Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex-specific RNA splicing patterns

Sex-specific alternative RNA splicing of the doublesex (dsx) pre-mRNA results in sex-specific polypeptides that regulate both male and female somatic sexual differentiation in Drosophila melanogaster. We have molecularly characterized a class of dsx mutations that act in cis to disrupt the regulation of dsx RNA processing, causing the dsx pre-mRNA to be spliced in the male-specific pattern regardless of the chromosomal sex of the fly. These dsx mutations are associated with rearrangements in the female-specific exon just 3' to the female-specific splice acceptor. The mutations do not affect the female-specific splice sites or intron that are identical to wild-type sequences. These results indicate that sequences in the female-specific exon are important for the regulation of sex-specific RNA splicing, perhaps by acting as sites of interaction with trans-acting regulators. Furthermore, the data suggest that female-specific regulation of dsx RNA processing occurs by promoting the usage of the female splice acceptor site, rather than by repressing the usage of the alternative male-specific splice acceptor.     

Suppression of gene expression by targeted disruption of messenger RNA: available options and current strategies

At least three different approaches may be used for gene targeting including: A) gene knockout by homologous recombination; B) employment of synthetic oligonucleotides capable of hybridizing with DNA or RNA, and C) use of polyamides and other natural DNA-bonding molecules called lexitropsins. Targeting mRNA is attractive because mRNA is more accessible than the corresponding gene. Three basic strategies have emerged for this purpose, the most familiar being to introduce antisense nucleic acids into a cell in the hopes that they will form Watson-Crick base pairs with the targeted gene's mRNA. Duplexed mRNA cannot be translated, and almost certainly initiates processes which lead to its destruction. The antisense nucleic acid can take the form of RNA expressed from a vector which has been transfected into the cell, or take the form of a DNA or RNA oligonucleotide which can be introduced into cells through a variety of means. DNA and RNA oligonucleotides can be modified for stability as well as engineered to contain inherent cleaving activity. It has also been proven that because RNA and DNA are very similar chemical compounds, DNA molecules with enzymatic activity could also be developed. This assumption proved correct and led to the development of a "general-purpose" RNA-cleaving DNA enzyme. The attraction of DNAzymes over ribozymes is that they are very inexpensive to make and that because they are composed of DNA and not RNA, they are inherently more stable than ribozymes. Although mRNA targeting is impeccable in theory, many additional considerations must be taken into account in applying these strategies in living cells including mRNA site selection, drug delivery and intracellular localization of the antisense agent. Nevertheless, the ongoing revolution in cell and molecular biology, combined with advances in the emerging disciplines of genomics and informatics, has made the concept of nontoxic, cancer-specific therapies more viable then ever and continues to drive interest in this field.     

Preclinical development strategies for novel gene therapeutic products.

With over 220 investigational new drug applications currently active, gene therapy represents one of the fastest growing areas in biotherapeutic research. Initially conceived for replacing defective genes in diseases such as cystic fibrosis or inborn errors of metabolism with genes encoding the normal, or wild-type, gene product, gene therapy has expanded into other novel applications such as treatment of cancer or cardiovascular disease, where the risk:benefit profiles may be more acceptable in relation to the severity of the disease. Different types of vectors, including modified retroviruses, adenoviruses, adenovirus-associated viruses, and herpesviruses and plasmid DNA, are used to transfer foreign genetic material into patients' cells or tissues. Developing a toxicology program to determine the safety of these agents, therefore, requires a modified approach that encompasses the pharmacology and toxicity of both the gene product itself and the vector system used for delivery in the context of the application for the clinical trial. In general, the issues involved in designing and developing appropriate preclinical testing to determine the safety of these products are similar to those encountered for other recombinant molecules, including protein biotherapeutics. Limitations to some of the typical toxicology studies conducted for a traditional drug development program may exist for these agents, and nontraditional approaches may be required to demonstrate their safety. Many factors may affect the safety and clinical activity of these agents, including the route, frequency, and duration of exposure and the type of vector employed. Other safety considerations include quantitation of the duration and degree of expression of the vector in target and other tissues, the effects of gene expression on organ pathology and/or histology, evaluation of trafficking of gene-transduced cells or vector after injection, and interactions of the host immune system with the transduced cell population. Because of the unique concerns regarding each of these therapies, the Center for Biologics Evaluation and Research encourages sponsors to obtain toxicity data whenever possible while evaluating the pharmacologic activity of the vector in a species or animal model relevant to their clinical indication. Sponsors are encouraged to discuss preclinical study design and results with the Center during product development to facilitate early identification of safety concerns prior to entry of these novel agents into the clinical setting and to ensure an uninterrupted course of development while addressing issues required for licensure.     

A cold-sensitive mRNA splicing mutant is a member of the RNA helicase gene family

We have isolated a cold-sensitive mutant of Saccharomyces cerevisiae in which the first step of mRNA splicing is inhibited. The growth and splicing defects are recessive and cosegregate, thus defining a single essential gene (PRP28). The wild-type PRP28 gene was cloned, and sequence analysis reveals extensive homology to a family of proteins that are thought to function as ATP-dependent RNA helicases. The cold sensitivity is caused by a glycine-to-glutamic acid change in a conserved sequence motif. Interestingly, double mutants containing conditional alleles of PRP28 and PRP24, which encodes a U6 snRNA-binding protein, are inviable. In addition, a suppressor of prp28-1 is a mutant allele of PRP8, which encodes a U5 protein, thus linking PRP28 with U5. These data are consistent with a scenario in which PRP28 acts to unwind the U4/U6 base-pairing interaction in the U4/U6/U5 snRNP, facilitating the first covalent step of splicing.     

The manipulation of brain histamine concentrations failed to modify voluntary alcohol intake by rats

Inflammation Research -     

Dysfunctional gene splicing as a potential contributor to neuropsychiatric disorders

Alternative pre‐mRNA splicing is a major mechanism by which the proteomic diversity of eukaryotic genomes is amplified. Much akin to neuropsychiatric disorders themselves, alternative splicing events can be influenced by genetic, developmental, and environmental factors. Here, we review the evidence that abnormalities of splicing may contribute to the liability toward these disorders. First, we introduce the phenomenon of alternative splicing and describe the processes involved in its regulation. We then review the evidence for specific splicing abnormalities in a wide range of neuropsychiatric disorders, including psychotic disorders (schizophrenia), affective disorders (bipolar disorder and major depressive disorder), suicide, substance abuse disorders (cocaine abuse and alcoholism), and neurodevelopmental disorders (autism). Next, we provide a theoretical reworking of the concept of “gene‐focused” epidemiologic and neurobiologic investigations. Lastly, we suggest potentially fruitful lines for future research that should illuminate the nature, extent, causes, and consequences of alternative splicing abnormalities in neuropsychiatric disorders. © 2011 Wiley‐Liss, Inc.     

Genome replication/expression strategies of positive-strand RNA viruses: A simple version of a combinatorial classification and prediction of new strategies

A combinatorial approach to the classification of replication/expression strategies of positive-strand RNA virus genomes is suggested. Eighteen genome strategies defined as combinations of distinct modes of expression and replication are briefly characterized, 10 of which have been actually found in diverse virus groups. The chances for realization of the remaining eight strategies are evaluated. It is demonstrated that positive-strand RNA virus genome strategies are not necessarily monophyletic characters and could, in some cases, evolve convergently.     

Identification of a splicing enhancer in MLH1 using COMPARE, a new assay for determination of relative RNA splicing efficiencies

Exonic splicing enhancers (ESEs) are sequences that facilitate recognition of splice sites and prevent exon-skipping. Because ESEs are often embedded within protein-coding sequences, alterations in them can also often be interpreted as nonsense, missense or silent mutations. To correctly interpret exonic mutations and their roles in diseases, it is important to develop strategies that identify ESE mutations. Potential ESEs can be found computationally in many exons but it has proven difficult to predict whether a given mutation will have effects on splicing based on sequence alone. Here, we describe a flexible in vitro method that can be used to functionally compare the effects of multiple sequence variants on ESE activity in a single in vitro splicing reaction. We have applied this method in parallel with conventional splicing assays to test for a splicing enhancer in exon 17 of the human MLH1 gene. Point mutations associated with hereditary non-polyposis colorectal cancer (HNPCC) have previously been found to correlate with exon-skipping in both lymphocytes and tumors from patients. We show that sequences from this exon can replace an ESE from the mouse IgM gene to support RNA splicing in HeLa nuclear extracts. ESE activity was reduced by HNPCC point mutations in codon 659, indicating that their primary effect is on splicing. Surprisingly, the strongest enhancer function mapped to a different region of the exon upstream of this codon. Together, our results indicate that HNPCC point mutations in codon 659 affect an auxillary element that augments the enhancer function to ensure exon inclusion.