共查询到20条相似文献,搜索用时 31 毫秒
1.
A series of HeLa cell lines which stably express beta-globin pre-mRNAs carrying point mutations at nt 654, 705, or 745 of intron 2 has been developed. The mutations generate aberrant 5' splice sites and activate a common 3' cryptic splice site upstream leading to aberrantly spliced beta-globin mRNA. Antisense oligonucleotides, which in vivo blocked aberrant splice sites and restored correct splicing of the pre-mRNA, revealed major differences in the sensitivity of these sites to antisense probes. Although the targeted pre-mRNAs differed only by single point mutations, the effective concentrations of the oligonucleotides required for correction of splicing varied up to 750-fold. The differences among the aberrant 5' splice sites affected sensitivity of both the 5' and 3' splice sites; in particular, sensitivity of both splice sites was severely reduced by modification of the aberrant 5' splice sites to the consensus sequence. These results suggest large differences in splicing of very similar pre-mRNAs in vivo. They also indicate that antisense oligonucleotides may provide useful tools for studying the interactions of splicing machinery with pre-mRNA. 相似文献
2.
Beta thalassemic mutations recognized by DNA mapping with Hph I and Rsa I in the Algerian population
G Kohen N Salome T Henni J Godet 《Biochemical and biophysical research communications》1983,113(1):269-272
By using Hph I and Rsa I restriction enzymes and beta globin large intervening sequence as a probe, we have investigated the DNA of 20 Algerian patients with beta(0) or beta(+) thalassemia. In any of them, we detected the nucleotide change which is known to generate an additional Hph I site at the 5' splice junction of the beta globin large intervening sequence and which yields a beta(0) phenotype. In one of them, we detected the nucleotide change which is known to generate an additional Rsa I site within the beta globin large intervening sequence and which is supposed to yield a beta(+) phenotype. These results indicate that these two types of mutation are relatively rare in the Algerian population. 相似文献
3.
Identification of a novel sequence that governs both polyadenylation and alternative splicing in region E3 of adenovirus. 总被引:13,自引:0,他引:13
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Region E3 encodes four major overlapping mRNAs with different splicing patterns. There are two poly(A) sites, an upstream site called E3A and a downstream site called E3B. We have analyzed virus mutants with deletions or insertions in E3 in order to identify sequences that function in the alternative processing of E3 pre-mRNAs, and to understand what determines which poly(A) sites and which splice sites are used. In previous studies we established that the 5' boundary of the E3A poly(A) signal is at an ATTAAA sequence. We now show, using viable virus mutants, that the 3' boundary of the E3A signal is located within 47-62 nucleotides (nt) downstream of the ATTAAA (17-32 nt downstream of the last microheterogenous poly(A) addition site). Our data further suggest that the spacing between the ATTAAA, the cleavage sites, and the essential downstream sequences may be important in E3A 3' end formation. Of particular interest, these mutants suggest a novel mechanism for the control of alternative pre-mRNA processing. Mutants which are almost completely defective in E3A 3' end formation display greatly increased use of a 3' splice site located 4 nt upstream of the ATTAAA. The mRNA that uses this 3' splice site is polyadenylated at the E3B poly(A) site. We suggest, for this particular case, that alternative pre-mRNA processing could be determined by a competition between trans-acting factors that function in E3A 3' end formation or in splicing. These factors could compete for overlapping sequences in pre-mRNA. 相似文献
4.
5.
High-affinity hnRNP A1 binding sites and duplex-forming inverted repeats have similar effects on 5' splice site selection in support of a common looping out and repression mechanism
下载免费PDF全文
![点击此处可从《RNA (New York, N.Y.)》网站下载免费的PDF全文](/ch/ext_images/free.gif)
High-affinity binding sites for the hnRNP A1 protein stimulate the use of a distal 5' splice site in mammalian pre-mRNAs. Notably, strong A1-mediated shifts in splice site selection are not accompanied by equivalent changes in the assembly of U1 snRNP-containing complexes on competing 5' splice sites. To explain the above results, we have proposed that an interaction between hnRNP A1 molecules bound to high-affinity sites loops out the internal 5' splice site. Here, we present additional evidence in support of the looping out model. First, replacing A1 binding sites with sequences that can generate a loop through RNA duplex formation activates distal 5' splice site usage in an equivalent manner. Second, increasing the distance between the internal 5' splice site and flanking A1 binding sites does not compromise activation of the distal 5' splice site. Similar results were obtained with pre-mRNAs carrying inverted repeats. Using a pre-mRNA containing only one 5' splice site, we show that splicing is repressed when flanked by two high-affinity A1 binding sites or by inverted repeats, and that inactivation of the internal 5' splice site is sufficient to elicit a strong increase in the use of the distal donor site. Our results are consistent with the view that the binding of A1 to high-affinity sites promotes loop formation, an event that would repress the internal 5' splice site and lead to distal 5' splice site activation. 相似文献
6.
7.
U5 snRNA interacts with exon sequences at 5' and 3' splice sites. 总被引:55,自引:0,他引:55
U5 snRNA is an essential pre-mRNA splicing factor whose function remains enigmatic. Specific mutations in a conserved single-stranded loop sequence in yeast U5 snRNA can activate cleavage of G1----A mutant pre-mRNAs at aberrant 5' splice sites and facilitate processing of dead-end lariat intermediates to mRNA. Activation of aberrant 5' cleavage sites involves base pairing between U5 snRNA and nucleotides upstream of the cleavage site. Processing of dead-end lariat intermediates to mRNA correlates with base pairing between U5 and the first two bases in exon 2. The loop sequence in U5 snRNA may therefore by intimately involved in the transesterification reactions at 5' and 3' splice sites. This pattern of interactions is strikingly reminiscent of exon recognition events in group II self-splicing introns and is consistent with the notion that U5 snRNA may be related to a specific functional domain from a group II-like self-splicing ancestral intron. 相似文献
8.
Selection of the bovine papillomavirus type 1 nucleotide 3225 3' splice site is regulated through an exonic splicing enhancer and its juxtaposed exonic splicing suppressor. 总被引:3,自引:1,他引:2
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Alternative splicing is an important mechanism for the regulation of bovine papillomavirus type 1 (BPV-1) gene expression during the virus life cycle. However, one 3' splice site, located at nucleotide (nt) 3225, is used for the processing of most BPV-1 pre-mRNAs in BPV-1-transformed C127 cells and at early to intermediate times in productively infected warts. At late stages of the viral life cycle, an alternative 3' splice site at nt 3605 is used for the processing of the late pre-mRNA. In this study, we used in vitro splicing in HeLa cell nuclear extracts to identify cis elements which regulate BPV-1 3' splice site selection. Two purine-rich exonic splicing enhancers were identified downstream of nt 3225. These sequences, designated SE1 (nt 3256 to 3305) and SE2 (nt 3477 to 3526), were shown to strongly stimulate the splicing of a chimeric Drosophila doublesex pre-mRNA, which contains a weak 3' splice site. A BPV-1 late pre-mRNA containing the nt 3225 3' splice site but lacking both SE1 and SE2 was spliced poorly, indicating that this 3' splice site is inherently weak. Analysis of the 3' splice site suggested that this feature is due to both a nonconsensus branch point sequence and a suboptimal polypyrimidine tract. Addition of SE1 to the late pre-mRNA dramatically stimulated splicing, indicating that SE1 also functions as an exonic splicing enhancer in its normal context. However, a late pre-mRNA containing both SE1 and SE2 as well as the sequence in between was spliced inefficiently. Further mapping studies demonstrated that a 48-nt pyrimidine-rich region immediately downstream of SE1 was responsible for this suppression of splicing. Thus, these data suggest that selection of the BPV-1 nt 3225 3' splice site is regulated by both positive and negative exonic sequences. 相似文献
9.
Isolation of the chicken beta-globin gene and a linked embryonic beta-like globin gene from a chicken DNA recombinant library. 总被引:74,自引:0,他引:74
A library of random chicken DNA fragments, 15-22 kb long, has been prepared in the vector lambda Charon 4A. This library was screened with combined adult and embryonic globin cDNA, and several independent globin gene-containing recombinants were isolated. One of these recombinants, lambda Chicken beta-globin 1 (lambda C beta G1), contains the adult chicken beta-globin gene and a closely linked embryonic beta-like globin gene. Both genes are transcribed in the same direction with the adult gene located 5' to the embryonic gene. Electron microscopic visualization of R loop structures generated by hybridization of globin RNA to lambda C beta G1 demonstrates that both globin genes contain major intervening sequences about 800 bp long, similar to those present in mammalian beta-globin genes. The adult beta-globin gene also contains a minor (approximately 100 bp long) intervening sequence analogous to the one observed in mammalian beta-globin genes. Restriction enzyme analysis of the adult beta-globin gene on lambda C beta G1 is consistent with the hypothesis that its two intervening sequences occur in the same positions with respect to the beta-globin amino acid sequence as do the corresponding mammalian intervening sequences. 相似文献
10.
Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing. 总被引:5,自引:1,他引:4
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Certain thalassemic human beta-globin pre-mRNAs carry mutations that generate aberrant splice sites and/or activate cryptic splice sites, providing a convenient and clinically relevant system to study splice site selection. Antisense 2'-O-methyl oligoribonucleotides were used to block a number of sequences in these pre-mRNAs and were tested for their ability to inhibit splicing in vitro or to affect the ratio between aberrantly and correctly spliced products. By this approach, it was found that (i) up to 19 nucleotides upstream from the branch point adenosine are involved in proper recognition and functioning of the branch point sequence; (ii) whereas at least 25 nucleotides of exon sequences at both 3' and 5' ends are required for splicing, this requirement does not extend past the 5' splice site sequence of the intron; and (iii) improving the 5' splice site of the internal exon to match the consensus sequence strongly decreases the accessibility of the upstream 3' splice site to antisense 2'-O-methyl oligoribonucleotides. This result most likely reflects changes in the strength of interactions near the 3' splice site in response to improvement of the 5' splice site and further supports the existence of communication between these sites across the exon. 相似文献
11.
Human β-globin mRNA precursors (pre-mRNAs) synthesized in vitro from a bacteriophage SP6 promoter/β-globin gene fusion are accurately and efficiently spliced when added to a HeLa cell nuclear extract. Under optimal conditions, the first intervening sequence (IVS 1) is removed by splicing in up to 90% of the input. pre-mRNA. Splicing requires ATP and in its absence the pre-mRNA is neither spliced nor cleaved at splice junctions. Splicing does not require that the pre-mRNA contain a correct 5′ or 3′ end, a 3′ poly A tail, or a 5′-terminal cap structure. However, capping of the pre-mRNA significantly affects the specificity of in vitro processing. In the absence of a cap approximately 30%–40% of the pre-mRNA is accurately spliced, and a number of aberrantly cleaved RNAs are also detected. In contrast, capped pre-mRNAs are spliced more efficiently and produce fewer aberrant RNA species. The specificity of splice-site selection in vitro was tested by analyzing pre-mRNAs that contain β-thalassemia splicing mutations in IVS 1. Remarkably, these mutations cause the same abnormal splicing events in vitro and in vivo. The ability to synthesize mutant pre-mRNAs and study their splicing in a faithful in vitro system provides a powerful approach to determine the mechanisms of RNA splice-site selection. 相似文献
12.
Le Guiner C Lejeune F Galiana D Kister L Breathnach R Stévenin J Del Gatto-Konczak F 《The Journal of biological chemistry》2001,276(44):40638-40646
TIA-1 has recently been shown to activate splicing of specific pre-mRNAs transcribed from transiently transfected minigenes, and of some 5' splice sites in vitro, but has not been shown to activate splicing of any endogenous pre-mRNA. We show here that overexpression of TIA-1 or the related protein TIAR has little effect on splicing of several endogenous pre-mRNAs containing alternative exons, but markedly activates splicing of some normally rarely used alternative exons on the TIA-1 and TIAR pre-mRNAs. These exons have weak 5' splice sites followed by U-rich stretches. When the U-rich stretch following the 5' splice site of a TIA-1 alternative exon was deleted, TIAR overexpression induced use of a cryptic 5' splice site also followed by a U-rich stretch in place of the original splice site. Using in vitro splicing assays, we have shown that TIA-1 is directly involved in activating the 5' splice sites of the TIAR alternative exons. Activation requires a downstream U-rich stretch of at least 10 residues. Our results confirm that TIA-1 activates 5' splice sites followed by U-rich sequences and show that TIAR exerts a similar activity. They suggest that both proteins may autoregulate their expression at the level of splicing. 相似文献
13.
14.
Nuclear pre-mRNA processing in plants: distinct modes of 3''-splice-site selection in plants and animals. 总被引:22,自引:4,他引:18
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The report that human growth hormone pre-mRNA is not processed in transgenic plant tissues (A. Barta, K. Sommergruber, D. Thompson, K. Hartmuth, M.A. Matzke, and A.J.M. Matzke, Plant Mol. Biol. 6:347-357, 1986) has suggested that differences in mRNA splicing processes exist between plants and animals. To gain more information about the specificity of plant pre-mRNA processing, we have compared the splicing of the soybean leghemoglobin pre-mRNA with that of the human beta-globin pre-mRNA in transfected plant (Orychophragmus violaceus and Nicotiana tabacum) protoplasts and mammalian (HeLa) cells. Of the three introns of leghemoglobin pre-mRNA, only intron 2 was correctly and efficiently processed in HeLa cells. The 5' splice sites of the remaining two introns were faithfully recognized, but correct processing of the 3' sites took place only rarely (intron 1) or not at all (intron 3); cryptic 3' splice sites were used instead. While the first intron in human beta-globin pre-mRNA was not spliced in transfected plant protoplasts, intron 2 processing occurred at a low level, indicating that some mammalian introns can be recognized by the plant intron-splicing machinery. However, excision of intron 2 proved to be incorrect, involving the authentic 5' splice site and a cryptic 3' splice site. Our results indicate that the mechanism of 3'-splice-site selection during intron excision differs between plants and animals. This conclusion is supported by analysis of the 3'-splice-site consensus sequences in animal and plant introns which revealed that polypyrimidine tracts, characteristic of animal introns, are not present in plant pre-mRNAs. It is proposed that an elevated AU content of plant introns is important for their processing. 相似文献
15.
SV40 recombinants carrying a functional RNA splice junction and polyadenylation site from the chromosomal mouse beta maj globin gene. 总被引:16,自引:0,他引:16
We have introduced a fragment of the chromosomal mouse beta major globin gene into SV40 and used the resultant hybrid virus to infect cultured monkey kidney cells. The mouse DNA fragment, which contains an intervening sequence and a poly(A) addition site, has been inserted in both possible orientations relative to the SV40 late region promoter. While the fragment is transcribed regardless of orientation, the RNA splice signal and poly(A) addition site are utilized only when the fragment is inserted in the "sense" orientation. Thus genomic mouse signals for both splicing and polyadenylation are recognized across species boundaries. Furthermore, since only an 18 bp segment was included on the 5' border of the intervening sequence, we can define a maximum length for this splice signal. 相似文献
16.
Five nucleotide changes in the large intervening sequence of a beta globin gene in a beta+ thalassemia patient. 总被引:3,自引:2,他引:1
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
S E Spence R G Pergolizzi M Donovan-Peluso K A Kosche C S Dobkin A Bank 《Nucleic acids research》1982,10(4):1283-1294
A beta globin gene from a patient with homozygous beta+ thalassemia has been cloned and completely sequenced. No changes from normal are found in the 200 nucleotides 5' to the cap site, in the 3' untranslated region up to the poly A addition site, in the small intervening sequence (IVS 1), or in the coding sequence except for a third base change in codon 2. The only other differences are in the large intervening sequence (IVS 2). One of these, at a position 16 nucleotides from the 5' end of IVS 2, has been reported previously in normal individuals, and is probably a polymorphism. Four other changes, at positions 74, 81, 666, and 705 are also seen in IVS 2. Abnormal beta globin mRNA precursors detected in the bone marrow cells of this patient, and abnormal beta globin RNA splicing observed when this gene is transcribed in a tissue culture system taken together with these IVS 2 changes, suggest that the beta+ thalassemia phenotype is produced by a decrease in normal beta globin mRNA processing. 相似文献
17.
Rabbit globin pseudogene psi beta 2 is a hybrid of delta- and beta-globin gene sequences 总被引:1,自引:0,他引:1
The evolutionary history of the rabbit globin pseudogene psi beta 2 was
studied by completing its nucleotide sequence and aligning the sequence
with that of the rabbit adult globin gene beta 1 and the human minor adult
globin gene delta. The 5' flanking region and exon 1 of psi beta 2 were
most similar to rabbit beta 1, but the large intervening sequence and the
3' untranslated region were most similar to human delta. Intron 1 and exon
2 were equally similar to both delta and beta 1. This pattern indicates
that psi beta 2 was originally a delta-like gene that acquired the 5'
portion of gene beta 1 by intrachromosomal gene conversion. The presence of
a delta-globin gene sequence in both rabbits and humans shows that it is an
ancient gene, predating the mammalian radiation that occurred over 85 Myr
ago. Delta has shown a pronounced tendency to be altered in its 5' end
during the course of mammalian evolution. Quantitative divergence analysis
shows that the ancestor to rabbit psi beta 2 was active until 20-30 Myr
ago, during which time the lagomorph beta-globin gene family apparently
functioned without a pseudogene.
相似文献
18.
Regulation of alternative 3' splice site selection by constitutive splicing factors. 总被引:13,自引:0,他引:13
下载免费PDF全文
![点击此处可从《RNA (New York, N.Y.)》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have devised an in vitro splicing assay in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. We have used this assay to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB could be partially reversed if pre-mRNAs were preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affected the joining of exons 1-3 with no dramatic increases in 1-2 splicing being detected. Preincubation of pre-mRNAs with SR proteins led to small increases in 1-2 splicing. However, if pre-mRNAs were preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there was a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus, multiple pairwise, and sometimes antagonizing, interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection. 相似文献
19.
Splice site selection is a key element of pre-mRNA splicing and involves specific recognition of consensus sequences at the 5(') and 3(') splice sites. Evidently, the compliance of a given sequence with the consensus 5(') splice site sequence is not sufficient to define it as a functional 5(') splice site, because not all sequences that conform with the consensus are used for splicing. We have previously hypothesized that the necessity to avoid the inclusion of premature termination codons within mature mRNAs may serve as a criterion that differentiates normal 5(') splice sites from unused (latent) ones. We further provided experimental support to this idea, by analyzing the splicing of pre-mRNAs in which in-frame stop codons upstream of a latent 5(') splice site were mutated, and showing that splicing using the latent site is indeed activated by such mutations. Here we evaluate this hypothesis by a computerized survey for latent 5(') splice sites in 446 protein-coding human genes. This data set contains 2311 introns, in which we found 10490 latent 5(') splice sites. The utilization of 10045 (95.8%) of these sites for splicing would have led to the inclusion of an in-frame stop codon within the resultant mRNA. The validity of this finding is confirmed here by statistical analyses. This finding, together with our previous experimental results, invokes a nuclear scanning mechanism, as part of the splicing machine, which identifies in-frame stop codons within the pre-mRNA and prevents splicing that could lead to the formation of a prematurely terminated protein. 相似文献
20.