1,721,005 research outputs found
Mutation analysis of large genomic regions in tumor DNA using single-strand conformation polymorphism. Lessons from the ATM gene
No full textIn recent years, we have seen a dramatic improvement in our ability to detect nucleotide changes in tumor DNA using a number of techniques for mutation detection that have become routine instruments in many laboratories. The choice of a suitable method or methods of mutation analysis is governed by many factors, including the costs, experimental sensitivity, expected mutation pattern in the target sequence and its functional consequences, as well as staff expertise, personal experience, and preference. The primary selection criterion for such a method is the ability of a technique to search for the presence of unknown mutations in the analyzed regions (scanning methods) as opposed to looking for known mutations already characterized at the nucleotide level. Scanning procedures represent a cost-effective alternative to nucleotide sequencing (see Chapter 13), but usually at a price of an inferior detection rate. The former group of techniques includes procedures based on conformation polymorphism changes, denaturing gradient gel electrophoresis (see Chapter 10), constant denaturant capillary electrophoresis, and mismatch repair and RNase cleavage methods (see Chapter 11). The latter group, exemplified by techniques using sequence-specific oligonucleotides, oligonucleotide liagation assay, and ligase chain reaction (1), is less frequently used for analyzing molecular changes in tumor samples. A wise choice of most appropriate procedures is a crucial step for the identification of molecular changes underlying cancer development and for the correct interpretation of mutation screening
Transposable elements in disease-associated cryptic exons
No full textTransposable elements (TEs) make up a half of the human genome, but the extent of their contribution to cryptic exon activation that results in genetic disease is unknown. Here, a comprehensive survey of 78 mutation-induced cryptic exons previously identified in 51 disease genes revealed the presence of TEs in 40 cases (51%). Most TE-containing exons were derived from short interspersed nuclear elements (SINEs), with Alus and mammalian interspersed repeats (MIRs) covering >18 and >16% of the exonized sequences, respectively. The majority of SINE-derived cryptic exons had splice sites at the same positions of the Alu/MIR consensus as existing SINE exons and their inclusion in the mRNA was facilitated by phylogenetically conserved changes that improved both traditional and auxiliary splicing signals, thus marking intronic TEs amenable for pathogenic exonization. The overrepresentation of MIRs among TE exons is likely to result from their high average exon inclusion levels, which reflect their strong splice sites, a lack of splicing silencers and a high density of enhancers, particularly (G)AA(G) motifs. These elements were markedly depleted in antisense Alu exons, had the most prominent position on the exon–intron gradient scale and are proposed to promote exon definition through enhanced tertiary RNA interactions involving unpaired (di)adenosines. The identification of common mechanisms by which the most dynamic parts of the genome contribute both to new exon creation and genetic disease will facilitate detection of intronic mutations and the development of computational tools that predict TE hot-spots of cryptic exon activatio
Aberrant 3' splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization
No full textThe frequency distribution of mutation-induced aberrant 3' splice sites (3'ss) in exons and introns is more complex than for 5' splice sites, largely owing to sequence constraints upstream of intron/exon boundaries. As a result, prediction of their localization remains a challenging task. Here, nucleotide sequences of previously reported 218 aberrant 3'ss activated by disease-causing mutations in 131 human genes were compared with their authentic counterparts using currently available splice site prediction tools. Each tested algorithm distinguished authentic 3'ss from cryptic sites more effectively than from de novo sites. The best discrimination between aberrant and authentic 3'ss was achieved by the maximum entropy model. Almost one half of aberrant 3'ss was activated by AG-creating mutations and approximately 95% of the newly created AGs were selected in vivo. The overall nucleotide structure upstream of aberrant 3'ss was characterized by higher purine content than for authentic sites, particularly in position -3, that may be compensated by more stringent requirements for positive and negative nucleotide signatures centred around position -11. A newly developed online database of aberrant 3'ss will facilitate identification of splicing mutations in a gene or phenotype of interest and future optimization of splice site prediction tool
Alternative splicing of U2AF1 reveals a shared repression mechanism for duplicated exons
No full textThe auxiliary factor of U2 small nuclear ribonucleoprotein (U2AF) facilitates branch point (BP) recognition and formation of lariat introns. The gene for the 35-kD subunit of U2AF gives rise to two protein isoforms (termed U2AF35a and U2AF35b) that are encoded by alternatively spliced exons 3 and Ab, respectively. The splicing recognition sequences of exon 3 are less favorable than exon Ab, yet U2AF35a expression is higher than U2AF35b across tissues. We show that U2AF35b repression is facilitated by weak, closely spaced BPs next to a long polypyrimidine tract of exon Ab. Each BP lacked canonical uridines at position -2 relative to the BP adenines, with efficient U2 base-pairing interactions predicted only for shifted registers reminiscent of programmed ribosomal frameshifting. The BP cluster was compensated by interactions involving unpaired cytosines in an upstream, EvoFold-predicted stem loop (termed ESL) that binds FUBP1/2. Exon Ab inclusion correlated with predicted free energies of mutant ESLs, suggesting that the ESL operates as a conserved rheostat between long inverted repeats upstream of each exon. The isoform-specific U2AF35 expression was U2AF65-dependent, required interactions between the U2AF-homology motif (UHM) and the ?6 helix of U2AF35, and was fine-tuned by exon Ab/3 variants. Finally, we identify tandem homologous exons regulated by U2AF and show that their preferential responses to U2AF65-related proteins and SRSF3 are associated with unpaired pre-mRNA segments upstream of U2AF-repressed 3?ss. These results provide new insights into tissue-specific subfunctionalization of duplicated exons in vertebrate evolution and expand the repertoire of exon repression mechanisms that control alternative splicing
SERPING1 rs2511988 and age-related macular degeneration
No full textUsing a two-stage case-control protocol, Sarah Ennis and colleagues (Nov 22, p 1828)1 show an association between the SERPING1 gene and age-related macular degeneration. Although this exciting study provides substantive evidence for association with several closely linked intragenic and extragenic variants, it is unclear which biological mechanisms are affected by the population variability in SERPING1 and which variants have a major role in genetic predisposition to this disease.We inspected gene sequences flanking the newly identified variants and found that one of them (rs2511988) was highly likely to have functional consequences. This single-nucleotide substitution is located 20 base pairs upstream of the 3? splice site of exon 7 and is much closer to the SERPING1 coding sequence than the rs2511989 variant initially discovered after completion of their first screen. The rs2511988 variant (A/G) is adjacent to a branch point sequence (TGTTAAG; branch point is underlined), which has been predicted computationally.2Mutations or variants located in the vicinity of this key exon recognition signal have been shown to promote or inhibit splicing efficiency in several human precursor messenger RNAs.3 In yeast, adenine at the same position relative to the branch site is the preferred base4 and facilitates interactions with the branch point binding protein.5 Although human branch point sequences are much more degenerate than those in yeast,[2] and [3] the rs2511988 variant is likely to alter efficiency of intron 6 removal by interfering with assembly of protein-RNA complexes at the 3? splice site and, ultimately, SERPING1 expression level
Position-dependent repression and promotion of DQB1 intron 3 splicing by GGGG motifs
No full textAlternative splicing of HLA-DQB1 exon 4 is allele-dependent and results in variable expression of soluble DQbeta. We have recently shown that differential inclusion of this exon in mature transcripts is largely due to intron 3 variants in the branch point sequence (BPS) and polypyrimidine tract. To identify additional regulatory cis-elements that contribute to haplotype-specific splicing of DQB1, we systematically examined the effect of guanosine (G) repeats on intron 3 removal. We found that the GGG or GGGG repeats generally improved splicing of DQB1 intron 3, except for those that were adjacent to the 5' splice site where they had the opposite effect. The most prominent splicing enhancement was conferred by GGGG motifs arranged in tandem upstream of the BPS. Replacement of a G-rich segment just 5' of the BPS with a series of random sequences markedly repressed splicing, whereas substitutions of a segment further upstream that lacked the G-rich elements and had the same size did not result in comparable splicing inhibition. Systematic mutagenesis of both suprabranch guanosine quadruplets (G4) revealed a key role of central G residues in splicing enhancement, whereas cytosines in these positions had the most prominent repressive effects. Together, these results show a significant role of tandem G4NG4 structures in splicing of both complete and truncated DQB1 intron 3, support position dependency of G repeats in splicing promotion and inhibition, and identify positively and negatively acting sequences that contribute to the haplotype-specific DQB1 expression
Copper-binding proteins and exonic splicing enhancers and silencers
No full textEukaryotic DNA codes not only for proteins but contains a wealth of information required for accurate splicing of messenger RNA precursors and inclusion of constitutively or alternatively spliced exons in mature transcripts. This “auxiliary” splicing code has been characterized as exonic splicing enhancers and silencers (ESE and ESS). The exact interplay between protein and splicing codes is, however, poorly understood. Here, we show that exons encoding copper-coordinating amino acids in human cuproproteins lack ESEs and/or have an excess of ESSs, yet RNA sequencing and expressed sequence tags data show that they are more efficiently included in mature transcripts by the splicing machinery than average exons. Their largely constitutive inclusion in messenger RNA is facilitated by stronger splice sites, including polypyrimidine tracts, consistent with an important role of the surrounding intron architecture in ensuring high expression of metal-binding residues during evolution. ESE/ESS profiles of codons and entire exons that code for copper-coordinating residues were very similar to those encoding residues that coordinate zinc but markedly different from those that coordinate calcium. Together, these results reveal how the traditional and auxiliary splicing motifs responded to constraints of metal coordination in proteins
Global control of aberrant splice-site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition
No full textAuxiliary splicing signals play a major role in the regulation of constitutive and alternative pre-mRNA splicing, but their relative importance in selection of mutation-induced cryptic or de novo splice sites is poorly understood. Here, we show that exonic sequences between authentic and aberrant splice sites that were activated by splice-site mutations in human disease genes have lower frequencies of splicing enhancers and higher frequencies of splicing silencers than average exons. Conversely, sequences between authentic and intronic aberrant splice sites have more enhancers and less silencers than average introns. Exons that were skipped as a result of splice-site mutations were smaller, had lower SF2/ASF motif scores, a decreased availability of decoy splice sites and a higher density of silencers than exons in which splice-site mutation activated cryptic splice sites. These four variables were the strongest predictors of the two aberrant splicing events in a logistic regression model. Elimination or weakening of predicted silencers in two reporters consistently promoted use of intron-proximal splice sites if these elements were maintained at their original positions, with their modular combinations producing expected modification of splicing. Together, these results show the existence of a gradient in exon and intron definition at the level of pre-mRNA splicing and provide a basis for the development of computational tools that predict aberrant splicing outcomes
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