Role of the 3′ splice site consensus sequence in mammalian pre-mRNA splicing

We studied the PK-LR gene in 15 unrelated Italian patients with congenital hemolytic anemia associated with erythrocyte pyruvate kinase (PK) deficiency. Fourteen different mutations were detected among 26 mutated alleles identified: a five-nucleotide (nt) deletion (227 to 231), two splice-site (1269C and IVS3(−2)c), 10 missense (514C, 787T, 823A, 993A, 994A, 1168A, 1456T, 1529A, 1552A, and 1594T) and one nonsense mutation(s) (721T). Eight of these (deletion 227-231, 1269C, IVS3(−2)c, 514C, 787T, 823A, 1168A, and 1552A) were novel. Moreover, a new polymorphic site was detected in the 3′ untranslated region of the mRNA (C/T, nucleotide 1738). The deletion 227-231 causes a stop codon after amino acid 77, probably resulting in an unstable gene product. Mutations 1269C and IVS3(−2)c lead to an alteration of the 5′ and 3′ splice-site consensus sequence, respectively; cDNA analysis failed to reveal any abnormal transcript, suggesting that these mutations generate an unstable mRNA that is rapidly degraded. Of the five new missense mutations, 823A (Gly275-Arg) and 1168A (Asp390-Asn) involve highly conserved amino acids, 514C (Glu172-Gln) and 1552A (Arg518-Ser), although found in less conserved regions, affect the balance of the electric charges of the protein. Mutation 787T (Gly263-Trp) is likely to determine strong modifications in the local structure of the molecule. The most frequent mutation in Italy appears to be 1456T (seven of 30 alleles), followed by 1529A (three of 30) and 994A (three of 30). A correlation was found between mutations, biochemical characteristics of the enzyme, and clinical course of the disease.

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Molecular Characterization of PK-LR Gene in Pyruvate Kinase–Deficient Italian Patients

Blood ◽

10.1182/blood.v89.10.3847 ◽

1997 ◽

Vol 89 (10) ◽

pp. 3847-3852 ◽

Cited By ~ 48

Author(s):

Alberto Zanella ◽

Paola Bianchi ◽

Luciano Baronciani ◽

Manuela Zappa ◽

Elena Bredi ◽

...

Keyword(s):

Pyruvate Kinase ◽

Splice Site ◽

Stop Codon ◽

Consensus Sequence ◽

Missense Mutations ◽

Frequent Mutation ◽

Unstable Gene ◽

Site Consensus ◽

Conserved Amino Acids

Abstract We studied the PK-LR gene in 15 unrelated Italian patients with congenital hemolytic anemia associated with erythrocyte pyruvate kinase (PK) deficiency. Fourteen different mutations were detected among 26 mutated alleles identified: a five-nucleotide (nt) deletion (227 to 231), two splice-site (1269C and IVS3(−2)c), 10 missense (514C, 787T, 823A, 993A, 994A, 1168A, 1456T, 1529A, 1552A, and 1594T) and one nonsense mutation(s) (721T). Eight of these (deletion 227-231, 1269C, IVS3(−2)c, 514C, 787T, 823A, 1168A, and 1552A) were novel. Moreover, a new polymorphic site was detected in the 3′ untranslated region of the mRNA (C/T, nucleotide 1738). The deletion 227-231 causes a stop codon after amino acid 77, probably resulting in an unstable gene product. Mutations 1269C and IVS3(−2)c lead to an alteration of the 5′ and 3′ splice-site consensus sequence, respectively; cDNA analysis failed to reveal any abnormal transcript, suggesting that these mutations generate an unstable mRNA that is rapidly degraded. Of the five new missense mutations, 823A (Gly275-Arg) and 1168A (Asp390-Asn) involve highly conserved amino acids, 514C (Glu172-Gln) and 1552A (Arg518-Ser), although found in less conserved regions, affect the balance of the electric charges of the protein. Mutation 787T (Gly263-Trp) is likely to determine strong modifications in the local structure of the molecule. The most frequent mutation in Italy appears to be 1456T (seven of 30 alleles), followed by 1529A (three of 30) and 994A (three of 30). A correlation was found between mutations, biochemical characteristics of the enzyme, and clinical course of the disease.

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Genes of nuclear encoded mitochondrial proteins: evidence for a variant of the 3′ splice-site consensus sequence

Nucleic Acids Research ◽

10.1093/nar/15.24.10083 ◽

1987 ◽

Vol 15 (24) ◽

pp. 10083-10086 ◽

Cited By ~ 11

Author(s):

Nenad Juretić ◽

Rolf Jausso ◽

Urs Mattes ◽

Philipp Christen

Keyword(s):

Splice Site ◽

Consensus Sequence ◽

Mitochondrial Proteins ◽

Site Consensus

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A 3′ splice site consensus sequence mutation in the cystic fibrosis gene

Human Genetics ◽

10.1007/bf02428306 ◽

1990 ◽

Vol 85 (4) ◽

pp. 450-453 ◽

Cited By ~ 38

Author(s):

H. Guillermit ◽

P. Fanent ◽

C. Ferec

Keyword(s):

Cystic Fibrosis ◽

Splice Site ◽

Consensus Sequence ◽

Cystic Fibrosis Gene ◽

Site Consensus

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Mutational analysis of pre-mRNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1.

Genetics ◽

10.1093/genetics/133.4.851 ◽

1993 ◽

Vol 133 (4) ◽

pp. 851-863 ◽

Cited By ~ 7

Author(s):

C F Lesser ◽

C Guthrie

Keyword(s):

Saccharomyces Cerevisiae ◽

Reporter Gene ◽

Splice Site ◽

Mutational Analysis ◽

Consensus Sequence ◽

Intron Position ◽

Mrna Splicing ◽

Copper Resistance ◽

Dependent Manner ◽

Fusion Construct

Abstract We have developed a new reporter gene fusion to monitor mRNA splicing in yeast. An intron-containing fragment from the Saccharomyces cerevisiae ACT1 gene has been fused to CUP1, the yeast metallothionein homolog. CUP1 is a nonessential gene that allows cells to grow in the presence of copper in a dosage-dependent manner. By inserting previously characterized intron mutations into the fusion construct, we have established that the efficiency of splicing correlates with the level of copper resistance of these strains. A highly sensitive assay for 5' splice site usage was designed by engineering an ACT1-CUP1 construct with duplicated 5' splice sites; mutations were introduced into the upstream splice site in order to evaluate the roles of these highly conserved nucleotides in intron recognition. Almost all mutations in the intron portion of the 5' consensus sequence abolish recognition of the mutated site, while mutations in the exon portion of the consensus sequence have variable affects on cleavage at the mutated site. Interestingly, mutations at intron position 4 demonstrate that this nucleotide plays a role in 5' splice site recognition other than by base pairing with U1 snRNA. The use of CUP1 as a reporter gene may be generally applicable for monitoring cellular processes in yeast.

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