splice site AG dinucleotide has no role in selection of these branchpoints. The second element is comprised of intron sequences located between the polypyrimidine tract and the 3' splice site of exon 7. It contains an important determinant in alternative splice site selection, because deletion of these sequences results in the use of the skeletal muscle-specific exon in nonmuscle cells. We propose that the use of lariat branchpoints located far upstream from a 3' splice site may be a general feature of some alternatively excised introns, reflecting the presence of regulatory sequences located between the lariat branch site and the 3' splice site. The data also indicate that alternative splicing of the rat beta-tropomyosin gene is regulated by a somewhat different mechanism from that described for rat alpha-tropomyosin gene and the transformer-2 gene of Drosophila melanogaster.     

Splicing and evolution of an unusually small group I intron

Introns are common in the rRNA gene loci of fungal genomes, but biochemical studies to investigate splicing are rare. Here, self-splicing of a very small (67 nucleotide) group I intron is demonstrated. The PaSSU intron (located within the rRNA small subunit gene of Phialophora americana) splices in vitro under group I intron conditions. Most group I ribozymes contain pairing regions P1-P10, with a conserved G.U pair at the 5' splice site, and a G at the 3' intron border. The PaSSU intron contains only P1, P7, and P10. While it contains the G.U pair at the 5' splice, a U is found at the 3' end of the intron instead of a G. Phylogenetic analysis places it within subgroup IC1, whose members are found in the nuclear rRNA genes of fungi. The structural elements are similar to those in the centermost regions of other group I introns. Its size can be explained by a single large deletion that removed P2 through much of P9. Part of the original P9 region has assumed the function of P7. Its small size and genealogy makes it an excellent model to study RNA catalysis and evolution.     

X-连锁迟发性脊椎骨骺发育不良SEDL基因剪接受体突变导致潜在剪接位点激活

目的 深入研究X-连锁迟发性脊椎骨骺发育不良（X-linked spondyloepiphyseal dysplasia tarda,SEDL）的发病机理，为最终防治本病提供依据。方法 应用逆转录-PCR及克隆测序方法对1例涉及SEDL基因第5内含子剪接受体缺失的SEDL患者进行mRNA表达研究。结果 该患者存在2个不同片段长度的mRNA表达产物，与GenBank正常序列进行BLAST比较后发现，393bp的表达产物是第6外显子内一个新的潜在剪接位点激活后形成的产物；433bp的表达产物与8号染色体的部分基因组序列完全一致。结论 SEDL基因第5内含子剪接受体位点及其后的第6外显子共13个碱基的缺失突变导致第6外显子内一个新的潜在剪接受体位点激活，使转录后的mRNA丢失了第6外显子内47bp的编码序列，并使紧接其后的2个密码子产生移码，导致翻译的提前终止（D109-S123del；S124fsX126）。另外，该突变可能激活了8号染色体上假基因SEDLP2的转录，从而部分地补偿了SEDL蛋白的功能。     

Alternative splicing of exon 37 of FBN1 deletes part of an 'eight-cysteine' domain resulting in the Marfan syndrome

A child and his father had the skeletal and cardiovascular manifestations of Marfan syndrome due to a heterozygous G+5 --> T transversion in intron 37 of the FBN1 gene. Cultured dermal fibroblasts preferentially used an alternative splice site in exon 37 that resulted in the loss of the 3' 48 nucleotides of this exon. The translational reading frame was maintained with deletion of lysine 1568 to threonine 1582 and splitting of the codons for glycine 1567 and serine 1583 to yield GCC for alanine. The deletion removed two cysteine residues as well as a potential N-linked oligosaccharide attachment site from the '8-cysteine' domain encoded by exons 37 and 38.     

Functional splicing assay shows a pathogenic intronic mutation in neurofibromatosis type 1 (NF1) due to intronic sequence exonization

Genomic variations with no apparent effect ("neutral polymorphisms") may have a significant effect on splicing. The effect of this type of mutation is difficult to spot, unless a functional assay is undertaken. In our study, DNA sequencing of a patient with clinically defined neurofibromatosis type 1 (NF1) showed only a single polymorphism in intron 30 due to an A>G transition 279 nucleotides from the 3' splice site. Using a minigene splicing assay we conclusively show that this change produces a cryptic exon with a 3' SS defined by the nucleotide change and the unexpected activation of a very weak 5'SS. Further site directed mutagenesis studies aimed at identifying the signals involved in the cryptic exon inclusion were carried out. Interestingly we find that particular characteristics of the cryptic 5' SS are essential for its inclusion. Significantly an additional single nucleotide change disrupting the cryptic 5'ss consensus sequence rescues the effect of the pathogenetic mutation resulting in normal splicing.     

Activation of a cryptic 5' splice site in the upstream exon of the phage T4 td transcript: exon context, missplicing, and mRNA deletion in a fidelity mutant

A collection of 100 td mutants defective in phage T4 thymidylate synthase (TS) production was screened for splicing impairments. Splicing-defective mutants were identified by a rapid assay developed to detect imbalances in the td protein products (TS, the exon ligation product, and NH2TS, encoded by the pre-mRNA). Thirteen selected mutants, confirmed to be splicing defective by an RNA-oligodeoxynucleotide hybridization assay, were all shown to be inhibited in the first step of the group I splicing pathway, cleavage at the 5' splice site. Of these, only one, SC99, appeared to be a specificity mutant. Whereas the 12 other mutants had sequence changes within the functionally important 5' and 3' domains of the intron, SC99 was shown to be an exon mutant. The G----A change at residue -3 of the upstream exon of SC99 resulted in loss of normal 5' splice site recognition. Furthermore, activation of a remote cryptic splice site at residue -29 of the upstream exon and missplicing of mRNA that is deleted for 29 nucleotides of the 5' exon are characteristic for this mutant. These results underscore the role of exon sequences in guiding the fidelity of the splicing reaction and they raise provocative questions about the alignment of introns within exon contexts that are consistent with accurate splicing and synthesis of an intact gene product.     

Functional analysis of cis-acting elements regulating the alternative splicing of human CFTR exon 9.

The rate of exon 9 exclusion from the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA is associated with monosymptomatic forms of cystic fibrosis. Exon 9 alternative splicing is modulated by a polymorphic polythymidine tract within its 3' splice site. We have generated a minigene carrying human CFTR exon 9 with its flanking intronic sequences and set up an in vivo model to study the cis-acting DNA elements which modulate its splicing. Transfections into human cell lines showed that T5, but not T9 or T7 alleles, significantly increases the alternative splicing of exon 9. Moreover, we found that another polymorphic locus juxtaposed upstream of the T tract, and constituted by (TG)(n)repeats, can further modulate exon 9 skipping but only when activated by the T5 allele. Then, we extended our studies to the mouse CFTR exon 9 which does not show alternative splicing. Comparison of human and mouse introns 8 and 9 revealed a low homology between the two sequences and the absence of the human polymorphic loci within the mouse intron 3' splice site. We have tested a series of constructs where the whole human exon 9 with its flanking intronic sequences was replaced partially or completely by the murine counterpart. The transfections of these constructs in human and murine cell lines reveal that also sequences of the downstream intron 9 affect exon 9 definition and co-modulate, with the UG/U 3' splice site sequences, the extent of exon 9 skipping in CFTR mRNA.     

Utilisation of a cryptic non-canonical donor splice site of the gene encoding PARAFIBROMIN is associated with familial isolated primary hyperparathyroidism

More than 99% of all splice sites conform to consensus sequences that usually include the invariant dinucleotides gt and ag at the 5' and 3' ends of the introns, respectively. We report on the utilisation of a non-consensus (non-canonical) donor splice site within exon 1 of the HRPT2 gene in familial isolated primary hyperparathyroidism (FIHP). HRPT2 mutations are more frequently associated with the hyperparathyroidism-jaw tumour syndrome (HPT-JT). Patients with FIHP were identified to have a donor splice site mutation, IVS1+1 g-->a, and the consequences of this for RNA processing were investigated. The mutant mRNA lacked 30 bp and DNA sequence analysis revealed this to result from utilisation of an alternative cryptic non-canonical donor splice site (gaatgt) in exon 1 together with the normally occurring acceptor splice site in intron 1. Translation of this mutant mRNA predicted the in-frame loss of 10 amino acids in the encoded protein, termed PARAFIBROMIN. Thus, these FIHP patients are utilising a ga-ag splice site pair, which until recently was considered to be incompatible with splicing but is now known to occur as a rare (<0.02%) normal splicing variant.     

Deletion of the C-terminal end of aspartylgiucosaminidase resulting in a lysosomal accumulation disease: evidence for a unique genomic rearrangement

Aspartyiglucosaminuria (AGU) is an inborn error of glycoproteincatabolism and represents the only known human deficiency ofan amidase, aspartyiglucosaminidase (AGA, EC 3.5.1.26 [EC] ). We reporthere a detailed characterization of a unique 2 kb deletion ofthe AGA gene in a North American AGU patient. To facilitatethe characterization of the deletion, genomic lamda clones spanningthe 3' flanking region of human AGA were isolated and sequenced.The breakpoint of the deletion was determined from the patient'sDNA by sequencing the genomic region containing the novel junction.The rearrangement involved a nonhomologous recombination withonly 2 bp of homology at the deletion breakpoint. The deletion's5' breakpoint was located in the last intron of AGA, thus abolishingthe normal C-terminal exon. This is in contrast to our previousfindings indicating that the deletion in the AGA gene wouldcontain only the complete 3' untranslated region and leave thecoding region intact (1). The unique feature of this deletionis a triplication of 19 thymidine nucleotides of an invertedAlu repeat, which is located at the deletion 3' breakpoint.The analysis of the patient's AGA cONA revealed an open readingframe containing a novel C-terminal exon, coding for a 64 aminoacid sequence, which has no homology to the normal exon 9 ofAGA. This new exon has a functional splice acceptor site atits 5' end, a stop codon, and a polyadenylation signal at the3' end. Expression of the mutant AGA cDNA in COS cells showedthat mutant mRNA is synthesized in equal amounts compared withnormal. However, the mutant polypeptide precursor is not processedinto the mature subunits of AGA, and is totally degraded within24 h of synthesis.     

Mutations causing defective splicing in the human hprt gene.

Ten intron mutations and one exon mutation giving rise to defective splicing in the human gene for hypoxanthine phosphoribosyl transferase (hprt) in T-lymphocytes have been characterized. The splicing mutants were detected by PCR amplification of hprt cDNA and direct sequencing. Nine of the mutants showed skipping of whole exons or parts of exons in the cDNA, one mutant had an inclusion of an intron sequence into the cDNA, and one mutant showed both inclusion of an intron sequence and skipping of exons as well as a normal cDNA. Genomic PCR and direct sequencing of the splice sites involved showed one deletion of three base pairs and 10 different single base alterations to be responsible for these splice alterations. One mutation in the last base pair of exon 6 causing skipping of the entire exon 6 was found, whereas an identical mutation in the last base pair of exon 2 caused no aberrant splicing. It was also found that a deletion mutation in the pyrimidine rich stretch of the acceptor site of intron 7 caused skipping of the entire exon 8, whereas a base substitution in the last base of intron 7 caused exclusion of only the first 21 base pairs of exon 8 as a result of the activation of a cryptic acceptor site in exon 8. The results show that many different types of mutations at several different sites can cause splicing errors in the hprt gene and that the sequence differences between the splice sites influence the possible spectrum of mutations in each site.     

Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome.

The highly conserved spliceosomal protein Prp8 is known to cross-link the critical sequences at both the 5' (GU) and 3' (YAG) ends of the intron. We have identified prp8 mutants with the remarkable property of suppressing exon ligation defects due to mutations in position 2 of the 5' GU, and all positions of the 3' YAG. The prp8 mutants also suppress mutations in position A51 of the critical ACAGAG motif in U6 snRNA, which has been observed previously to cross-link position 2 of the 5' GU. Other mutations in the 5' splice site, branchpoint, and neighboring residues of the U6 ACAGAG motif are not suppressed. Notably, the suppressed residues are specifically conserved from yeast to man, and from U2- to U12-dependent spliceosomes. We propose that Prp8 participates in a previously unrecognized tertiary interaction between U6 snRNA and both the 5' and 3' ends of the intron. This model suggests a mechanism for positioning the 3' splice site for catalysis, and assigns a fundamental role for Prp8 in pre-mRNA splicing.