End Of Intron Research Articles

Neuronal SH-SY5Y cells use the C-dystrophin promoter coupled with exon 78 skipping and display multiple patterns of alternative splicing including two intronic insertion events.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by mutations in the dystrophin gene. One-third of DMD cases are complicated by mental retardation. Here, we used reverse transcription PCR to analyze the pattern of dystrophin transcripts in neuronal SH-SY5Y cells. Among the three alternative promoters/first exons at the 5'-end, only transcripts containing the brain cortex-specific C1 exon could be amplified. The C-transcript appeared as two products: a major product of the expected size and a minor larger product that contained the cryptic exon 1a between exons C1 and 2. At the 3'-end there was complete exon 78 skipping. Together, these findings indicate that SH-SY5Y cells have neuron-specific characteristics with regard to both promoter activation and alternative splicing. We also revealed partial skipping of exons 9 and 71. Four amplified products were obtained from a fragment covering exons 36-41: a strong expected product, two weak products lacking either exon 37 or exon 38, and a second strong larger product with a 568-bp insertion between exons 40 and 41. The inserted sequence matched the 3'-end of intron 40 perfectly. We concluded that a cryptic splice site was activated in SH-SY5Y cells to create the novel, unusually large, exon 41e (751bp). In total, we identified seven alternative splicing events in neuronal SH-SY5Y cells, and calculated that 32 dystrophin transcripts could be produced. Our results may provide clues in the analysis of transcriptype-phenotype correlations as regards mental retardation in DMD.

Yeast Assay Highlights the Intrinsic Genomic Instability of Human PML Intron 6 over Intron 3 and the Role of Replication Fork Proteins.

Human acute promyelocytic leukemia (APL) is characterized by a specific balanced translocation t(15;17)(q22;q21) involving the PML and RARA genes. In both de novo and therapy-related APL, the most frequent PML breakpoints are located within intron 6, and less frequently in intron 3; the precise mechanisms by which these breakpoints arise and preferentially in PML intron 6 remain unsolved. To investigate the intrinsic properties of the PML intron sequences in vivo, we designed Saccharomyces cerevisiae strains containing human PML intron 6 or intron 3 sequences inserted in yeast chromosome V and measured gross chromosomal rearrangements (GCR). This approach provided evidence that intron 6 had a superior instability over intron 3 due to an intrinsic property of the sequence and identified the 3’ end of intron 6 as the most susceptible to break. Using yeast strains invalidated for genes that control DNA replication, we show that this differential instability depended at least upon Rrm3, a DNA helicase, and Mrc1, the human claspin homolog. GCR induction by hydrogen peroxide, a general genotoxic agent, was also dependent on genetic context. We conclude that: 1) this yeast system provides an alternative approach to study in detail the properties of human sequences in a genetically controlled situation and 2) the different susceptibility to produce DNA breaks in intron 6 versus intron 3 of the human PML gene is likely due to an intrinsic property of the sequence and is under replication fork genetic control.

Evolutionary emergence of a novel splice variant with an opposite effect on the cell cycle.

Alternative splicing contributes greatly to the diversification of mammalian proteomes, but the molecular basis for the evolutionary emergence of splice variants remains poorly understood. We have recently found a novel class of splicing regulatory elements between the polypyrimidine tract (Py) and 3' AG (REPA) at intron ends in many human genes, including the multifunctional PRMT5 (for protein arginine methyltransferase 5) gene. The PRMT5 element is comprised of two G tracts that arise in most mammals and accompany significant exon skipping in human transcripts. The G tracts inhibit splicing by recruiting heterogeneous nuclear ribonucleoprotein (hnRNP) H and F (H/F) to reduce U2AF65 binding to the Py, causing exon skipping. The resulting novel shorter variant PRMT5S exhibits a histone H4R3 methylation effect similar to that seen with the original longer PRMT5L isoform but exhibits a distinct localization and preferential control of critical genes for cell cycle arrest at interphase in comparison to PRMT5L. This report thus provides a molecular mechanism for the evolutionary emergence of a novel splice variant with an opposite function in a fundamental cell process. The presence of REPA elements in a large group of genes implies their wider impact on different cellular processes for increased protein diversity in humans.

Genetic mutations potentially cause two novel NCF1 splice variants up-regulated in the mammary gland, blood and neutrophil of cows infected by Escherichia coli

Neutrophil cytosolic factor 1 (NCF1) plays a crucial role in host defense against microbial pathogens. In this study, we investigated the potential alternative splicing patterns, expression and splice-relevant single nucleotide polymorphisms (SNPs) of the bovine NCF1 gene to increase insights into its potential role against bovine mastitis caused by Escherichia coli infection. Using RT-PCR and clone sequencing methods, we found two novel splice variants designed as NCF1-TV1 (retained intron 6) and NCF1-TV2 (retained part of intron 8), respectively, encoding two putative truncated proteins (239AA and 283AA). Two splice variants were drastically up-regulated in the mastitis-infected cows’ mammary tissues, blood and neutrophils compared with these of healthy cows using real-time RT-PCR. Genomic sequencing analysis identified four novel SNPs g.10112 G>A, g.10766 T>C, SNPs g.12085 G>A and g.12430 T>C at the ends of intron 6 and intron 8 of NCF1. ESE motif predicted that the SNP (g.10766 T>C) might affect the binding with splicing-related factors and subsequently caused the production of aberrant splice variant NCF1-TV1, which is one of the potential reasons that the functional SNP was associated with increased milk somatic cell score in cow. Our results would help in better understanding the NCF1 gene function in the process against pathogen infection, and the effect of splicing-related SNP on the production of aberrant splice variant and careful functional characterization in dairy cattle.

The Human Nuclear Exosome Targeting Complex Is Loaded onto Newly Synthesized RNA to Direct Early Ribonucleolysis

The RNA exosome complex constitutes the major nuclear eukaryotic 3'-5' exonuclease. Outside of nucleoli, the human nucleoplasmic exosome is directed to some of its substrates by the nuclear exosome targeting (NEXT) complex. How NEXT targets RNA has remained elusive. Using an in vivo crosslinking approach, we report global RNA binding sites of RBM7, a key component of NEXT. RBM7 associates broadly with RNA polymerase II-derived RNA, including pre-mRNA and short-lived exosome substrates such as promoter upstream transcripts (PROMPTs), enhancer RNAs (eRNAs), and 3'-extended products from snRNA and replication-dependent histone genes. Within pre-mRNA, RBM7 accumulates at the 3' ends of introns, and pulse-labeling experiments demonstrate that RBM7/NEXT defines an early exosome-targeting pathway for 3'-extended snoRNAs derived from such introns. We propose that RBM7 is generally loaded onto newly synthesized RNA to accommodate exosome action in case of available unprotected RNA 3' ends.

Different patterns of A*80:01:01:01 allele generation based on exon or intron sequences.

Generation of the HLA-A*80:01:01:01 allele has been analysed using its complete sequence. Direct comparison of the sequences and phylogenetic trees using the human leukocyte antigen (HLA)-A representative alleles and the major histocompatibility complex (MHC)-A sequences of non-human primates has been made. Results based on exon sequences confirm previously published, but considering only the sequences of the introns, two distinct regions can be differentiated. The first one comprises from the 5' untranslated region region to the first part of intron 3 sequence (shared with A2 family), and the second one includes the sequence from the end of intron 3 to intron 7 (shared with A1/A3/A11/A36/A30 family). Each of them clusters with Gorilla and Chimpanzee MHC-A sequences, respectively, suggesting an origin coming from a common ancestor to Gorilla and Chimpanzee.

Evolutionarily emerged G tracts between the polypyrimidine tract and 3' AG are splicing silencers enriched in genes involved in cancer.

BackgroundThe 3′ splice site (SS) at the end of pre-mRNA introns has a consensus sequence (Y)nNYAG for constitutive splicing of mammalian genes. Deviation from this consensus could change or interrupt the usage of the splice site leading to alternative or aberrant splicing, which could affect normal cell function or even the development of diseases. We have shown that the position “N” can be replaced by a CA-rich RNA element called CaRRE1 to regulate the alternative splicing of a group of genes.ResultsTaking it a step further, we searched the human genome for purine-rich elements between the -3 and -10 positions of the 3′ splice sites of annotated introns. This identified several thousand such 3′SS; more than a thousand of them contain at least one copy of G tract. These sites deviate significantly from the consensus of constitutive splice sites and are highly associated with alterative splicing events, particularly alternative 3′ splice and intron retention. We show by mutagenesis analysis and RNA interference that the G tracts are splicing silencers and a group of the associated exons are controlled by the G tract binding proteins hnRNP H/F. Species comparison of a group of the 3′SS among vertebrates suggests that most (~87%) of the G tracts emerged in ancestors of mammals during evolution. Moreover, the host genes are most significantly associated with cancer.ConclusionWe call these elements together with CaRRE1 regulatory RNA elements between the Py and 3′AG (REPA). The emergence of REPA in this highly constrained region indicates that this location has been remarkably permissive for the emergence of de novo regulatory RNA elements, even purine-rich motifs, in a large group of mammalian genes during evolution. This evolutionary change controls alternative splicing, likely to diversify proteomes for particular cellular functions.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1143) contains supplementary material, which is available to authorized users.

G-Quadruplex (G4) Motifs in the Maize (Zea mays L.) Genome Are Enriched at Specific Locations in Thousands of Genes Coupled to Energy Status, Hypoxia, Low Sugar, and Nutrient Deprivation

The G-quadruplex (G4) elements comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This G4 DNA can form within or across single-stranded DNA molecules and is mutually exclusive with duplex B-form DNA. The reversibility and structural diversity of G4s make them highly versatile genetic structures, as demonstrated by their roles in various functions including telomere metabolism, genome maintenance, immunoglobulin gene diversification, transcription, and translation. Sequence motifs capable of forming G4 DNA are typically located in telomere repeat DNA and other non-telomeric genomic loci. To investigate their potential roles in a large-genome model plant species, we computationally identified 149,988 non-telomeric G4 motifs in maize (Zea mays L., B73 AGPv2), 29% of which were in non-repetitive genomic regions. G4 motif hotspots exhibited non-random enrichment in genes at two locations on the antisense strand, one in the 5′ UTR and the other at the 5′ end of the first intron. Several genic G4 motifs were shown to adopt sequence-specific and potassium-dependent G4 DNA structures in vitro. The G4 motifs were prevalent in key regulatory genes associated with hypoxia (group VII ERFs), oxidative stress (DJ-1/GATase1), and energy status (AMPK/SnRK) pathways. They also showed statistical enrichment for genes in metabolic pathways that function in glycolysis, sugar degradation, inositol metabolism, and base excision repair. Collectively, the maize G4 motifs may represent conditional regulatory elements that can aid in energy status gene responses. Such a network of elements could provide a mechanistic basis for linking energy status signals to gene regulation in maize, a model genetic system and major world crop species for feed, food, and fuel.

An ARHGEF10 deletion is highly associated with a juvenile-onset inherited polyneuropathy in Leonberger and Saint Bernard dogs.

An inherited polyneuropathy (PN) observed in Leonberger dogs has clinical similarities to a genetically heterogeneous group of peripheral neuropathies termed Charcot-Marie-Tooth (CMT) disease in humans. The Leonberger disorder is a severe, juvenile-onset, chronic, progressive, and mixed PN, characterized by exercise intolerance, gait abnormalities and muscle atrophy of the pelvic limbs, as well as inspiratory stridor and dyspnea. We mapped a PN locus in Leonbergers to a 250 kb region on canine chromosome 16 (Praw = 1.16×10−10, Pgenome, corrected = 0.006) utilizing a high-density SNP array. Within this interval is the ARHGEF10 gene, a member of the rho family of GTPases known to be involved in neuronal growth and axonal migration, and implicated in human hypomyelination. ARHGEF10 sequencing identified a 10 bp deletion in affected dogs that removes four nucleotides from the 3′-end of exon 17 and six nucleotides from the 5′-end of intron 17 (c.1955_1958+6delCACGGTGAGC). This eliminates the 3′-splice junction of exon 17, creates an alternate splice site immediately downstream in which the processed mRNA contains a frame shift, and generates a premature stop codon predicted to truncate approximately 50% of the protein. Homozygosity for the deletion was highly associated with the severe juvenile-onset PN phenotype in both Leonberger and Saint Bernard dogs. The overall clinical picture of PN in these breeds, and the effects of sex and heterozygosity of the ARHGEF10 deletion, are less clear due to the likely presence of other forms of PN with variable ages of onset and severity of clinical signs. This is the first documented severe polyneuropathy associated with a mutation in ARHGEF10 in any species.

Conservation in first introns is positively associated with the number of exons within genes and the presence of regulatory epigenetic signals.

BackgroundGenomes of higher eukaryotes have surprisingly long first introns and in some cases, the first introns have been shown to have higher conservation relative to other introns. However, the functional relevance of conserved regions in the first introns is poorly understood. Leveraging the recent ENCODE data, here we assess potential regulatory roles of conserved regions in the first intron of human genes.ResultsWe first show that relative to other downstream introns, the first introns are enriched for blocks of highly conserved sequences. We also found that the first introns are enriched for several chromatin marks indicative of active regulatory regions and this enrichment of regulatory marks is correlated with enrichment of conserved blocks in the first intron; the enrichments of conservation and regulatory marks in first intron are not entirely explained by a general, albeit variable, bias for certain marks toward the 5’ end of introns. Interestingly, conservation as well as proportions of active regulatory chromatin marks in the first intron of a gene correlates positively with the numbers of exons in the gene but the correlation is significantly weakened in second introns and negligible beyond the second intron. The first intron conservation is also positively correlated with the gene’s expression level in several human tissues. Finally, a gene-wise analysis shows significant enrichments of active chromatin marks in conserved regions of first introns, relative to the conserved regions in other introns of the same gene.ConclusionsTaken together, our analyses strongly suggest that first introns are enriched for active transcriptional regulatory signals under purifying selection.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-526) contains supplementary material, which is available to authorized users.

A novel mutation in the thyroglobulin gene that causes goiter and dwarfism in Wistar Hannover GALAS rats

Outbred stocks of rats have been used extensively in biomedical, pharmaceutical and/or toxicological studies as a model of genetically heterogeneous human populations. One of such stocks is the Wistar Hannover GALAS rat. However, the colony of Wistar Hannover GALAS rat has been suspected of keeping a problematic mutation that manifests two distinct spontaneous abnormalities, goiter and dwarfism, which often confuses study results. We have successfully identified the responsible mutation, a guanine to thymine transversion at the acceptor site (3′ end) of intron 6 in the thyroglobulin (Tg) gene (Tgc.749−1G>T), that induces a complete missing of exon 7 from the whole Tg transcript by mating experiments and subsequent molecular analyses. The following observations confirmed that Tgc.749−1G>T/Tgc.749−1G>T homozygotes manifested both dwarfism and goiter, while Tgc.749−1G>T/+ heterozygotes had only a goiter with normal appearance, suggesting that the mutant phenotypes inherit as an autosomal semi-dominant trait. The mutant phenotypes, goiter and dwarfism, mimicked those caused by typical endocrine disrupters attacking the thyroid. Hence a simple and reliable diagnostic methodology has been developed for genomic DNA-based genotyping of animals. The diagnostic methodology reported here would allow users of Wistar Hannover GALAS rats to evaluate their study results precisely by carefully interpreting the data obtained from Tgc.749−1G>T/+ heterozygotes having externally undetectable thyroidal lesions.

Two novel mutations in glucocerebrosidase, C23W and IVS7 − 1 G > A, identified in Type 1 Gaucher patients heterozygous for N370S

Gaucher disease is an autosomal recessive lysosomal storage disorder resulting from deficient glucocerebrosidase activity. There have been nearly 300 mutations described to date. Novel mutations can potentially provide insight into the biochemical basis of the disease. Two novel mutations are described in two Type 1 Gaucher patients with N370S compound heterozygosity; a point mutation that causes an amino acid substitution at cysteine residue 23 for tryptophan, and a second point mutation within the splicing element at the 3′ end of intron 7. Both mutations were identified by PCR amplification and sequence analysis of patient glucocerebrosidase genomic DNA. Restriction fragment length polymorphism analysis was established for both novel mutations for efficient identification in future patients. Past literature suggests that mutations affecting cysteine residues involved in disulfide bridges, as well as mutations affecting splicing patterns of the glucocerebrosidase transcript, are detrimental to enzyme activity. However, compound heterozygosity with N370S, a mild mutation, will lead to a mild phenotype. The cases reported here support these past findings.

Distribution of Conventional and Nonconventional Introns in Tubulin (α and β) Genes of Euglenids

The nuclear genomes of euglenids contain three types of introns: conventional spliceosomal introns, nonconventional introns for which a splicing mechanism is unknown (variable noncanonical borders, RNA secondary structure bringing together intron ends), and so-called intermediate introns, which combine features of conventional and nonconventional introns. Analysis of two genes, tubA and tubB, from 20 species of euglenids reveals contrasting distribution patterns of conventional and nonconventional introns—positions of conventional introns are conserved, whereas those of the nonconventional ones are unique to individual species or small groups of closely related taxa. Moreover, in the group of phototrophic euglenids, 11 events of conventional intron loss versus 15 events of nonconventional intron gain were identified. A comparison of all nonconventional intron sequences highlighted the most conserved elements in their sequence and secondary structure. Our results led us to put forward two hypotheses. 1) The first one posits that mutational changes in intron sequence could lead to a change in their excision mechanism—intermediate introns would then be a transitional form between the conventional and nonconventional introns. 2) The second hypothesis concerns the origin of nonconventional introns—because of the presence of inverted repeats near their ends, insertion of MITE-like transposon elements is proposed as a possible source of new introns.

A naturally occurring long insertion in the first intron in the Brassica rapa FLC2 gene causes delayed bolting

We investigated the molecular basis of an extremely late bolting, non-heading ‘Leafy Green Parental Line No. 2 (Tsukena No. 2)’, to obtain suitable DNA markers for breeding the late bolting trait in Chinese cabbage (Brassica rapa L. ssp. pekinensis). We found that Tsukena No. 2 contains a ~5 kbp large insertion near the 5′ end of the first intron of BrFLC2, BrFLC3 and BrFLC3′, which are homologs of an Arabidopsis repressor gene for floral transition, FLOWERING LOCUS C (FLC). The transcript abundance of BrFLC1 in Tsukena No. 2 was repressed during cold exposure to the same level as found in a mid-season bolting commercial F1 variety “Muso” (heading Chinese cabbage) and an early-bolting parent of commercial F1 varieties, “Early” (Sakata Co.), whereas repression of BrFLC2 and BrFLC3 containing the large insertion was weak. Furthermore, QTL analysis of a F2 population derived from the Tsukena No. 2 × “Early” revealed that polymorphisms at the BrFLC2 and BrFLC3 loci explained 46.0 and 9.9 % of the phenotypic variation in the bolting time of vernalized plants, respectively. In Arabidopsis, cold-induced repression of FLC and maintenance of that repression are associated with the first intron of FLC. Our study suggests that a naturally occurring large insertion in the first intron resulted in weak repression of BrFLC2 and BrFLC3 during cold exposure and therefore explains the extremely late bolting of the Tsukena No. 2 cultivar.

Transcriptional Analysis Of a Tissue-Specific Factor VIII Knockout Model Demonstrates The Importance Of Endothelial Factor VIII Synthesis

Definitively identifying the cells responsible for synthesis of coagulation factor VIII (F8) has proven to be a challenge. Transplantation studies demonstrate that as an organ liver is the major, but not exclusive source of plasma F8. Within the liver F8 expression has been variously attributed to hepatocytes, and/or liver sinusoidal endothelial cells, and/or Kupffer cells. Extrahepatic transcription of F8 mRNA appears to be nearly ubiquitous at a low level throughout the body. Previous studies have relied upon retrospective post-expression detection of F8 protein or mRNA using a variety of immunochemical, in situ, and cell isolation techniques, but continuing controversy speaks to the difficulties in localizing expression of a trace protein such as F8.We used a rather different, pre-emptive approach to address the question of F8 synthesis. We developed a conditional F8 knockout (KO) mouse model that allows inactivation of the F8 gene, thus preventing expression, in specific cell types. Exons 17/18 of the F8 gene were flanked by LoxP sites (floxed) resulting in their excision in cells expressing Cre recombinase. Tissue-specific Cre-expressing mouse strains were cross-bred with floxed (F8F) mice to generate tissue-specific F8-KO models. Embryonic Cre expression resulted in a new F8KOstrain displaying a severe hemophilia A phenotype. A hepatocyte-specific F8-KO has completely normal plasma F8 levels, while each of 3 endothelial cell (EC)-Cre models displays a reduced-F8 phenotype that correlates in severity with endothelial Cre efficiency. Presumably due to a shared hemangioblast progenitor, Cre is expressed with similar efficiency in both EC and hematopoietic cells in these models. Plasma F8 is undetectable in the most efficient EC-KO model. In contrast, a highly efficient hematopoietic F8-KO model presents with only modestly reduced F8 levels, likely due to off-target effects.RNA analysis revealed that the F8KO allele produces 2 alternatively spliced transcripts in roughly equivalent amounts. The 1st transcript represents the predicted exon 16/19 splicing event. In the 2nd transcript, 46bp at the 5' end of intron 16 are retained due to the same cryptic splice site observed in the Kazazian exon 17-disrupted F8null model. Combined, the 2 F8KO allele transcripts are present at ∼1/8 to 1/5 of normal levels in the F8KO strain. No normal F8 transcripts are present. In the phenotypically normal hepatocyte-KO model ∼70% of total liver gDNA is converted to the F8KO allele, indicative of very efficient hepatocyte Cre activity, yet almost exclusively normal F8 mRNA is present, with only traces of F8KO message. This is consistent with endothelial synthesis as our further results indicate. For the 3 EC-KO models, plasma F8 levels were correlated with hepatic levels of normal F8 mRNA, and inversely correlated with F8KO transcripts. Excessive F8F to F8KOconversion in the hematopoietic-Cre model suggests variable loss of tissue-specificity.In the most efficient, functionally hemophilic EC-KO model, ∼20% of liver gDNA is converted to the F8KO allele, in good agreement with the expected number of hepatic EC, and F8KO mRNA is present at ∼10% of normal liver levels. With undetectable plasma F8, the continued production of normal F8 mRNA at a similar low level (∼10%) by the remaining 80% of Cre-negative, presumably non-endothelial hepatic cells, was unexpected. In addition to liver we found both normal and F8KO message only in kidney and perhaps brain. As expected, only F8KOmRNA was found in spleen and bone marrow, but the presence of exclusively normal mRNA in heart, intestine, testis, lung, and thymus, at relatively normal (low) levels, was surprising. The persistence of widespread transcriptional “expression” of F8, albeit in a functionally hemophilic mouse, is reminiscent of the near-ubiquitous presence of low level F8 transcription in normal mice. This low level transcription apparently does not support functional plasma F8 production, at least not in these EC-KO mice.In summary, our results support the hypothesis that synthesis of F8 is a function of endothelial cells, both in the liver and presumably elsewhere. Neither hepatocytes nor hematopoietic cells appear to contribute significantly to steady-state plasma F8 levels. Transcriptional analysis of normal and F8KO-specific transcripts provides further support for the localization of F8 expression to endothelial cells. Disclosures:No relevant conflicts of interest to declare.

P.6.a.008 Neuropeptide S receptor gene variant and stressful life events: associations with alcohol use disorder and alcohol consumption

Genetic variation in the Protein C gene (PROC) is associated with altered risk of adverse outcome for a number of diseases. Common single nucleotide polymorphisms (SNPs) in the promoter region and the adjacent 5’ region of PROC are associated with Protein C expression. We tested the hypothesis that common SNPs (minor allele frequency > 10%) between the frequently studied promoter SNPs -1654 (rs1799808) and -1641 (rs1799809), and the end of PROC intron 2 alter nuclear transcription factor binding.We used electrophoretic mobility shift assays with 25-mer oligonucleotides centered on each of the 10 SNPs assessed in this potential regulatory region of the Protein C gene to test for differential binding to nuclear factors isolated from Hep-G2 cells.We found that the G-allele oligo of the intron 2 SNP rs2069915[G/A] bound nuclear factors more avidly than the A-allele (p = 1.9 × 10-9, n = 24). Similarly, we found that the C-allele oligo of the intron 2 SNP rs2069916[C/T] bound nuclear factors more avidly than the T-allele, (p = 3.7 × 10-6, n = 19). Cold competition and supershift assays suggested that the protein differentially binding to the C-allele of rs2069916 was USF1. Notably, we observed minimal nuclear factor binding to oligos containing haplotypes of the previously reported -1654 and -1641 SNPs. Luciferase reporter assays that showed the A-T haplotype of rs2069915 and rs2069916 drives transcription significantly more than the C-G haplotype (t-test, P = 0.015, n = 12).Differential transcription factor binding occurs for common SNPs in the 5’ intronic regions of PROC which may contribute to PROC regulation and reported PROC SNP – phenotype associations.

New insights into the melanophilin (MLPH) gene controlling coat color phenotypes in American mink

The mutation causing the Silverblue color type (pp) is one of the most used recessive mutations within American mink (Neovison vison) fur farming, since it is involved in some of the popular color types such as Violet and Saphire which originate from a combination of recessive mutations. In the present study, the genomic and mRNA sequences of the melanophilin (MLPH) gene were studied in Violet, Silverblue and wild-type (wt) mink animals. Although breeding schemes and previous literature indicates that the Violet (aammpp) phenotype is a triple recessive color type involving the same locus as the Silverblue (pp) color type, our findings indicate different genotypes at the MLPH locus. Upon comparison at genomic level, we identified two deletions of the entire intron 7 and of the 5′ end of intron 8 in the sequence of the Silverblue MLPH gene. When investigating the mRNA, the Silverblue animals completely lack exon 8, which encodes 65 residues, of which 47 define the Myosin Va (MYO5A) binding domain. This may cause the incorrect anchoring of the MLPH protein to MYO5A in Silverblue animals, resulting in an improper pigmentation as seen in diluted phenotypes. Additionally, in the MLPH mRNA of wt, Violet and Silverblue phenotypes, part of intron 8 is retained resulting in a truncated MLPH protein, which is 359 residues long in wt and Violet and 284 residues long in Silverblue. Subsequently, our findings point out that the missing actin-binding domain, in neither of the 3 analyzed phenotypes affects the transport of melanosomes or the consequent final pigmentation. Moreover, the loss of the major part of the MYO5A domain in the Silverblue MLPH protein seems to be the responsible for the dilute phenotype. Based on our genomic DNA data, genetic tests for selecting Silverblue and Violet carrier animals can be performed in American mink.

Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site

Removal of introns from the precursors to messenger RNA (pre-mRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5' splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites.

MLPA-based genotype–phenotype analysis in 1053 Chinese patients with DMD/BMD

BackgroundLarge-scale analysis of the transmission, mutation characteristics and the relationship between the reading frame and phenotype of the DMD gene has previously been performed in several countries, however, analogous studies have yet to be performed in Chinese populations.MethodsClinical data from 1053 Chinese patients with DMD/BMD were collected, and the DMD gene was tested by MLPA in all patients and 400 proband mothers. In 20 patients with negative MLPA, sequencing was also performed.ResultsWe found that 27.50% of cases had a family medical history of DMD/BMD, and large rearrangements were identified in 70.56% of the probands, of which 59.35% and 11.21% were deletions or duplications, respectively. The carrier status of the mothers in the study was determined to be 50.75%, and it was established that the DMD mutation was inherited from the mother in 51.72% of the probands. Exons 45–54 and 3–22 were the most frequently deleted regions, and exons 3–11 and 21–37 were the most prevalently duplicated regions of the gene. Breakpoints mainly occurred in introns 43–55 for deletion mutations and in introns 2 and 7 for duplication mutations. No breakpoints were found at the 5′ or 3′ end of introns 31, 35, 36, 40, 65, 68, and 74–78 in any of the deletion or duplication mutations. The reading frame rule held true for 86.4% of the DMD patients and 74.55% of the BMD patients.ConclusionIt is essential to increase physicians’ understanding of DMD/BMD, to promote scientific information, and to increase awareness in regards to genetic counseling and prenatal diagnosis in pedigrees with a family history of the disease, particularly in families with small DMD lesions in China. In addition, such a large-scale analysis will prove to be instructive for leading translational studies between basic science and clinical medicine.

Exon Skipping of Hepatic APOB Pre-mRNA With Splice-switching Oligonucleotides Reduces LDL Cholesterol In Vivo

Familial hypercholesterolemia (FH) is a genetic disorder characterized by extremely high levels of plasma low-density lipoprotein (LDL), due to defective LDL receptor-apolipoprotein B (APOB) binding. Current therapies such as statins or LDL apheresis for homozygous FH are insufficiently efficacious at lowering LDL cholesterol or are expensive. Treatments that target APOB100, the structural protein of LDL particles, are potential therapies for FH. We have developed a series of APOB-directed splice-switching oligonucleotides (SSOs) that cause the expression of APOB87, a truncated isoform of APOB100. APOB87, like similarly truncated isoforms expressed in patients with a different condition, familial hypobetalipoproteinemia, lowers LDL cholesterol by inhibiting very low-density lipoprotein (VLDL) assembly and increasing LDL clearance. We demonstrate that these "APO-skip " SSOs induce high levels of exon skipping and expression of the APOB87 isoform, but do not substantially inhibit APOB48 expression in cell lines. A single injection of an optimized APO-skip SSO into mice transgenic for human APOB resulted in abundant exon skipping that persists for >6 days. Weekly treatments generated a sustained reduction in LDL cholesterol levels of 34-51% in these mice, superior to pravastatin in a head-to-head comparison. These results validate APO-skip SSOs as a candidate therapy for FH.