Cryptic Splice Acceptor Research Articles

Complete androgen insensitivity syndrome caused by c.1769-1G > C mutation and activation of a cryptic splice acceptor site in the androgen receptor gene

Androgen insensitivity syndrome (AIS) is the most common cause of 46,XY disorders of sex development (46,XY DSD). This syndrome is an X-linked recessive genetic disease characterized by resistance to the actions of androgens in an individual with a male karyotype and it is caused by mutations in the androgen receptor (AR) gene. We evaluated two siblings with primary amenorrhea, normal secondary sex characteristics, absence of uterus and ovaries, intra-abdominal testis, and elevated testosterone levels. Sequence analysis of the AR gene revealed a splice acceptor site mutation in intron 2 (c.1769-1G > C). The analysis of mRNA showed that this mutation resulted in the activation of a cryptic splice acceptor site located in intron 2 and in the synthesis of an aberrant mRNA transcript with 69 nucleotides insertion between exon 2 and exon 3, leading to an insertion of 23 amino acids in the AR protein instead of generating a premature termination codon. The additional 23 amino acids insertion affects AR intracellular trafficking by impairing its translocation from the cytoplasm to the nucleus after hormone stimulation. The c.1769-1G > C mutation provides new insights into the molecular mechanism involved in splicing defects and expands the spectrum of mutations associated with the androgen insensitivity syndrome.

GAA Deficiency in Pompe Disease Is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells.

Pompe disease is a metabolic myopathy caused by deficiency of the acid α-glucosidase (GAA) enzyme and results in progressive wasting of skeletal muscle cells. The c.-32-13T>G (IVS1) GAA variant promotes exon 2 skipping during pre-mRNA splicing and is the most common variant for the childhood/adult disease form. We previously identified antisense oligonucleotides (AONs) that promoted GAA exon 2 inclusion in patient-derived fibroblasts. It was unknown how these AONs would affect GAA splicing in skeletal muscle cells. To test this, we expanded induced pluripotent stem cell (iPSC)-derived myogenic progenitors and differentiated these to multinucleated myotubes. AONs restored splicing in myotubes to a similar extent as in fibroblasts, suggesting that they act by modulating the action of shared splicing regulators. AONs targeted the putative polypyrimidine tract of a cryptic splice acceptor site that was part of a pseudo exon in GAA intron 1. Blocking of the cryptic splice donor of the pseudo exon with AONs likewise promoted GAA exon 2 inclusion. The simultaneous blocking of the cryptic acceptor and cryptic donor sites restored the majority of canonical splicing and alleviated GAA enzyme deficiency. These results highlight the relevance of cryptic splicing in human disease and its potential as therapeutic target for splicing modulation using AONs.

Identification of Novel and Recurrent Disease-Causing Mutations in Retinal Dystrophies Using Whole Exome Sequencing (WES): Benefits and Limitations

Inherited retinal dystrophies (IRDs) are Mendelian diseases with tremendous genetic and phenotypic heterogeneity. Identification of the underlying genetic basis of these dystrophies is therefore challenging. In this study we employed whole exome sequencing (WES) in 11 families with IRDs and identified disease-causing variants in 8 of them. Sequence analysis of about 250 IRD-associated genes revealed 3 previously reported disease-associated variants in RHO, BEST1 and RP1. We further identified 5 novel pathogenic variants in RPGRIP1 (p.Ser964Profs*37), PRPF8 (p.Tyr2334Leufs*51), CDHR1 (p.Pro133Arg and c.439-17G>A) and PRPF31 (p.Glu183_Met193dup). In addition to confirming the power of WES in genetic diagnosis of IRDs, we document challenges in data analysis and show cases where the underlying genetic causes of IRDs were missed by WES and required additional techniques. For example, the mutation c.439-17G>A in CDHR1 would be rated unlikely applying the standard WES analysis. Only transcript analysis in patient fibroblasts confirmed the pathogenic nature of this variant that affected splicing of CDHR1 by activating a cryptic splice-acceptor site. In another example, a 33-base pair duplication in PRPF31 missed by WES could be identified only via targeted analysis by Sanger sequencing. We discuss the advantages and challenges of using WES to identify mutations in heterogeneous diseases like IRDs.

Revealing the function of a novel splice-site mutation of CHD7 in CHARGE syndrome

Most cases of CHARGE syndrome are sporadic and autosomal dominant. CHD7 is a major causative gene of CHARGE syndrome. In this study, we screened CHD7 in two Turkish patients demonstrating symptoms of CHARGE syndrome such as coloboma, heart defect, choanal atresia, retarded growth, genital abnomalities and ear anomalies. Two mutations of CHD7 were identified including a novel splice-site mutation (c.2443-2A>G) and a previously known frameshift mutation (c.2504_2508delATCTT). We performed exon trapping analysis to determine the effect of the c.2443-2A>G mutation at the transcriptional level, and found that it caused a complete skip of exon 7 and splicing at a cryptic splice acceptor site. Our current study is the second study demonstrating an exon 7 deficit in CHD7. Results of previous studies suggest that the c.2443-2A>G mutation affects the formation of nasal tissues and the neural retina during early development, resulting in choanal atresia and coloboma, respectively. The findings of the present study will improve our understanding of the genetic causes of CHARGE syndrome.

The origin of the p.E180 growth hormone receptor gene mutation

Laron syndrome, an autosomal recessive condition of extreme short stature, is caused by the absence or dysfunction of the growth hormone receptor. A recurrent mutation in the GHR gene, p.E180, did not alter the encoded amino acid, but activated a cryptic splice acceptor resulting in a receptor protein with an 8-amino acid deletion in the extracellular domain. This mutation has been observed among Sephardic Jews and among individuals in Ecuador, Brazil and Chile, most notably in a large genetic isolate in Loja, Ecuador. A common origin has been postulated based on a shared genetic background of markers flanking this mutation, suggesting that the Lojanos (and others) may have Sephardic (Converso) Jewish ancestry. Analysis of the population structure of Lojanos based on genome-wide analysis demonstrated European, Sephardic Jewish and Native American ancestry in this group. X-autosomal comparison and monoallelic Y chromosomal and mitochondrial genetic analysis demonstrated gender-biased admixture between Native American women and European and Sephardic Jewish men. These findings are compatible with the co-occurrence of the Inquisition and the colonization of the Americas, including Converso Jews escaping the Inquisition in the Iberian Peninsula. Although not found among Lojanos, Converso Jews also brought founder mutations to contemporary Hispanic and Latino populations in the BRCA1 (c.68_69delAG) and BLM (c.2207_2212delATCTGAinsTAGATTC) genes.

A novel FOXP1-PDGFRA fusion gene in myeloproliferative neoplasm with eosinophilia

We identified a novel fusion gene, FOXP1-PDGFRA, in a patient with myeloproliferative neoplasm (MPN) with eosinophilia, harboring the chromosome abnormality t(3;4)(p13;q12). The patient responded well to imatinib and has remained in molecular remission for 3 years. This is the seventh fusion gene involving PDGFRA in MPN with eosinophilia. PDGFRA was truncated in its autoinhibitory domain, as in other PDGFRA-related MPNs, and was fused to FOXP1 at its functional forkhead domain. Comparing genomic DNA with mRNA sequences provides the possibility that the splicing process near the breakpoint junction in the FOXP1-PDGFRA fusion gene may use the normal splice donor site for intron 23a of FOXP1 and the cryptic splice acceptor site in exon 12 of PDGFRA. This is the first report to describe the FOXP1-PDGFRA fusion gene in MPN.

Splice-acceptor site mutation in p53 gene of hu888 zebrafish line.

The p53 transcription factor is a key tumor suppressor and a central regulator of the stress response, which has been a subject of intense research for over 30 years. Recently, a zebrafish line which carries splice site mutation (G>T) in intron 8 of p53 gene (p53 hu888), encoding the p53 paralogue, was developed (The Zebrafish Mutation Project). To uncover molecular effects of the mutation, we raised hu888 zebrafish line to adulthood and analyzed DNA, mRNA data, and protein levels of p53 to assess their potential contribution in molecular mechanisms of the mutant fish. To obtain zebrafish individuals homozygous for the point mutation, p53 hu888 carriers were repeatedly incrossed but only heterozygous mutants (p53 hu888/+) or p53-wild type hu888 zebrafish (p53 +/+) were identified in their progeny. By evaluation of p53 expression changes in the liver of mutant and wild type hu888 zebrafish as well as of Tübingen reference strain, we demonstrated that two types of splicing occurred in each case: a classical one and the alternative splicing which involves the activation of cryptic splice-acceptor site in the exon 9 of zebrafish p53 pre-mRNA. The alternative splicing event results in a deletion 12 nucleotides in the mature mRNA, and produces a shortened variant of p53 protein. Interestingly, expression of p53 protein in liver of both heterozygous and wild type hu888 zebrafish was highly reduced compared to that in the reference strain.

Functional Studies of p.R132C, p.R149C, p.M283V, p.E431K, and a Novel c.652-2A>G Mutations of the CYP21A2 Gene

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism and accounts for 90–95% of CAH cases. In the present work, we analyzed the functional consequence of four novel previously reported point CYP21A2 mutations -p.R132C, p.R149C, p.M283V, p.E431K- found in Argentinean 21-hydroxylase deficient patients. In addition, we report an acceptor splice site novel point mutation, c.652-2A>G, found in a classical patient in compound heterozygosity with the rare p.R483Q mutation. We performed bioinformatic and functional assays to evaluate the biological implication of the novel mutation. Our analyses revealed that the residual enzymatic activity of the isolated mutants coding for CYP21A2 aminoacidic substitutions was reduced to a lesser than 50% of the wild type with both progesterone and 17-OH progesterone as substrates. Accordingly, all the variants would predict mild non-classical alleles. In one non-classical patient, the p.E431K mutation was found in cis with the p.D322G one. The highest decrease in enzyme activity was obtained when both mutations were assayed in the same construction, with a residual activity most likely related to the simple virilizing form of the disease. For the c.652-2A>G mutation, bioinformatic tools predicted the putative use of two different cryptic splicing sites. Nevertheless, functional analyses revealed the use of only one cryptic splice acceptor site located within exon 6, leading to the appearance of an mRNA with a 16 nt deletion. A severe allele is strongly suggested due to the presence of a premature stop codon in the protein only 12 nt downstream.

Intronic splicing mutations in PTCH1 cause Gorlin syndrome

Gorlin syndrome is an autosomal dominant disorder characterized by multiple early-onset basal cell carcinoma, odontogenic keratocysts and skeletal abnormalities. It is caused by heterozygous mutations in the tumour suppressor PTCH1. Routine clinical genetic testing, by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to confirm a clinical diagnosis of Gorlin syndrome, identifies a mutation in 60-90% of cases. We undertook RNA analysis on lymphocytes from ten individuals diagnosed with Gorlin syndrome, but without known PTCH1 mutations by exonic sequencing or MLPA. Two altered PTCH1 transcripts were identified. Genomic DNA sequence analysis identified an intron 7 mutation c.1068-10T>A, which created a strong cryptic splice acceptor site, leading to an intronic insertion of eight bases; this is predicted to create a frameshift p.(His358Alafs*12). Secondly, a deep intronic mutation c.2561-2057A>G caused an inframe insertion of 78 intronic bases in the cDNA transcript, leading to a premature stop codon p.(Gly854fs*3). The mutations are predicted to cause loss of function of PTCH1, consistent with its tumour suppressor function. The findings indicate the importance of RNA analysis to detect intronic mutations in PTCH1 not identified by routine screening techniques.

Abstract 1125: Spliceosome-targeted antitumor drug discovery.

Abstract Over the last several years there has been a remarkable convergence of research related to oncogenic aberrations of the spliceosome, which has linked the identification of recurrent spliceosome mutations in tumors to medicinal chemistry. We designed synthetic natural product analogs (the sudemycins) that modulate the activity of the spliceosome and show potent and selective antitumor activity. We hypothesized that splicing gene mutations cause alterations in splice isoforms, which provide a selective advantage to tumor cells, and that perturbation of this environment with spliceosome modulators may cause a selective disadvantage for tumors. To test this hypothesis, we generated MSCV-based retroviruses encoding the wildtype or S34F mutant U2AF1 alleles. We infected c-kit+ primary C57BL/6J murine bone marrow cells with these recombinant retroviruses and cultured them in vitro with cytokine-supplemented media. Vehicle-treated cells expressing the mutant allele had reduced growth over 5 days, compared to cells expressing the wildtype allele or empty vector (P<0.0001, n=5 technical replicates, two experiments). At doses of 10-150 nM, sudemycin D1 (but not the inactive analog sudemycin E-OH) induced a reproducible dose-dependent decrease in cell growth and increase in apoptosis, as measured by Annexin V, in cells expressing the mutant, compared to the wildtype allele or empty vector. To study the effect of the U2AF1 mutation and sudemycin exposure on splicing, we analyzed RNA isoforms of Fmr1, a gene known to undergo alternative splicing. After retroviral transduction, GFP+ cells were sorted and RNA was isolated for RT-PCR analysis at baseline or 2h-6h after exposure to sudemycin D1 (75 nM) or vehicle. The U2AF1 S34F mutation increased the abundance of Fmr1 transcripts utilizing a cryptic splice acceptor, compared to cells transduced with empty vector or wildtype U2AF1 (P<0.05, n=3 technical replicates). These experiments provide proof-of-concept that cells expressing mutant U2AF1 have increased sensitivity to spliceosome modulators. The sudemycins show a short half-life (<15 min) when dosed IV via a bolus tail vein injection. So we have developed a jugular continuous infusion model to achieve therapeutic drug levels, and found that sudemycin D6 is tolerated at doses up to 200 mg/kg/h in mice. Additionally we have investigated numerous drug combinations and found significant cytotoxic co-treatment synergism with the experimental dual mTOR and PI3K inhibitor BEZ235, which suggests opportunities for multi-agent animal studies as a prelude to the design of human clinical trials. In this presentation we will also report in vitro and in vivo data on new sudemycin analogs along with additional work on the pharmacology, pharmacokinetics, toxicity and in vivo efficacy. Our results indicate a maturing therapeutic potential for sudemycin D6, which is in preclinical development for the treatment of subsets of MDS, melanoma and numerous other human cancers. Citation Format: Timothy A. Graubert, Sharyn Baker, Philip M. Potter, Manorama Tripathi, Matthew Walter, Gustavo Palacios, Thomas R. Webb. Spliceosome-targeted antitumor drug discovery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1125. doi:10.1158/1538-7445.AM2013-1125

Sudemycin Selectively Inhibits Growth of Primary Murine Hematopoietic Cells Expressing Mutant U2AF1

Abstract 554Core components of the pre-mRNA splicing complex (the “spliceosome”) are targets of recurrent somatic mutation in patients with myelodysplastic syndrome (MDS). Using whole genome and targeted resequencing, we previously identified recurrent missense mutations in U2AF1 (encoding U2AF35, the 35 kDa U2 accessory factor) at serine 34 (S34F or S34Y) or glutamine 157 (Q157R) in 8.7% of de novo MDS patients. The mechanism by which mutations in U2AF1 and other recurrently mutated spliceosome components contribute to MDS pathogenesis is unknown, but recurrent mutations in this pathway suggest that it may provide a novel therapeutic target. Pladienolide B and FR901464 are two bacterial products that modulate activity of the spliceosome. The derivatives, E7107 and spliceostatin A, and synthetic analogs (sudemycins), bind SF3B, a complex that includes SF3B1 and six other subunits of the spliceosome. Treatment of cell lines with these agents causes accumulation of aberrantly spliced gene products. Sudemycins have favorable pharmacokinetic properties (improved solubility, chemical stability, and plasma half-life) and induce cell cycle arrest and apoptosis in low nanomolar concentrations in cell lines in vitro. They also inhibit growth in xenograft models in vivo, with no evidence of toxicity in mice treated with pharmacologically active doses. We hypothesize that splicing gene mutations cause alterations in splice isoforms that provide a selective advantage to tumor cells, and that perturbation of this environment with small molecule spliceosome modulators may cause a selective disadvantage for cells with these mutations. To test this hypothesis, we generated MSCV-based retroviruses encoding the wildtype (MSCV-WT-ires-GFP) or S34F mutant (MSCV-MT- ires-GFP) U2AF1 alleles. We infected c-kit+ primary C57BL/6J murine bone marrow cells with these recombinant retroviruses (or empty vector) and cultured them in vitro with cytokine-supplemented media. Consistent with previous reports, vehicle-treated cells (0.1% DMSO) expressing the mutant allele had reduced growth over 5 days, compared to cells expressing the wildtype allele or empty vector (P<0.0001, n=5 technical replicates, two experiments). At doses between 10–150 nM, sudemycin (but not an inactive sudemycin analog) induced a reproducible dose-dependent decrease in cell growth, most significant at 75 nM (p<0.0001, n=5 technical replicates, two experiments), and an increase in apoptosis (p=<0.05, n=3 technical replicates), as measured by annexin V positivity, in cells expressing the mutant, compared to the wildtype allele or empty vector. To study the effect of the U2AF1 mutation and sudemycin exposure on splicing, we analyzed RNA isoforms of Mdm2 and Fmr1, two genes known to undergo alternative splicing. After retroviral transduction, GFP+ cells were sorted and RNA was isolated for RT-PCR analysis at baseline or 2h–6h after exposure to sudemycin (75 nM) or vehicle. The U2AF1 S34F mutation increased the abundance of Fmr1 transcripts utilizing a cryptic splice acceptor, compared to cells transduced with empty vector or wildtype U2AF1 (P<0.05, n=3 technical replicates). Sudemycin treatment decreased Fmr1 cryptic splice site utilization (p<0.05, n=3 technical replicates) and increased Mdm2 exon skipping (p<0.05, n=3 technical replicates) at 2h and 6hr, compared to vehicle-treated cells, with the most significant differences in cells expressing mutant U2AF1. These experiments provide proof-of-concept that cells expressing mutant U2AF1 have increased sensitivity to spliceosome modulators and warrant further investigation as a possible treatment for patients with these mutations. Ongoing experiments include in vivo experiments in mice transplanted with transduced cells, and in vitro treatment of primary clinical samples. Small molecule modulators of the spliceosome may provide a novel therapeutic strategy for MDS/AML patients with spliceosome mutations. Disclosures:No relevant conflicts of interest to declare.

Incidence of the BCR-ABL Kinase Domain Mutations in Imatinib Treated CML Patients: Russian Experience

Abstract 4420In the period from 18.01.06 to 25.05.11 we have performed mutation analysis in CML pts with the obvious manifestation of resistance against imatinib. Chronic phase CML patients have been treated by imatinib and monitored by means of RQ-PCR using IS units. At first signs of the evident BCR-ABL level increase (more than 5–10% IS), the mutation analysis has been performed by means of direct PCR fragment sequencing. We have used original primers that enable to search for mutations in the area spanning from a3 to a11 ABL exons of BCR-ABL. Sequencing has been performed for 274 CML pts that were suspected to acquire imatinib resistance. Among them there were 47,45% (130/274) of females (median age 51 years, from 15 to 73), 52,55% (144/274) of males (median age 50 years, from 15 to 74). In the group of mutation negative pts (80/274 – 29,2%) the BCR-ABL level was more than 9% (IS). Interestingly, in this group there were a little bit more females than in the total cohort of pts (58,7% against 47,45%, p=0,034). The median age of the mutation negative females (51 years) was quite the same as in the total cohort, whereas the median age of mutation negative males significantly differed from the one in the total cohort (58 vs. 51 year, p=0,041). 70,8% (194/274) pts tested occurred to be mutation positive (42,8% females (83/194), 57,2% males (111/194)). 214 point mutations of 38 different types have been detected, among them T315I (26/194 – 12%), G250E (26/194 – 12%), T317L (16/194 – 7,4%), F359V (15/194 – 7,0%), H396R (15/194 – 7,0%), M244V (15/194 – 7,0%), E255K (12/194 – 5,6%), Y253H (12/194 – 5,6%), E255V (9/194 – 4,2%), L248V (8/194 – 3,7%), E355G (6/194 – 2,8%), M315T (6/194 – 2,8%), Q252H (5/194 – 2,3%), L387F (4/194 – 1,8%), S348L (4/194 – 1,8%), F311I (3/194 – 1,4%), F359C (3/194 – 1,4%), E255D (2/194 – 0,9%), E275K (2/194 – 0,9%), E279A (2/194 – 0,9%), K247R (2/194 – 0,9%), E292V (1/194 – 0,46%), E334G (1/194 – 0,46%), E450K (1/194 – 0,46%), E459A (1/194 – 0,46%), E459K (1/194 – 0,46%), F359I (1/194 – 0,46%), F486S (1/194 – 0,46%), L383F (1/194 – 0,46%), P441L (1/194 – 0,46%), Q252M (1/194 – 0,46%), Q491L (1/194 – 0,46%), T305I (1/194 – 0,46%), T345I (1/194 – 0,46%), V299A (1/194 – 0,46%), Y312C (1/194 – 0,46%), T520S (1/194 – 0,46%). As rare events we have observed some other types of mutations. In one CML patient with primary imatinib resistance there was a G425Stop (1/194 – 0,46%) mutation which caused a significant truncation of the C-end of the BCR-ABL protein. There were also 2 highly imatinib resistant pts with deletions of exon a7 ABL (2/194 – 0,9%). One more unusual mutation was an insertion of 72 bp between the a8 and a9 exons of ABL. The origin of this insertion was a middle part of the 8th intron of the ABL gene. This sequence is arranged as a typical human exon because it is flanked by donor and acceptor splicing signals. We found this mutation in 3 imatinib resistant CML pts (3/194 – 1,4%). This mutation gives rise to the abnormal stop-codon within the exon a9 due to a CD's frameshift. In all pts with point L248V mutation (8/194 – 3,7%) we have also observed additional abnormal variant of BCR-ABL gene as a splicing isoform lacking 81 bp of exon a4. The point mutation L248V (742C>G) exchange the motif CAAGCT for CAAGGT and the latter is a strong splice acceptor site. It seems that this cryptic splice acceptor site in the middle of 4a exon carrying 742C>G point mutation may compete with natural splice acceptor site from 4th intron. It gives rise to two BCR-ABL transcripts: one carrying a point mutation and the other one with 81 bp's deletion of the 3'end of exon 4a, without frameshift. These findings suggest that in the case of mutational analysis the considerable attention should be paid not only to BCR-ABL point mutations but also to the possible emergence of the other changes of the BCR-ABL mRNA structure which may occur due to arbitrary activity of splicing machinery. Our findings also suggest that among imatinib resistant CML pts some of them already acquired mutations that prevented adequate response for the 2nd generation of TKI. In our study 12% of BCR-ABL mutation positive CML pts resistant to imatinib (n=26/194) had T315I which also render nilotinib and dasatinib resistance. 23,8% (n=51/194) were positive for mutations that decreases nilotinib response (E255K/V, F359C/V, Y253H) and 7,5% (n=16/194) were positive for T317L that hinders dasatinib response. Disclosures:No relevant conflicts of interest to declare.

The Arabic allele: A single base pair substitution activates a 10-base downstream cryptic splice acceptor site in exon 12 of LDLR and severely decreases LDLR expression in two unrelated Arab families with familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a monogenic autosomal dominant disorder caused by defects in LDLR. Few reports describe FH mutations among Arabs. We describe a mutation in LDLR of two unrelated Arab families. We investigated 19 patients using DNA sequencing, RFLP, and real-time (RT) PCR. DNA sequencing showed a base pair substitution (c.1706-2 A>T) in the splice acceptor site of LDLR intron 11. Our results were confirmed by RFLP on 2% agarose gel. In silico analysis predicted a new cryptic splice site downstream of the original position generating a 10-base deletion from the beginning of exon 12; (c.1706-1715del.ATCTCCTCAG). cDNA sequencing of exon 12 confirmed the computational analysis. The deletion was visualized on 4% agarose gel. The deletion generates a frameshift and a premature termination codon (c.1991-1993; p.(Asp569Valfs*93). RT-PCR revealed that LDLR mRNA is 9.3% ± 6.5 and 17.9% ± 8.0 for FH homozygote and heterozygote individuals respectively, compared to a healthy family control. We predict a class II LDLR mutation that leads to a truncated receptor missing exons 14–18. We called this mutation “the Arabic allele”. We expect a significant contribution of this mutation to the prevalence of FH among Arabs. Also, we propose that the severe down regulation of LDLR mRNA expression is due to nonsense-mediated-decay

Dissection of Splicing Regulation at an Endogenous Locus by Zinc-Finger Nuclease-Mediated Gene Editing

Sequences governing RNA splicing are difficult to study in situ due to the great difficulty of traditional targeted mutagenesis. Zinc-finger nuclease (ZFN) technology allows for the rapid and efficient introduction of site-specific mutations into mammalian chromosomes. Using a ZFN pair along with a donor plasmid to manipulate the outcomes of DNA repair, we introduced several discrete, targeted mutations into the fourth intron of the endogenous BAX gene in Chinese hamster ovary cells. Putative lariat branch points, the polypyrimidine tract, and the splice acceptor site were targeted. We recovered numerous otherwise isogenic clones carrying the intended mutations and analyzed the effect of each on BAX pre-mRNA splicing. Mutation of one of three possible branch points, the polypyrimidine tract, and the splice acceptor site all caused exclusion of exon five from BAX mRNA. Interestingly, these exon-skipping mutations allowed usage of cryptic splice acceptor sites within intron four. These data demonstrate that ZFN-mediated gene editing is a highly effective tool for dissection of pre-mRNA splicing regulatory sequences in their endogenous context.

Presenilin-Dependent Receptor Processing Is Required for Axon Guidance

The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloid-β precursor protein, contributing to the pathogenesis of neurodegeneration that is characterized by loss of neuronal connections, but the role of Presenilin in establishing neuronal connections is less clear. Through a forward genetic screen in mice for recessive genes affecting motor neurons, we identified the Columbus allele, which disrupts motor axon projections from the spinal cord. We mapped this mutation to the Presenilin-1 gene. Motor neurons and commissural interneurons in Columbus mutants lacking Presenilin-1 acquire an inappropriate attraction to Netrin produced by the floor plate because of an accumulation of DCC receptor fragments within the membrane that are insensitive to Slit/Robo silencing. Our findings reveal that Presenilin-dependent DCC receptor processing coordinates the interplay between Netrin/DCC and Slit/Robo signaling. Thus, Presenilin is a key neural circuit builder that gates the spatiotemporal pattern of guidance signaling, thereby ensuring neural projections occur with high fidelity.

Ca2+/Calmodulin-dependent Protein Kinase II Binds to and Phosphorylates a Specific SAP97 Splice Variant to Disrupt Association with AKAP79/150 and Modulate α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid-type Glutamate Receptor (AMPAR) Activity

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) promotes trafficking and activation of the GluR1 subunit of alpha-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) during synaptic plasticity. GluR1 is also modulated in parallel by multiprotein complexes coordinated by synapse-associated protein 97 (SAP97) that contain A-kinase anchoring protein 79/150 (AKAP79/150), protein kinase A, and protein phosphatase 2B. Here we show that SAP97 is present in CaMKII immune complexes isolated from rodent brain as well as from HEK293 cells co-expressing CaMKIIalpha and SAP97. CaMKIIalpha phosphorylated recombinant SAP97 within immune complexes in vitro and in intact cells. Four alternative mRNA splice variants of SAP97 expressing combinations of four inserts (I2, I3, I4, I5) in the U5 region between Src homology 3 (SH3) and guanylyl kinase-like (GK) domains were identified in rat brain at postnatal day 21. CaMKIIalpha preferentially phosphorylated a full-length SAP97 and a glutathione S-transferase (GST) fusion protein containing the I3 and I5 inserts (SAP97-I3I5 and GST-SH3-I3I5-GK, respectively) and also specifically interacted with GST-SH3-I3I5-GK compared with GST proteins containing other naturally occurring insert combinations. AKAP79/150 also directly and specifically bound only to GST-SH3-I3I5-GK, but CaMKII phosphorylation of GST-SH3-I3I5-GK prevented this interaction. AKAP79-dependent down-regulation of GluR1 AMPAR currents was ablated by overexpression of SAP97-I2I5 (which does not bind AKAP79) or by infusion of active CaMKIIalpha. Collectively, the data suggest that CaMKIIalpha targets a specific SAP97 splice variant to disengage AKAP79/150 from regulating GluR1 AMPARs, providing new insight into protein-protein interactions and phosphorylation events that are required for normal regulation of glutamatergic synaptic transmission, learning, and memory.

Dual effect of a single nucleotide polymorphism in the first intron of the porcine Secreted phosphoprotein 1 gene: allele-specific binding of C/EBP beta and activation of aberrant splicing

BackgroundSecreted phosphoprotein 1 (SPP1 or Osteopontin, OPN) is a multifunctional matricellular glycoprotein involved in development and regeneration of skeletal muscle. Previously, we have demonstrated that porcine SPP1 shows breed-related differential mRNA expression during myogenesis. With the aim to identify putative contributing cis-regulatory DNA variation we resequenced the 5' upstream region of the gene in the respective breeds Pietrain and Duroc. We found two single nucleotide polymorphisms (SNP; [GenBank:M84121]: g.1804C>T and g.3836A>G). We focused our investigation on the SNP g.3836A>G, because in silico analysis and knowledge about the regulation of SPP1 suggested an effect of this SNP on a CCAAT/enhancer binding protein beta (C/EBPβ) responsive transcriptional enhancer.ResultsUsing electrophoretic mobility shift assay we demonstrated that, similar to human SPP1, the 3' terminal end of the first intron of porcine SPP1 harbors a C/EBPβ binding site and showed that this binding site is negatively affected by the mutant G allele. Genotyping of 48 fetuses per breed revealed that the G allele segregated exclusively in Duroc fetuses with a frequency of 57 percent. Using real-time quantitative PCR we showed that, consistent with its negative effect on a transcriptional enhancer element, the G allele tends to decrease mRNA abundance of SPP1 in the fetal musculus longissimus dorsi (~1.3 fold; P ≥ 0.1).Moreover, we showed that the SNP g.3836A>G leads to ubiquitous aberrant splicing of the first intron by generating a de novo and activating a cryptic splice acceptor site. Aberrantly spliced transcripts comprise about half of the SPP1 messages expressed by the G allele. Both aberrant splice variants differ from the native transcript by insertions in the leader sequences which do not change the reading frame of SPP1.ConclusionAt the 3' terminal end of the first intron of the porcine SPP1 we identified a unique, dually functional SNP g.3836A>G. This SNP affects the function of the SPP1 gene at the DNA level by affecting a C/EBPβ binding site and at the RNA level by activating aberrant splicing of the first intron, and thus represents an interesting DNA-marker to study phenotypic effects of SPP1 DNA-variation.

The RNA-binding Motif Protein 15B (RBM15B/OTT3) Acts as Cofactor of the Nuclear Export Receptor NXF1

The human SPEN family proteins SHARP, RBM15/OTT1, and RBM15B/OTT3 share the structural domain architecture but show distinct functional properties. Here, we examined the function of OTT3 and compared it with its paralogues RBM15 and SHARP. We found that OTT3, like RBM15, has post-transcriptional regulatory activity, whereas SHARP does not, supporting a divergent role of RBM15 and OTT3. OTT3 shares with RBM15 the association with the splicing factor compartment and the nuclear envelope as well as the binding to mRNA export factors NXF1 and Aly/REF. Mutational analysis revealed direct interaction of OTT3 and RBM15 with NXF1 via their C-terminal regions. Biochemical and subcellular localization studies showed that OTT3 and RBM15 also interact with each other in vivo, further supporting a shared function. Genetic knockdown of RBM15 in mouse is embryonically lethal, indicating that OTT3 cannot compensate for the RBM15 loss, which supports the notion that these proteins, in addition to sharing similar activities, likely have distinct biological roles.

A new mutation in COG7 extends the spectrum of COG subunit deficiencies

We describe a patient homozygous for a novel mutation in COG7, coding for one of the subunits of the Conserved Oligomeric Golgi complex, involved in retrograde vesicular trafficking. His brother showed a similar clinical syndrome and glycosylation defect but no DNA could be obtained from this patient. This mutation, c.170-7A > G, activates a cryptic splice acceptor and leads to the insertion of 2 amino acids at protein level (p.56–57insAT). The insertion disturbs the structure and function of the Conserved Oligomeric Golgi complex. In comparison to the previously described patients with a different COG7 mutation, intrauterine growth retardation and dysmorphic features were absent and there was a longer survival.

Neurodegenerative lysosomal storage disease in European Burmese cats with hexosaminidase β-subunit deficiency

GM2 gangliosidosis is a fatal, progressive neuronopathic lysosomal storage disease resulting from a deficiency of β- N-acetylhexosaminidase (EC 3.2.1.52) activity. GM2 gangliosidosis occurs with varying degrees of severity in humans and in a variety of animals, including cats. In the current research, European Burmese cats presented with clinical neurological signs and histopathological features typical of a lysosomal storage disease. Thin layer chromatography revealed substantial storage of GM2 ganglioside in brain tissue of affected cats, and assays with a synthetic fluorogenic substrate confirmed the absence of hexosaminidase activity. When the hexosaminidase β-subunit cDNA was sequenced from affected cats, a 91 base pair deletion constituting the entirety of exon 12 was documented. Subsequent sequencing of introns 11 and 12 revealed a 15 base pair deletion at the 3′ end of intron 11 that included the preferred splice acceptor site, generating two minor transcripts from cryptic splice acceptor sites in affected Burmese cats. In the cerebral cortex of affected cats, hexosaminidase β-subunit mRNA levels were approximately 1.5 times higher than normal ( P < 0.001), while β-subunit protein levels were substantially reduced on Western blots.