For more than three decades, researchers have known that consensus splice sites alone are not sufficient regulatory elements to provide complex splicing regulation. Other regulators, so-called ...splicing regulatory elements (SREs) are needed. Most importantly, their sequence variants often underlie the development of various human disorders. However, due to their variable location and high degeneracy, these regulatory sequences are also very difficult to recognize and predict. Many different approaches aiming to identify SREs have been tried, often leading to the development of in silico prediction tools. While these tools were initially expected to be helpful to identify splicing-affecting mutations in genetic diagnostics, we are still quite far from meeting this goal. In fact, most of these tools are not able to accurately discern the SRE-affecting pathological variants from those not affecting splicing. Nonetheless, several recent evaluations have given appealing results (namely for EX-SKIP, ESRseq and Hexplorer predictors). In this review, we aim to summarize the history of the different approaches to SRE prediction, and provide additional validation of these tools based on patients' clinical data. Finally, we evaluate their usefulness for diagnostic settings and discuss the challenges that have yet to be met.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Tandem donor splice sites (5'ss) are unique regions with at least two GU dinucleotides serving as splicing cleavage sites. The Δ3 tandem 5'ss are a specific subclass of 5'ss separated by 3 ...nucleotides which can affect protein function by inserting/deleting a single amino acid. One 5'ss is typically preferred, yet factors governing particular 5'ss choice are not fully understood. A highly conserved exon 21 of the STAT3 gene was chosen as a model to study Δ3 tandem 5'ss splicing mechanisms. Based on multiple lines of experimental evidence, endogenous U1 snRNA most likely binds only to the upstream 5'ss. However, the downstream 5'ss is used preferentially, and the splice site choice is not dependent on the exact U1 snRNA binding position. Downstream 5'ss usage was sensitive to exact nucleotide composition and dependent on the presence of downstream regulatory region. The downstream 5'ss usage could be best explained by two novel interactions with endogenous U6 snRNA. U6 snRNA enables the downstream 5'ss usage in STAT3 exon 21 by two mechanisms: (i) binding in a novel non-canonical register and (ii) establishing extended Watson-Crick base pairing with the downstream regulatory region. This study suggests that U6:5'ss interaction is more flexible than previously thought.
•SERPING1 exon 3 is alternatively spliced.•Highly abundant exon3 skipping isoform is not degraded by NMD.•Newly identified isoforms -15 and +38 comprise appreciable proportion of all SERPING1 ...transcripts.•Close acceptor splice sites are tightly co-regulated.
Mutations in the C1 inhibitor (C1INH) encoding gene, SERPING1, are associated with hereditary angioedema (HAE) which manifests as recurrent submucosal and subcutaneous edema episodes. The major C1INH function is the complement system inhibition, preventing its spontaneous activation. The presented study is focused on SERPING1 exon 3, an alternative and extraordinarily long exon (499 bp). Endogenous expression analysis performed in the HepG2, human liver, and human peripheral blood cells revealed several exon 3 splicing variants alongside exon inclusion: a highly prevalent exon skipping variant and less frequent +38 and -15 variants with alternative 3′ splice sites (ss) located 38 and 15 nucleotides downstream and upstream from the authentic 3′ ss, respectively. An exon skipping variant introducing a premature stop codon, represented nearly one third of all splicing variants and surprisingly appeared not to be degraded by NMD. The alternative -15 3′ ss was used to a small extent, although predicted to be extremely weak. Its use was shown to be independent of its strength and highly sensitive to any changes in the surrounding sequence. -15 3′ ss seems to be co-regulated with the authentic 3′ ss, whose use is dependent mainly on its strength and less on the presence of intronic regulatory motifs. Subtle SERPING1 exon 3 splicing regulation can contribute to overall C1INH plasma levels and HAE pathogenesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Pre-mRNA splicing is an essential step in gene expression, when introns are removed and exons joined by the complex of proteins called spliceosome. Correct splicing requires a precise exon/intron ...junction definition, which is determined by a consensual donor and acceptor splice site at the 5′ and 3′ end, respectively. An acceptor splice site (3′ss) consists of highly conserved AG nucleotides in positions E
−2
and E
−1
. These nucleotides can appear in tandem, located 3 bp from each other. Then they are referred to as NAGNAG or tandem 3′ss, which can be alternatively spliced. NAG/TAG 3′ss motif abundance is extremely low and cannot be easily explained by just a nucleotide preference in this position. We tested artificial NAG/TAG motif’s potential negative effect on exon recognition using a minigene assay. Introducing the NAG/TAG motif into seven different exons revealed no general negative effect on exon recognition. The only observed effect was the partial use of the newly formed distal 3′ss. We can conclude that this motif’s extremely low preference in a natural 3′ss is not a consequence of the NAG/TAG motif’s negative effect on exon recognition, but more likely the result of other RNA processing aspects, such as an alternative 3′ss choice, decreased 3′ss strength, or incorporating an amber stop codon.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Abstract The low density lipoprotein receptor (LDLR) is a transmembrane protein that plays a key role in cholesterol metabolism. It contains 860 amino acids including a 21 amino acid long signal ...sequence, which directs the protein into the endoplasmic reticulum. Mutations in the LDLR gene lead to cholesterol accumulation in the plasma and results in familial hypercholesterolemia (FH). Knowledge of the impact of a mutation on the LDLR protein structure and function is very important for the diagnosis and management of FH. Unfortunately, for a large proportion of mutations this information is still missing. In this study, we focused on the LDLR signal sequence and carried out functional and in silico analyses of two sequence changes, p.(Gly20Arg) and p.(Leu15Pro), localized in this part of the LDLR. Our results revealed that the p.(Gly20Arg) change, previously described as disease causing, has no detrimental effect on protein expression or LDL particle binding. In silico analysis supports this observation, showing that both the wt and p.(Gly20Arg) signal sequences adopt an expected α-helix structure. In contrast, the mutation p.(Leu15Pro) is not associated with functional protein expression and exhibits a structure with disrupted a α-helical arrangement in the signal sequence, which most likely affects protein folding in the endoplasmic reticulum.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK, ZRSKP
Selective immunoglobulin A deficiency (IgAD) is the most common primary immunodeficiency in Europeans. Our genome-wide association study (GWAS) meta-analysis of 1,635 patients with IgAD and 4,852 ...controls identified four new significant (P < 5 × 10
) loci and association with a rare IFIH1 variant (p.Ile923Val). Peak new variants (PVT1, P = 4.3 × 10
; ATG13-AMBRA1, P = 6.7 × 10
; AHI1, P = 8.4 × 10
; CLEC16A, P = 1.4 × 10
) overlapped with autoimmune markers (3/4) and correlated with 21 putative regulatory variants, including expression quantitative trait loci (eQTLs) for AHI1 and DEXI and DNase hypersensitivity sites in FOXP3
regulatory T cells. Pathway analysis of the meta-analysis results showed striking association with the KEGG pathway for IgA production (pathway P < 0.0001), with 22 of the 30 annotated pathway genes containing at least one variant with P ≤ 0.05 in the IgAD meta-analysis. These data suggest that a complex network of genetic effects, including genes known to influence the biology of IgA production, contributes to IgAD.
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IJS, NUK, SBMB, UL, UM, UPUK
The aim of this study was to analyse genotypes, antimicrobial susceptibility patterns and serotypes in
Pseudomonas aeruginosa
clinical strains, including the clonal dissemination of particular ...strains throughout various intensive care units in one medical centre. Using random amplified polymorphic DNA (RAPD–PCR) and
P. aeruginosa
antisera, 22 different genotypes and 8 serotypes were defined among 103 isolates from 48 patients. No direct association between
P. aeruginosa
strain genotypes and serotypes was observed. RAPD typing in strains with the same serotype revealed different genotypes and, on the contrary, most strains with a different serotype displayed the same amplification pattern. The resulting banding patterns showed a high degree of genetic heterogeneity among all isolates from the patients examined, suggesting a non-clonal relationship between isolates from these patients. A higher degree of antibiotic resistance and stronger biofilm production in common genotypes compared to rare ones and genetic homogeneity of the most resistant strains indicated the role of antibiotic pressure in acquiring resistant and more virulent strains in our hospital. In conclusion, genetic characterisation of
P. aeruginosa
strains using RAPD method was shown to be more accurate in epidemiological analyses than phenotyping.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Our understanding of human gut microbiota in health and disease depends on accurate and reproducible microbial data acquisition. The critical step in this process is to apply an appropriate ...methodology to extract microbial DNA, since biases introduced during the DNA extraction process may result in inaccurate microbial representation. In this study, we attempted to find a DNA extraction protocol which could be effectively used to analyze both the bacterial and fungal community. We evaluated the effect of five DNA extraction methods (QIAamp DNA Stool Mini Kit, PureLink
Microbiome DNA Purification Kit, ZR Fecal DNA MiniPrep
Kit, NucleoSpin
DNA Stool Kit, and IHMS protocol Q) on bacterial and fungal gut microbiome recovery using (i) a defined system of germ-free mice feces spiked with bacterial or fungal strains, and (ii) non-spiked human feces. In our experimental setup, we confirmed that the examined methods significantly differed in efficiency and quality, which affected the identified stool microbiome composition. In addition, our results indicated that fungal DNA extraction might be prone to be affected by reagent/kit contamination, and thus an appropriate blank control should be included in mycobiome research. Overall, standardized IHMS protocol Q, recommended by the International Human Microbiome Consortium, performed the best when considering all the parameters analyzed, and thus could be applied not only in bacterial, but also in fungal microbiome research.
Among alternative splicing events in the human transcriptome, tandem NAGNAG acceptor splice sites represent an appreciable proportion. Both proximal and distal NAG can be used to produce two splicing ...isoforms differing by three nucleotides. In some cases, the upstream exon can be alternatively spliced as well, which further increases the number of possible transcripts. In this study, we showed that NAG choice in tandem splice site depends considerably not only on the concerned acceptor, but also on the upstream donor splice site sequence. Using an extensive set of experiments with systematically modified two-exonic minigene systems of
AFAP1L2
or
CSTD
gene, we recognized the third and fifth intronic upstream donor splice site position and the tandem acceptor splice site region spanning from −10 to +2, including NAGNAG itself, as the main drivers. In addition, competition between different branch points and their composition were also shown to play a significant role in NAG choice. All these nucleotide effects appeared almost additive, which explained the high variability in proximal versus distal NAG usage.
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EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Large deletions and duplications within the low-density lipoprotein receptor (LDLR) gene make up approximately 10% of LDLR pathogenic variants found in Czech patients with familial ...hypercholesterolemia. The goal of this study was to test the hypothesis that all probands with each rearrangement share identical breakpoints inherited from a common ancestor and to determine the role of Alu repetitive elements in the generation of these rearrangements.
The breakpoint sequence was determined by PCR amplification and Sanger sequencing. To confirm the breakpoint position, an NGS analysis was performed. Haplotype analysis of common LDLR variants was performed using PCR and Sanger sequencing.
The breakpoints of 8 rearrangements within the LDLR gene were analysed, including the four most common LDLR rearrangements in the Czech population (number of probands ranging from 8 to 28), and four less common rearrangements (1-4 probands). Probands with a specific rearrangement shared identical breakpoint positions and haplotypes associated with the rearrangement, suggesting a shared origin from a common ancestor. All breakpoints except for one were located inside an Alu element. In 6 out of 8 breakpoints, there was high homology (≥ 70%) between the two Alu repeats in which the break occurred.
The most common rearrangements of the LDLR gene in the Czech population likely arose from one mutational event. Alu elements likely played a role in the generation of the majority of rearrangements inside the LDLR gene.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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