Cloning and nucleotide sequences of cDNAs spanning the splice junctions of Rous sarcoma virus mRNAs.

The cDNAs corresponding to the 5' ends of the mRNAs coding for the envelope protein precursor (gPr92env) of the B77 strain and the transforming protein (pp60src) of the Prague B strain of Rous sarcoma virus were cloned into pBR322, and the nucleotide sequences surrounding the splice junctions were determined. Both mRNAs are products of single splicing events from a common donor splice site at nucleotide 398 from the 5' end of the RNA to acceptor splice sites at nucleotides 5078 and 7054 for the env and src mRNAs, respectively. These results confirm and extend previous conclusions based on peptide mapping and single-strand nuclease mapping. Compared with the sequence of the Prague C genome RNA, the B77 strain contains a 6-nucleotide deletion in the sequence corresponding to the hydrophobic portion of the signal peptide of the envelope protein precursor.     

Transduction of c-src coding and intron sequences by a transformation-defective deletion mutant of Rous sarcoma virus.

The mechanism of cellular src (c-src) transduction by a transformation-defective deletion mutant, td109, of Rous sarcoma virus was studied by sequence analysis of the recombinational junctions in three td109-derived recovered sarcoma viruses (rASVs). Our results show that two rASVs have been generated by recombination between td109 and c-src at the region between exons 1 and 2 defined previously. Significant homology between td109 and c-src sequences was present at the sites of recombination. The viral and c-src sequence junction of the third rASV was formed by splicing a cryptic donor site at the 5' region of env of td109 to exon 1 of c-src. Various lengths of c-src internal intron 1 sequences were incorporated into all three rASV genomes, which resulted from activation of potential splice donor and acceptor sites. The incorporated intron 1 sequences were absent in the c-src mRNA, excluding its being the precursor for recombination with td109 and implying that initial recombinations most likely took place at the DNA level. A potential splice acceptor site within the incorporated intron 1 sequences in two rASVs was activated and was used for the src mRNA synthesis in infected cells. The normal env mRNA splice acceptor site was used for src mRNA synthesis for the third rASV.     

Three novel types of splicing aberrations in the tuberous sclerosis TSC2 gene caused by mutations apart from splice consensus sequences

Disease causing aberrations in both tuberous sclerosis predisposing genes, TSC1 and TSC2, comprise nearly every type of alteration with a predominance of small truncating mutations distributed over both genes. We performed an RNA based screening of the entire coding regions of both TSC genes applying the protein truncation test (PTT) and identified a high proportion of unusual splicing abnormalities affecting the TSC2 gene. Two cases exhibited different splice acceptor mutations in intron 9 (IVS9-15G-->A and IVS9-3C-->G) both accompanied by exon 10 skipping and simultaneous usage of a cryptic splice acceptor in exon 10. Another splice acceptor mutation (IVS38-18A-->G) destroyed the putative polypyrimidine structure in intron 38 and resulted in simultaneous intron retention and usage of a downstream cryptic splice acceptor in exon 39. Another patient bore a C-->T transition in intron 8 (IVS8+281C-->T) activating a splice donor site and resulting in the inclusion of a newly recognised exon in the mRNA followed by a premature stop. These splice variants deduced from experimental results are additionally supported by RNA secondary structure analysis based on free energy minimisation. Three of the reported splicing anomalies are due to sequence changes remote from exon/intron boundaries, described for the first time in TSC. These findings highlight the significance of investigating intronic changes and their consequences on the mRNA level as disease causing mutations in TSC.     

Two alternative exons can result from activation of the cryptic splice acceptor site deep within intron 2 of the dystrophin gene in a patient with as yet asymptomatic dystrophinopathy

Intron 2 of the dystrophin gene is unusually large, extending 157 kb on the X-chromosome, and is known to contain one cryptic exon 2a. Here, we report that a single nucleotide change in the middle of this huge intron is a source of two novel extra exons. A novel point mutation changing T to A nucleotide was identified at 5591 bp downstream from the 3' end of exon 2 (T310+5591A) in genomic DNA of an asymptomatic dystrophinopathy case. The mutation identification was initiated by detection of two novel dystrophin mRNAs containing a 132-nucleotide or 46-nucleotide insertion between exons 2 and 3 in lymphocytes but one with a 132-nucleotide insertion in skeletal muscle. It was concluded that T310+5591A created a novel consensus sequence for a splice acceptor site leading to the formation of two novel exon structures by using two cryptic splice donor sites at 132 bp or 46 bp downstream. The former maintained the dystrophin reading frame and was expected to insert 44 amino acids in the N-terminal domain of dystrophin, whereas the latter created a premature stop codon. An immunohistochemical study of the skeletal muscle of the patient disclosed that the N-terminal domain of dystrophin was not stained, but the rod- and C-terminal domains were stained in a patchy and discontinuous manner, indicating that the in-frame mRNA was functional. Creation of a splice acceptor site by a single nucleotide change leading to extra exon structures is a novel molecular mechanism in human disease.     

Novel compound heterozygous mutations for lipoprotein lipase deficiency. A G-to-T transversion at the first position of exon 5 causing G154V missense mutation and a 5' splice site mutation of intron 8.

We systematically investigated the molecular defects causing a primary LPL deficiency in a Japanese male infant (patient DI) with fasting hyperchylomicronemia (type I hyperlipoproteinemia) and in his parents. Patient DI had neither LPL activity nor immunoreactive LPL mass in the pre- and post-heparin plasma. The patient was a compound heterozygote for novel mutations consisting of a G-to-T transversion at the first nucleotide of exon 5 [+1 position of 3' acceptor splice site (3'-ass) of intron 4] and a T-to-C transition in the invariant GT at position +2 of the 5' donor splice site (5'-dss) of intron 8 (Int8/5'-dss/t(+2)c). The G-to-T transversion, although affecting the 11 nucleotide of the 3'-consensus acceptor splice site, resulted in a substitution of Gly(154) to Val (G154V; GG(716)C(-->)GTC). The mutant G154V LPL expressed in COS-1 cells was catalytically inactive and hardly released from the cells by heparin. The Int8/5'-dss/t(+2)c mutation inactivated the authentic 5' splice site of intron 8 and led to the utilization of a cryptic 5'-dss in exon 8 as an alternative splice site 133 basepairs upstream from the authentic splice site, thereby causing joining of a part of exon 8 to exon 9 with skipping of a 134-bp fragment of exon 8 and intron 8. These additional mutations in the consensus sequences of the 3' and 5' splice sites might be useful for better understanding the factors that are involved in splice site selection in vivo.     

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.     

A catalogue of splice junction sequences.

Splice junction sequences from a large number of nuclear and viral genes encoding protein have been collected. The sequence CAAG/GTAGAGT was found to be a consensus of 139 exon-intron boundaries (or donor sequences) and (TC)nNCTAG/G was found to be a consensus of 130 intron-exon boundaries (or acceptor sequences). The possible role of splice junction sequences as signals for processing is discussed.