pre-mRNA Levels Research Articles

Rapid muscle atrophy response to unloading: pretranslational processes involving MHC and actin

Skeletal muscles, especially weight-bearing muscles, are very sensitive to changes in loading state. The aim of this paper was to characterize the dynamic changes in the unloaded soleus muscle in vivo following a short bout of hindlimb suspension (HS), testing the hypothesis that transcriptional events respond early to the atrophic stimulus. In fact, we observed that after only 1 day of HS, primary transcript levels of skeletal alpha-actin and type I myosin heavy chain (MHC) genes were significantly reduced by more than 50% compared with ground control levels. The degree of the decline for the mRNA expression of actin and type I MHC lagged behind that of the pre-mRNA levels after 1 day of HS, but by 2 and 7 days of HS, large decreases were observed. Although the faster MHC isoforms, IIx and IIb, began to be expressed in soleus after 1 day of HS, a relatively significant shift in mRNA expression from the slow MHC isoform type I toward these fast MHC isoforms did not emerge until 7 days of HS. One day of HS was sufficient to show significant decreases in mRNA levels of putative signaling factors serum response factor (SRF), suppressor of cytokine signaling-3 (SOCS3), and striated muscle activator of Rho signaling (STARS), although transcription factors yin-yang-1 (YY1) and transcriptional enhancing factor-1 (TEF-1) were not significantly affected by HS. The protein levels of actin and type I MHC were significantly decreased after 2 days of HS, and SRF protein was significantly decreased after 7 days HS. Our results show that after only 1 day of unloading, pre-mRNA and mRNA expression of muscle proteins and muscle-specific signaling factors are significantly reduced, suggesting that the downregulation of the synthesis side of the protein balance equation that occurs in atrophying muscle is initiated rapidly.

Cytoprotective Nrf2 Pathway Is Induced In Chronically Txnrd 1-Deficient Hepatocytes

BackgroundMetabolically active cells require robust mechanisms to combat oxidative stress. The cytoplasmic thioredoxin reductase/thioredoxin (Txnrd1/Txn1) system maintains reduced protein dithiols and provides electrons to some cellular reductases, including peroxiredoxins.Principal FindingsHere we generated mice in which the txnrd1 gene, encoding Txnrd1, was specifically disrupted in all parenchymal hepatocytes. Txnrd1-deficient livers exhibited a transcriptome response in which 56 mRNAs were induced and 12 were repressed. Based on the global hybridization profile, this represented only 0.3% of the liver transcriptome. Since most liver mRNAs were unaffected, compensatory responses were evidently effective. Nuclear pre-mRNA levels indicated the response was transcriptional. Twenty-one of the induced genes contained known antioxidant response elements (AREs), which are binding sites for the oxidative and chemical stress-induced transcription factor Nrf2. Txnrd1-deficient livers showed increased accumulation of nuclear Nrf2 protein and chromatin immunoprecipitation on the endogenous nqo1 and aox1 promoters in fibroblasts indicated that Txnrd1 ablation triggered in vivo assembly of Nrf2 on each.ConclusionsChronic deletion of Txnrd1 results in induction of the Nrf2 pathway, which contributes to an effective compensatory response.

Breeding and Field Performance of Novel Soft and Waxy Transgenic Rice Lines Without Selectable Marker

Amylose content is one of the important factors affecting rice cooking and eating quality, and is controlled by Waxy ( Wx) gene encoding a granule-bound starch synthase. High amylose level is usually associated with poor cooking and eating quality. Several homozygous transgenic lines with soft or waxy grain property were selected from the offspring of selectable marker-free (SMF) transformants containing the antisense Wx gene. The results from Northern blotting analysis indicated that the levels of both Wx mature mRNA and pre-mRNA were reduced in the endosperm of different transgenic lines. The Wx protein content in mature seeds of the transgenic lines also decreased. The amylose contents of 2 transgenic lines were reduced to that of the soft rice (about 10%), and one of them was even reduced to the level of waxy rice (lower than 2%). The gel consistency of these transgenic lines was softer than that of the wild type. The variance of gelatinization temperature between the transgenic lines and the wild type was significant. Most of the main agronomic traits of these transgenic lines were similar to those of the wild type. Therefore, the 3 SMF transgenic homozygous lines with improved quality were successfully developed.

First-in-human clinical, pharmacokinetic (PK), and pharmacodynamic (PD) phase I study of the first-in-class spliceosome inhibitor E7107 administered IV (bolus) on days 1, 8, and 15 every 28 days to patients with solid tumors

3508 Background: E7107 is a potent first-in-class inhibitor of the spliceosome, most likely by interacting with spliceosome-associated protein-130 (SAP 130). Splicing removes intron sequences from pre-mRNA and exons are fused resulting in the formation of mature mRNA. Alternative splicing frequently encodes oncoproteins. E7107 interferes the maturation process of pre-mRNA to mRNA, with consequent changes in protein expression profiles. Methods: Objectives of this study were to explore (1) tolerability and safety profile, (2) PK, (3) PD effects on pre-mRNA splicing, and (4) anti tumor activity of E7107 administered as bolus infusion on days 1, 8, 15 of a 28-day schedule Results: 36 patients (21M/15F, median age 61yrs (45–79)) received E7107 doses of 0.6 mg/m2 (n=4), 0.9 mg/m2 (n=3), 1.3 mg/m2 (n=3), 2.0 mg/m2 (n=3), 3.0 mg/m2 (n=4), 4.5 mg/m2 (n=3) and 4.0 mg/m2 (n=16). At 4.5 mg/m2 two episodes of DLT (grade 3 and 4 diarrhea) and at 4.0 mg/m2 one episode of DLT (a combination of grade 3 nausea, vomiting and abdominal cramps) were observed. Other frequently occurring side effects were mainly gastrointestinal. The maximum tolerable dose (MTD) is 4.0 mg/m2. No complete or partial responses were observed. Pharmacokinetic analysis revealed large volume of distribution (Vss: 279 to 1369 L), high systemic clearance (CL: 111 to 253 L/hr), and moderate elimination half-life (t1/2: 5.3 to 15.1 hr). Systemic exposure on Days 1 and 15 (Cmax, AUC0-∞) increased in a dose-dependent manner. At the MTD, mRNA levels of selected target genes (TRAPPC4, SLC25A19, GTF2H1), monitored in PBMC's, showed a 15–25-fold decrease, whereas unspliced pre-mRNA levels of DNAJB1 and EIF4A1 showed a 10–25-fold increase. Notably, at days 1 and 15, modulations generally peaked at 2–6 hr after end of the infusion and almost completely recovered to base-line levels at 24–48 hr. Conclusions: The MTD for E7107 using this schedule is 4.0 mg/m2. PK is dose-dependent and reproducible within subjects. PD analysis revealed dose-dependent reversible inhibition of pre-mRNA processing of target genes, confirming proof-of-principle activity of E7107. [Table: see text]

Mechanism for decrease of glucocorticoid receptor mRNA in human hepatic cancer cell line SMMC-7721 during heat shock

Objective:To investigate the mechanism for decrease of glucocorticoid receptor mRNA(GR mRNA)during heat shock response.Methods:The changes of GR mRNA level in SMMC-7721 cells were examined by semi-quantitative RT-PCR during heat shock response.We designed 2 RNA fragments paired with GR gene intron E and cross exon 3,4,and 5,which were used as probes for in situ hybridization with the sequences in SMMC-7721 cells at different periods after heat shock.The result was subjected to confocol microscope observation and computer image analysis.The transcription,degradation,and splicing of GR mRNA were investigated.Results:RT-PCR showed that the GR mRNA level was decreased during heat shock response.In situ hybridization revealed that both GR mRNA and GR pre-mRNA levels were lower in the heat shock group than in the non-heat shock group(P0.05).GR pre-mRNA level was higher in cells treated with actinomycine D before heat shock than in cells only treated with actinomycine D,while the GR mRNA level was lower in cells treated with actinomycine D before heat shock(P0.05).Conclusion:During heat shock response,GR mRNA transcription is suppressed,the splicing of GR pre-mRNA is suppressed,and GR mRNA degradation is accelerated.

A Generalizable Strategy for Imaging pre-mRNA Levels in Living Subjects Using Spliceosome-Mediated RNA Trans-Splicing

Molecular imaging of gene expression is currently hindered by the lack of a generalizable platform for probe design. For any gene of interest, a probe that targets protein levels must often be generated empirically. Targeting gene expression at the level of mRNA, however, would allow probes to be built on the basis of sequence information alone. Presented here is a class of generalizable probes that can image pre-mRNA in a sequence-specific manner, using signal amplification and a facile method of delivery. Pre-trans-splicing molecules (PTMs) were engineered to capitalize on the phenomenon of spliceosome-mediated RNA trans-splicing. Using a modular binding domain that confers specificity by base-pair complementarity to the target pre-mRNA, PTMs were designed to target a chimeric target mini gene and trans-splice the Renilla luciferase gene onto the end of the target. PTMs and target genes were transfected in cell culture and assessed by luciferase assay, reverse-transcriptase polymerase chain reaction, Western blot, and rapid analysis of 5' cDNA ends. PTMs and target genes were also assessed in vivo by hydrodynamic delivery in mice. Efficiency and specificity of the trans-splicing reaction were found to vary depending on the binding domain length and structure. Specific trans-splicing was observed in living animals (P = 0.0862, Kruskal-Wallis test). Described here is a model system used to demonstrate the feasibility of spliceosome-mediated RNA trans-splicing for imaging gene expression at the level of pre-mRNA using optical imaging techniques in living animals. The experiments reported here show proof of principle for a generalizable imaging probe against RNA that can amplify signal on detection and be delivered using existing gene delivery methodology.

Developmental changes in expression and self-editing of adenosine deaminase type 2 pre-mRNA and mRNA in rat brain and cultured cortical neurons

Adenosine deaminase-1 and -2 (ADAR-1 and -2) are double-stranded RNA-specific enzymes involved in the editing of genes including serotonin 2C receptor (5-HT2CR) mRNA and ADAR-2 pre-mRNA. We reported that the editing efficacy of 5-HT2CR mRNA altered during brain development in rats. The present study aimed to clarify if changes in the expression of ADAR genes and the editing of ADAR-2 pre-mRNA occur during development. The expression level of ADAR-1 mRNA was constant during development, whereas the expression levels of ADAR-2 mRNA and ADAR-2 pre-mRNA markedly increased during development. ADAR-2 pre-mRNA possesses six editing sites. Editing of these sites did not occur during the embryonic period; however, the number of edited sites and the editing frequency at these sites increased after birth and cultivation period. These results suggest that the increases in ADAR-2 pre-mRNA editing and mRNA expression of the enzyme may play a role in development. We also discuss the relationship between 5-HT2CR mRNA editing and the expression/RNA editing of ADAR-1 and ADAR-2 mRNA.

Intergenic bidirectional promoter and cooperative regulation of the IIx and IIb MHC genes in fast skeletal muscle

This study investigated the dynamic regulation of IIx-IIb MHC genes in the fast white medial gastrocnemius (WMG) muscle in response to intermittent resistance exercise training (RE), a model associated with a rapid shift from IIb to IIx expression (11). We investigated the effect of 4 days of RE on the transcriptional activity across the skeletal MHC gene locus in the WMG in female Sprague-Dawley rats. Our results show that RE resulted in significant shifts from IIb to IIx observed at both the pre-mRNA and mRNA levels. An antisense RNA (xII NAT) was detected in the intergenic (IG) region between IIx and IIb, extending across the entire IIx gene and into its promoter. The expression of the xII NAT was positively correlated with IIb pre-mRNA (R = +0.8), and negatively correlated with IIx pre-mRNA (R = -0.8). Transcription mapping of the IIx-IIb IG region revealed the generation of sense IIb and xII NATs from a single promoter region. This bidirectional promoter is highly conserved among species and contains several regulatory elements that may be implicated in its regulation. These results suggest that the IIx and the IIb genes are physically and functionally linked via the bidirectional promoter. In order for the IIx MHC gene to be regulated, a feedback mechanism from the IG xII NAT is needed. In conclusion, the IG bidirectional promoter generating antisense RNA appears to be essential for the coordinated regulation of the skeletal muscle MHC genes during dynamic phenotype shifts.

A Novel Bone Morphogenetic Protein Signaling in Heterotypic Cell Interactions in Prostate Cancer

We examined the effect of the extracellular bone morphogenetic protein (BMP) 2 and 7, which are up-regulated in the prostate adenocarcinomas of the conditional Pten deletion mouse model, on primary cultures of cancer-associated fibroblasts (CAF) derived from these tumors. In the CAF, we show that BMP2 or BMP7, but not transforming growth factor beta-1, can strikingly stimulate secretion of stromal cell-derived factor-1 (SDF-1), also known as CXCL12. The CAF cells express type I and type II BMP receptors as well as the receptor for SDF-1, CXCR4. SDF-1 activation is associated with BMP-induced Smad phosphorylation, and the stimulatory effect is blocked by BMP antagonist, noggin. The findings that BMP treatment can increase SDF-1 pre-mRNA levels in a time-dependent manner and actinomycin D treatment can abolish stimulatory effect of BMP suggest a transcriptional modulation of SDF-1 by BMP signaling. Using a human microvascular endothelial cell line, we show that SDF-1 present in the conditioned medium from the stimulated CAF can significantly induce tube formation, an effect relating to angiogenic function. Furthermore, we found that BMP2 can also protect the CAF from serum starvation-induced apoptosis independent of SDF-1, implying that BMP may induce other factors to sustain the survival of these cells. In short, this report establishes a novel BMP-SDF-1 axis in the prostate tumor along with a new prosurvival effect of BMP that when considered together with our previously described oncogenic properties of BMP indicate a circuitry for heterotypic cell interactions potentially critical in prostate cancer.

Intergenic transcription and developmental regulation of cardiac myosin heavy chain genes

Cardiac myosin heavy chain (MHC) gene expression undergoes a rapid transition from beta- to alpha-MHC during early rodent neonatal development (0-21 days of age). Thyroid hormone (3,5,3'-triiodothyronine, T(3)) is a major player in this developmental shift; however, the exact mechanism underlying this transition is poorly understood. The goal of this study was to conduct a more thorough analysis of transcriptional activity of the cardiac MHC gene locus during the early postnatal period in the rodent, in order to gain further insight on the regulation of cardiac MHC genes. We analyzed the expression of alpha- and beta-MHC at protein, mRNA, and pre-mRNA levels at birth and 7, 10, 15, and 21 days after birth in euthyroid and hypothyroid rodents. Using novel technology, we also analyzed RNA expression across the cardiac gene locus, and we discovered that the intergenic (IG) region between the two cardiac genes possesses bidirectional transcriptional activity. This IG transcription results in an antisense RNA product as described previously, which is thought to exert an inhibitory effect on beta-MHC gene transcription. On the second half of the IG region, sense transcription occurs, resulting in expression of a sense IG RNA that merges with the alpha-MHC pre-mRNA. This sense IG RNA transcription was detected in the alpha-MHC gene promoter, approximately -1.8 kb relative to the alpha-MHC transcription start site. Both sense and antisense IG RNAs were developmentally regulated and responsive to a hypothyroid state (11, 14). This novel observation provides more complexity to the cooperative regulation of the two genes, suggesting the involvement of epigenetic processes in the regulation of cardiac MHC gene locus.

Androgen receptor‐mediated repression of novel target genes

The androgen receptor (AR) plays a pivotal role in prostate cancer (PCa) initiation and progression. To date, studies have focused disproportionately on androgen-stimulated genes such as prostate-specific antigen (PSA), while repressed genes have gained little attention, even though they too may be involved in regulating cell growth, differentiation, and apoptosis. ChIP Display was used to identify putative AR target genes in the ablation-resistant human PCa cell line, C4-2B. Quantitative real-time reverse transcription-PCR analysis was used to measure gene expression in cells subjected to dihydrotestosterone (DHT) timecourse and dose-response, as well as AR knock-down and bicalutamide-treatments. We report on three genes, KIAA1217, CHRM1, and WBSCR28, which were newly identified in a screen for AR-occupied regions in C4-2B PCa cells, and which were repressed by treatment with DHT. AR knock-down resulted in increased KIAA1217, CHRM1, and WBSCR28 mRNA, indicating that, like PSA stimulation, AR represses these three genes even in the absence of added ligand. DHT decreased KIAA1217 and CHRM1 pre-mRNA levels, suggesting AR-mediated transcriptional inhibition. Cycloheximide attenuated DHT-mediated repression of CHRM1, suggesting the requirement of new protein synthesis. Furthermore, bicalutamide treatment did not mimic, but rather antagonized DHT-mediated KIAA1217 repression. Unlike the handful of androgen-repressed genes studied thus far, AR occupancy at KIAA1217, CHRM1, and WBSCR28 was mapped outside their respective 5'-promoter regions. Many more genes likely share AR-mediated gene repression through distal regulatory elements. Further study of such targets and their transcriptional regulation may help explain the receptor's tumorigenicity in PCa.

ROS mediate the hypoxic repression of the hepcidin gene by inhibiting C/EBPα and STAT-3

Hepcidin, a liver peptide, systemically inhibits iron utilization and is downregulated under hypoxic conditions. However, little is known about the mechanism underlying the hypoxic suppression of hepcidin. Here, we tested the possibility that HIF-1 and ROS are involved in hepcidin regulation. Hepcidin mRNA, pre-mRNA, and protein levels were reduced in mouse livers and in HepG2 cells after hypoxic incubation, and HIF-1 overexpression and knock-down studies showed that hepcidin regulation is independent of HIF-1. On the other hand, ROS levels were significantly elevated in hypoxic HepG2 cells, and anti-oxidants prevented the hypoxic down-regulation of hepcidin. Conversely, a prooxidant, H 2O 2, suppressed hepcidin expression in these cells even in normoxia. Of the various transcription factors examined, C/EBPα and STAT-3 were found to dissociate from hepcidin promoter under hypoxia, but to become fully engaged after anti-oxidant treatment. These results suggest that ROS repress the hepcidin gene by preventing C/EBPα and STAT-3 binding to hepcidin promoter during hypoxia.

Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes

Little is known about the higher-order structure of prespliceosomal A complexes, in which pairing of the pre-mRNA's splice sites occurs. Here, human A complexes were isolated under physiological conditions by double-affinity selection. Purified complexes contained stoichiometric amounts of U1, U2 and pre-mRNA, and crosslinking studies indicated that these form concomitant base pairing interactions with one another. A complexes contained nearly all U1 and U2 proteins plus approximately 50 non-snRNP proteins. Unexpectedly, proteins of the hPrp19/CDC5 complex were also detected, even when A complexes were formed in the absence of U4/U6 snRNPs, demonstrating that they associate independent of the tri-snRNP. Double-affinity purification yielded structurally homogeneous A complexes as evidenced by electron microscopy, and allowed for the first time the generation of a three-dimensional structure. A complexes possess an asymmetric shape (approximately 260 x 200 x 195 angstroms) and contain a main body with various protruding elements, including a head-like domain and foot-like protrusions. Complexes isolated here are well suited for in vitro assembly studies to determine factor requirements for the A to B complex transition.

Activity of the β-myosin heavy chain antisense promoter responds to diabetes and hypothyroidism

Two genes encoding cardiac myosin heavy chain (MHC) isoforms, beta and alpha, are arranged in tandem 4.5 kb apart. We examined pre-mRNA and mature mRNA levels of beta and alpha genes in control, diabetic (streptozotocin), hypothyroid (propylthiouracil), and hyperthyroid rat hearts and analyzed the naturally occurring antisense (AS) beta RNA species that starts in the middle of the 4.5-kb intergenic region and extends upstream to the beta-gene promoter. The beta and alpha genes are expressed antithetically in control, diabetic, hypothyroid, and hyperthyroid hearts. Expression of AS beta-RNA was positively correlated with alpha-mRNA and negatively correlated with sense beta mRNA. These results support the novel idea of common promoter-regulatory elements situated in the intergenic region that likely control transcription of both sense alpha and AS beta genes and that AS beta transcription negatively regulates beta-MHC gene expression. To test whether an intergenic promoter drives transcription of AS beta RNA, a 1340-bp sequence of the intergenic region was inserted into a luciferase plasmid in the 3'-to-5' AS direction and was injected into rat ventricle. This promoter was activated in control heart and decreased greatly in response to propylthiouracil and streptozotocin and increased in hyperthyroid rats, similar in pattern to the endogenous AS beta RNA. When a putative retinoic acid receptor (RAR) site (a known thyroid hormone receptor cofactor) in this promoter was mutated, the reporter activity was almost abolished in control, propylthiouracil, and streptozotocin hearts. We conclude that there is an intergenic promoter that is active in the AS direction and that the putative RAR element is a vital regulatory site.

A missense mutation in the chloride/proton ClC-5 antiporter gene results in increased expression of an alternative mRNA form that lacks exons 10 and 11. Identification of seven new CLCN5 mutations in patients with Dent’s disease

Mutations in the voltage-gated chloride/proton antiporter ClC-5 gene, CLCN5, are associated with Dent's disease, an X-linked renal tubulopathy. Our interest is to identify and characterize disease-causing CLCN5 mutations, especially those that alter the splicing of the pre-mRNA. We analyzed the CLCN5 gene from nine unrelated Spanish Dent's disease patients and their relatives by DNA sequencing. Pre-mRNA splicing analysis was performed by RT-PCR. Seven new mutations were identified, consisting of three missense mutations (C219R, F273L, and W547G), one splice-site mutation (IVS-2A > G), one deletion (976delG), and two non-sense mutations (Y140X and W314X). We found that missense mutation W547G also led to increased expression of a new alternative isoform lacking exons 10 and 11 that was expressed in several human tissues. In addition, we describe another novel CLCN5 splicing variant lacking exon 11 alone, which was expressed only in human skeletal muscle. We conclude that missense mutation W547G can also alter the expression levels of a CLCN5 mRNA splicing variant. This type of mutation has not been previously described in the CLCN5 gene. Our results support the importance of a routine analysis at the pre-mRNA level of mutations that are commonly assumed to cause single amino acids alterations.

Hu Proteins Regulate Polyadenylation by Blocking Sites Containing U-rich Sequences

A recent genome-wide bioinformatic analysis indicated that 54% of human genes undergo alternative polyadenylation. Although it is clear that differential selection of poly(A) sites can alter gene expression, resulting in significant biological consequences, the mechanisms that regulate polyadenylation are poorly understood. Here we report that the neuron-specific members of a family of RNA-binding proteins, Hu proteins, known to regulate mRNA stability and translation in the cytoplasm, play an important role in polyadenylation regulation. Hu proteins are homologs of the Drosophila embryonic lethal abnormal visual protein and contain three RNA recognition motifs. Using an in vitro polyadenylation assay with HeLa cell nuclear extract and recombinant Hu proteins, we have shown that Hu proteins selectively block both cleavage and poly(A) addition at sites containing U-rich sequences. Hu proteins have no effect on poly(A) sites that do not contain U-rich sequences or sites in which the U-rich sequences are mutated. All three RNA recognition motifs of Hu proteins are required for this activity. Overexpression of HuR in HeLa cells also blocks polyadenylation at a poly(A) signal that contains U-rich sequences. Hu proteins block the interaction between the polyadenylation cleavage stimulation factor 64-kDa subunit and RNA most likely through direct interaction with poly(A) cleavage stimulation factor 64-kDa subunit and cleavage and polyadenylation specificity factor 160-kDa subunit. These studies identify a novel group of mammalian polyadenylation regulators. Furthermore, they define a previously unknown nuclear function of Hu proteins.

Transcriptional activity of the vasopressin gene measured by onestep quantitative RT‐PCR.

Traditional methods for detecting transcriptional activity of neuropeptide genes include in situ hybridization histochemistry and classical transcriptional run-on assays for examining pre-mRNA, and northern blots, ribonuclease protection assays and gene chip arrays for mRNA. These methods are labor intensive, costly, can require much tissue and are often too insensitive to detect small changes in low abundant transcripts. Here we describe a method for measuring changes in vasopressin (VP) pre-mRNA using one-step quantitative reverse-transcription PCR (qRT-PCR), with intron-specific primers. Supraoptic nuclei were collected 30 minutes following an intraperitoneal injection of iso- or hypertonic saline. Rats injected with 900 mOsm saline expressed an increase in VP pre-mRNA (173% above 290 mOsm isotonic controls, n=12). Injections of 2M NaCl, the strongest stimulus tested, resulted in pre-mRNA levels 204% above control levels, n=6. These results agree with earlier studies and suggest qRT-PCR can be used to measure transcription of VP and other neuropeptides in both quantitative and qualitative studies.

Repair of von Willebrand Factor (vWF) Gene Defect by Targeted RNA Trans-Splicing.

RNA repair based on spliceosome-mediated RNA trans-splicing allows for direct repair or reprogramming of endogenous genetic defects at the pre-mRNA level. Trans-splicing has significant therapeutic and safety advantages over traditional gene therapy including the ability to correct defects in large gene. Progress has been made during the past few years toward developing trans-splicing for the treatment of variety of hematological diseases. von Willebrand disease (vWD) is the most common inherited bleeding disorder. Due to the large size of von Willebrand factor (vWF) gene (approximately 180kb, cDNA 9kb) and packaging size restrictions of most gene delivery vectors, methods for genetic correction of vWD are limited. We developed a spliceosome-mediated RNA trans-splicing strategy to correct a murine model of vWD. The vWD knockout mice were created with a neomycin gene insertion at vWF gene intron 5. We designed pre-mRNA trans-splicing molecules (PTMs) that bind to intron 6 and carry the normal vWD gene sequence from exon 1–5. The 5′ trans-splicing of the pre-mRNA generated from the PTM is designed to hybridize with the endogenous vWD pre-mRNA at intron 6. Before testing in animals, we developed several PTMs with different binding domains to murine vWF intron 6. These PTMs carry a GFP hemi-reporter that includes the 5′ half of the GFP coding region followed by a spacer sequence and a binding domain sequence that is complementary to vWF gene intron-6. The target for these PTMs was a murine vWD mini-gene expressing pre-mRNA containing vWF gene intron-6 followed by sequences encoding 3′GFP. The expression of both PTMs and mini-gene target were driven by CMV IE promoter/enhancer. Both PTM and mini-gene target were co-transfected into 293 cells. Trans-splicing in this in vitro model produces an intact GFP gene easily measured by fluorescence. Two of the 15 PTMs restored full-length GFP mRNA as measured RT-PCR, FACS and sequence analysis of splice junction. No positive results were obtained in controls (either PTM or target alone). These results encouraged us to design murine vWF gene specific PTMs that encode for vWF exons 1–6. A new vWF mini-gene target was also generated by replacing 3′GFP coding region of hemi-reporter mini-gene with murine vWF coding sequences from exon 7–51. To assess specific trans-splicing between 5′ splice donor of vWF PTM and 3′ splice acceptor of vWF mini-gene target, both PTMs and target were co-delivered into 293 cells by transient transfection. Total RNA of transfected cells was isolated and a 411bp PCR fragment representing specific exon 6 and 7 splice junction trans-spliced mRNA was detected by RT-PCR and confirmed by sequencing. The level of trans-splicing was estimated to be at 0.1–1%. Our results demonstrate the repair of defective murine vWF mini-gene pre-mRNA was achieved in our in vitro model established to mimic the genetic defect in vWF−/− KO mice. The use of 2 selected PTMs in vWD KO mice is now ongoing. The fact that only a few rational designed PTMs (2 out 15) were functional also suggested that a systemic and versatile approach is needed in order to identify the most efficient PTM with high specificity. Such an effort is on going in our lab. Overall, These data and our previous results for identifying optimal PTM provide strong evidence to support the use of trans-splicing to correct vWF pre-mRNA in vWF−/− KO mice.

The conserved protein kinase-A target motif in synapsin of Drosophilais effectively modified by pre-mRNA editing

BackgroundSynapsins are abundant synaptic vesicle associated phosphoproteins that are involved in the fine regulation of neurotransmitter release. The Drosophila member of this protein family contains three conserved domains (A, C, and E) and is expressed in most or all synaptic terminals. Similar to mouse mutants, synapsin knock-out flies show no obvious structural defects but are disturbed in complex behaviour, notably learning and memory.ResultsWe demonstrate that the N-terminal phosphorylation consensus motif RRxS that is conserved in all synapsins investigated so far, is modified in Drosophila by pre-mRNA editing. In mammals this motif represents the target site P1 of protein kinase A (PKA) and calcium/calmodulin dependent protein kinase I/IV. The result of this editing, by which RRFS is modified to RGFS, can be observed in cDNAs of larvae and adults and in both isolated heads and bodies. It is also seen in several newly collected wild-type strains and thus does not represent an adaptation to laboratory culture conditions. A likely editing site complementary sequence is found in a downstream intron indicating that the synapsin pre-mRNA can form a double-stranded RNA structure that is required for editing by the adenosine deaminase acting on RNA (ADAR) enzyme. A deletion in the Drosophila Adar gene generated by transposon remobilization prevents this modification, proving that the ADAR enzyme is responsible for the pre-mRNA editing described here. We also provide evidence for a likely function of synapsin editing in Drosophila. The N-terminal synapsin undeca-peptide containing the genomic motif (RRFS) represents an excellent substrate for in-vitro phosphorylation by bovine PKA while the edited peptide (RGFS) is not significantly phosphorylated. Thus pre-mRNA editing by ADAR could modulate the function of ubiquitously expressed synapsin in a cell-specific manner during development and adulthood.ConclusionSimilar to several other neuronal proteins of Drosophila, synapsin is modified by ADAR-mediated recoding at the pre-mRNA level. This editing likely reduces or abolishes synapsin phosphorylation by PKA. Since synapsin in Drosophila is required for various forms of behavioural plasticity, it will be fascinating to investigate the effect of this recoding on learning and memory.

Multiexon skipping leading to an artificial DMD protein lacking amino acids from exons 45 through 55 could rescue up to 63% of patients with Duchenne muscular dystrophy

Approximately two-thirds of Duchenne muscular dystrophy (DMD) patients show intragenic deletions ranging from one to several exons of the DMD gene and leading to a premature stop codon. Other deletions that maintain the translational reading frame of the gene result in the milder Becker muscular dystrophy (BMD) form of the disease. Thus the opportunity to transform a DMD phenotype into a BMD phenotype appeared as a new treatment strategy with the development of antisense oligonucleotides technology, which is able to induce an exon skipping at the pre-mRNA level in order to restore an open reading frame. Because the DMD gene contains 79 exons, thousands of potential transcripts could be produced by exon skipping and should be investigated. The conventional approach considers skipping of a single exon. Here we report the comparison of single- and multiple-exon skipping strategies based on bioinformatic analysis. By using the Universal Mutation Database (UMD)-DMD, we predict that an optimal multiexon skipping leading to the del45-55 artificial dystrophin (c.6439_8217del) could transform the DMD phenotype into the asymptomatic or mild BMD phenotype. This multiple-exon skipping could theoretically rescue up to 63% of DMD patients with a deletion, while the optimal monoskipping of exon 51 would rescue only 16% of patients.