Effect of 5' splice site mutations on splicing of the preceding intron

1990 ◽  
Vol 10 (12) ◽  
pp. 6299-6305
Author(s):  
M Talerico ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factors ◽  
Exon 2 ◽  
Intron 1 ◽  
Direct Assembly ◽  
Internal Exon ◽  
Exon 1 ◽  
Lariat Rna

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.

scholarly journals Effect of 5' splice site mutations on splicing of the preceding intron.

1990 ◽  
Vol 10 (12) ◽  
pp. 6299-6305 ◽  
Author(s):  
M Talerico ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factors ◽  
Exon 2 ◽  
Intron 1 ◽  
Direct Assembly ◽  
Internal Exon ◽  
Exon 1 ◽  
Lariat Rna

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.

Exon definition may facilitate splice site selection in RNAs with multiple exons

1990 ◽  
Vol 10 (1) ◽  
pp. 84-94 ◽  
Author(s):  
B L Robberson ◽  
G J Cote ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Exon Skipping ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Spliceosome Assembly ◽  
Exon Definition ◽  
Exon 2 ◽  
Correct Orientation

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.

scholarly journals Exon definition may facilitate splice site selection in RNAs with multiple exons.

1990 ◽  
Vol 10 (1) ◽  
pp. 84-94 ◽  
Author(s):  
B L Robberson ◽  
G J Cote ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Exon Skipping ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Spliceosome Assembly ◽  
Exon Definition ◽  
Exon 2 ◽  
Correct Orientation

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.

scholarly journals Complete Thyroxine-Binding Globulin (TBG) Deficiency Produced by a Mutation in Acceptor Splice Site Causing Frameshift and Early Termination of Translation (TBG-Kankakee)12

1998 ◽  
Vol 83 (10) ◽  
pp. 3604-3608
Author(s):  
Gisah A. Carvalho ◽  
Roy E. Weiss ◽  
Samuel Refetoff
Keyword(s):  
Splice Site ◽  
Messenger Rna ◽  
Restriction Site ◽  
Splice Junction ◽  
Early Termination ◽  
Coding Region ◽  
Acceptor Splice Site ◽  
Exon 2 ◽  
Gene Level ◽  
Exon 1

Fourteen T4-binding globulin (TBG) variants have been identified at the gene level. They are all located in the coding region of the gene and 6 produce complete deficiency of TBG (TBG-CD). We now describe the first mutation in a noncoding region producing TBG-CD. The proband was treated for over 20 yr with L-T4 because of fatigue associated with a low concentration of serum total T4. Fifteen family members were studied showing low total T4 inherited as an X chromosome-linked trait, and affected males had undetectable TBG in serum. Sequencing of the entire coding region and promoter of the TBG gene revealed no abnormality. However, an A to G transition was found in the acceptor splice junction of intron II that produced a new HaeIII restriction site cosegregating with the TBG-CD phenotype. Sequencing exon 1 to exon 3 of TBG complementary DNA reverse transcribed from messenger RNA of skin fibroblasts from an affected male, confirmed a shift in the ag acceptor splice site. This results in the insertion of a G in exon 2 and causes a frameshift and a premature stop at codon 195. This early termination of translation predicts a truncated TBG lacking 201 amino acids.

scholarly journals GEN-03 GENOTYPE-PHENOTYPE CORRELATION IN 111 FAMILIES OF VON HIPPEL-LINDAU DISEASE IN JAPAN

2019 ◽  
Vol 1 (Supplement_2) ◽  
pp. ii8-ii8
Author(s):  
Hiroshi Kanno ◽  
Tetsuya Yoshizumi ◽  
Masamichi Shinonaga ◽  
Masahiro Yao
Keyword(s):  
Amino Acids ◽  
Missense Mutation ◽  
Splice Site ◽  
Phenotype Correlation ◽  
Exon 2 ◽  
Endolymphatic Sac Tumor ◽  
Von Hippel Lindau ◽  
Genotype Phenotype Correlation ◽  
Exon 1 ◽  
Vhl Disease

Abstract BACKGROUND AND AIM von Hippel-Lindau (VHL) disease is a hereditary disease which manifest central nervous system (CNS) hemangioblastoma, retinal angioma, renal cell carcinoma (RCC), pheochromocytoma, endolymphatic sac tumor, and pancreas cyst. The VHL gene is located at 3p25.3 and is corresponding to 213 amino acids. Genotype-phenotype correlation analyses of VHL disease have been recently reported from several foreign countries, but the genotype-phenotype correlation has not been characterized since above 10 years ago. Therefore, this study aimed to evaluate the VHL mutation spectrum and genotype-phenotype correlations in Japanese VHL patients. METHODS Blood samples of 111 unrelated families of VHL disease were collected and DNAs were extracted. Direct sequencing and real-time PCR analysis were performed. Consequently, the clinical manifestations and family histories of the subjects were evaluated. RESULTS We identified VHL mutations as follows: missense 47; deletion 17; insertion 5; nonsense 8; splice-site 9; larger deletion 25. At hot-spot codon 167, 4 minsense mutations were identified, with Arg167Trp, 4 cases; Arg167Gln2, 2 cases. At codon 155, splice-site mutations were identified at 6 cases. Mutation sites were distributed in exon 1, 45; exon 2, 21; exon 3, 36. Large deletions were distributed in exon 1 & 2, 1; exon 2& 3, 1; all exons, 11. Genotype-phenotype correlation analysis revealed that age-specific risk and number of CNS hemangioblastoma were significantly higher in subjects carrying missense mutation within HIF-α binding site or non-missense mutation (P < 0.05). In addition, penetrance of RCC was significantly higher in subjects carrying non-missense mutation (P < 0.05). CONCLUSIONS The results of this study were similar to the previous foreign studies. This study provides insight into the genotype-phenotype correlation in that amino acids substitutions in the HIF- α binding and non-sense mutations may predispose VHL patients to age-related risk and number of CNS hemangioblastoma.

In-Vitro Cytotoxicity of Tipifarnib (Zarnestra TM) in Pediatric AML and ALL Samples Is Independent of RAS Mutations.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1172-1172 ◽  
Author(s):  
Bianca F. Goemans ◽  
Christian M. Zwaan ◽  
Gertjan J.L. Kaspers ◽  
Karel Hählen ◽  
Dirk Reinhardt ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Direct Sequencing ◽  
Farnesyltransferase Inhibitors ◽  
Exon 2 ◽  
Ras Mutations ◽  
Lc50 Value ◽  
Significant Difference ◽  
Exon 1 ◽  
Pediatric Aml

Abstract The farnesyltransferase inhibitor tipifarnib (Zarnestra™) was originally developed to target malignancies harbouring RAS mutations. In the first clinical studies with tipifarnib, in adults with leukemia, it was found that patients who responded did not harbour any RAS mutations, suggesting a different mechanism of response. In a previous study we showed that 18% of 150 untreated pediatric AML patients harbour mutations in RAS, of which 30% were CBF-AML. We now studied 44 untreated and 13 relapsed pediatric AML, as well as 22 untreated ALL samples for mutations in RAS, using D-HPLC and direct sequencing. In vitro tipifarnib resistance was determined by a 4-day MTT assay (concentration 0.016-51μM, kindly provided by Janssen Research). The LC50 value, the concentration at which 50% of cells are killed by tipifarnib, was used as a measure of resistance. Patient characteristics were; for untreated AML: 64% boys; median age 9.3 years; median WBC 74.8x109/L; FAB 2xM0, 2xM1, 8xM2, 3xM3, 16xM4, 8xM5, 5x unclassified; for relapsed AML: 77% boys; median age 4.0 years; median WBC 41.6x109/L; FAB 2xM0, 2xM2, 3xM4, 2xM5, 2xM7, 2x unclassified; for untreated ALL: 73%boys; median age 6.0 years; median WBC 10.2x109/L; 15 B-cell precursor (BCP) ALL and 7 T-ALL. We found RAS mutations in 14 (32%) untreated AML samples (N-RAS : 8 samples exon 1, 1 sample exon 2; K-RAS: 5 samples exon 1 mutations). In relapsed AML 2 samples showed an N-RAS exon 1 mutation (15.4%). In ALL 18.2% had a RAS mutation: an N-RAS exon 1 mutation was found in 2 patients (9.1%) and a K-RAS exon 1 mutation in another 2 patients (9.1%). The distribution of tipifarnib sensitivity was similar in RAS mutated- and non-mutated untreated AML patients [median LC50 RAS mutated 7.1μM (P25-P75: 6.0-9.6μM) vs. non-mutated 4.9μM (P25-P75 2.3-8.2μM); p=0.199]. When we compared N-RAS mutated samples with K-RAS mutated samples there was no statistically significant difference in sensitivity to tipifarnib (median LC50 [p25-p75] 3.2μM [2.9-3.9μM] and 4.9μM [3.7-23.1μM], p=0.20), and comparing them separately with non-mutated AML did not show differences in sensitivity to tipifarnib (p=0.172 and p=0.463 respectively). One out of 9 (11%) N-RAS mutated and 3 out of 5 (60%) K-RAS mutated samples had an LC50 value above the 75th percentile for non-mutated AML and were considered resistant. Within relapsed AML the 2 RAS mutated samples had LC50 values of 0.83 and 6.3μM, versus a median value of 6.9μM for non-mutated relapsed AML. In ALL, we found similar results [median LC50 RAS mutated 7.8μM (P25-75: 4.1-12.8μM) vs. non-mutated 17.4μM (P25-75: 4.5-22.9μM), p=0.3], but the groups were very small. In conclusion, primary pediatric AML and ALL samples withRAS mutations show similar distributions of tipifarnib sensitivity as samples withoutRAS mutations. Hence, some RAS mutated samples may be relatively in-vitro resistant to tipifarnib, and some non-mutated samples may be relatively sensitive. Therefore, clinical studies with these compounds should not be restricted to RAS-mutated leukemia. Further studies are necessary to determine the molecular targets of farnesyltransferase inhibitors.

scholarly journals Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles.

1988 ◽  
Vol 8 (2) ◽  
pp. 814-821 ◽  
Author(s):  
M Zillmann ◽  
M L Zapp ◽  
S M Berget
Keyword(s):  
Gel Electrophoresis ◽  
Splice Junction ◽  
Rnase H ◽  
Exon 2 ◽  
Small Nuclear Ribonucleoprotein ◽  
Complementary Oligonucleotide ◽  
Exon 1 ◽  
Precursor Rna ◽  
Splice Junctions

Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H cleavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions.

scholarly journals Splicing Functions and Global Dependency on Fission Yeast Slu7 Reveal Diversity in Spliceosome Assembly

2013 ◽  
Vol 33 (16) ◽  
pp. 3125-3136 ◽  
Author(s):  
Shataparna Banerjee ◽  
Piyush Khandelia ◽  
Geetha Melangath ◽  
Samirul Bashir ◽  
Vijaykrishna Nagampalli ◽  
...  
Keyword(s):  
Splice Site ◽  
Genetic Interactions ◽  
Splicing Factors ◽  
Spliceosome Assembly ◽  
Content Type ◽  
Missense Mutant ◽  
Stable Association ◽  
Content Factor ◽  
The Impact

The multiple short introns inSchizosaccharomyces pombegenes with degeneratecissequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of theS. pombe slu7+(spslu7+) gene product, known fromSaccharomyces cerevisiaeand humanin vitroreactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3′ splice site choice during the second reaction. By using a missense mutant of this essentialS. pombefactor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3′ splice site distance, intron length, and the impact of its A/U content at the 5′ end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest inspslu7-2revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions withspprp1+, a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly.

scholarly journals In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit.

1993 ◽  
Vol 13 (5) ◽  
pp. 2677-2687 ◽  
Author(s):  
D A Sterner ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Troponin I ◽  
Exon Skipping ◽  
Splice Sites ◽  
Small Vertebrate ◽  
Upstream Exon ◽  
I Gene

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

scholarly journals Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF

1993 ◽  
Vol 13 (5) ◽  
pp. 2993-3001
Author(s):  
A Mayeda ◽  
D M Helfman ◽  
A R Krainer
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factor ◽  
Alternative Exon ◽  
Hnrnp A1 ◽  
Exon Inclusion ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.

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