Alternative Transcript Processing Research Articles

Isoform-specific NF1 mRNA levels correlate with disease severity in Neurofibromatosis type 1

BackgroundNeurofibromatosis type 1 (NF1) is characterized by an extreme clinical variability both within and between families that cannot be explained solely by the nature of the pathogenic NF1 gene mutations. A proposed model hypothesizes that variation in the levels of protein isoforms generated via alternative transcript processing acts as modifier and contributes to phenotypic variability.ResultsHere we used real-time quantitative PCR to investigate the levels of two major NF1 mRNA isoforms encoding proteins differing in their ability to control RAS signaling (isoforms I and II) in the peripheral blood leukocytes of 138 clinically well-characterized NF1 patients and 138 aged-matched healthy controls. As expected, expression analysis showed that NF1 isoforms I and II levels were significantly lower in patients than controls. Notably, these differences were more evident when patients were stratified according to the severity of phenotype. Moreover, a correlation was identified when comparing the levels of isoform I mRNA and the severity of NF1 features, with statistically significant lower levels associated with a severe phenotype (i.e., occurrence of learning disability/intellectual disability, optic gliomas and/or other neoplasias, and/or cerebrovascular disease) as well as in patients with cognitive impairment.ConclusionsThe present findings provide preliminary evidence for a role of circuits controlling NF1 transcript processing in modulating NF1 expressivity, and document an association between the levels of neurofibromin isoform I mRNA and the severity of phenotype and cognitive impairment in NF1.

The role of isoforms in the evolution of cryptic coloration in Peromyscus mice.

A central goal of evolutionary biology is to understand the molecular mechanisms underlying phenotypic adaptation. While the contribution of protein-coding and cis-regulatory mutations to adaptive traits has been well documented, additional sources of variation - such as the production of alternative RNA transcripts from a single gene, or isoforms - have been understudied. Here, we focus on the pigmentation gene Agouti, known to express multiple alternative transcripts, to investigate the role of isoform usage in the evolution of cryptic colour phenotypes in deer mice (genus Peromyscus). We first characterize the Agouti isoforms expressed in the Peromyscus skin and find two novel isoforms not previously identified in Mus. Next, we show that a locally adapted light-coloured population of P.maniculatus living on the Nebraska Sand Hills shows an upregulation of a single Agouti isoform, termed 1C, compared with their ancestral dark-coloured conspecifics. Using invitro assays, we show that this preference for isoform 1C may be driven by isoform-specific differences in translation. In addition, using an admixed population of wild-caught mice, we find that variation in overall Agouti expression maps to a region near exon 1C, which also has patterns of nucleotide variation consistent with strong positive selection. Finally, we show that the independent evolution of cryptic light pigmentation in a different species, P.polionotus, has been driven by a preference for the same Agouti isoform. Together, these findings present an example of the role of alternative transcript processing in adaptation and demonstrate molecular convergence at the level of isoform regulation.

Thioredoxin and glutathione systems differ in parasitic and free-living platyhelminths

BackgroundThe thioredoxin and/or glutathione pathways occur in all organisms. They provide electrons for deoxyribonucleotide synthesis, function as antioxidant defenses, in detoxification, Fe/S biogenesis and participate in a variety of cellular processes. In contrast to their mammalian hosts, platyhelminth (flatworm) parasites studied so far, lack conventional thioredoxin and glutathione systems. Instead, they possess a linked thioredoxin-glutathione system with the selenocysteine-containing enzyme thioredoxin glutathione reductase (TGR) as the single redox hub that controls the overall redox homeostasis. TGR has been recently validated as a drug target for schistosomiasis and new drug leads targeting TGR have recently been identified for these platyhelminth infections that affect more than 200 million people and for which a single drug is currently available. Little is known regarding the genomic structure of flatworm TGRs, the expression of TGR variants and whether the absence of conventional thioredoxin and glutathione systems is a signature of the entire platyhelminth phylum.ResultsWe examine platyhelminth genomes and transcriptomes and find that all platyhelminth parasites (from classes Cestoda and Trematoda) conform to a biochemical scenario involving, exclusively, a selenium-dependent linked thioredoxin-glutathione system having TGR as a central redox hub. In contrast, the free-living platyhelminth Schmidtea mediterranea (Class Turbellaria) possesses conventional and linked thioredoxin and glutathione systems. We identify TGR variants in Schistosoma spp. derived from a single gene, and demonstrate their expression. We also provide experimental evidence that alternative initiation of transcription and alternative transcript processing contribute to the generation of TGR variants in platyhelminth parasites.ConclusionsOur results indicate that thioredoxin and glutathione pathways differ in parasitic and free-living flatworms and that canonical enzymes were specifically lost in the parasitic lineage. Platyhelminth parasites possess a unique and simplified redox system for diverse essential processes, and thus TGR is an excellent drug target for platyhelminth infections. Inhibition of the central redox wire hub would lead to overall disruption of redox homeostasis and disable DNA synthesis.

Regulation of gene expression in plants: the role of transcript structure and processing

This book deals with the fundamentals and intricacies of the regulation of gene expression in plants, and the impact that this regulation has on plant development. Although the book mostly presents facts at an in-depth, molecular level, focusing in particular on the role of RNA, it does not lose sight of the cellular, organellar and physiological consequences of the various regulatory mechanisms. The reader is provided with a fascinating and structured account of the subject area: the book starts with basic introductory facts as well as practical applications (e.g. how RNA is used to measure gene expression), before moving on to discuss in detail the regulation of different cellular reactions by a multitude of diverse mechanisms (e.g. through alternative transcript initiation and processing, polyadenylation, RNA interference, as well as the transport and turnover of RNA). The book is composed of six well-written chapters by different authors, which all begin with a helpful, basic introduction to the relevant topic, before heading into more detailed territory. Even though the volume has less than 200 pages it manages very well to highlight most of the main subjects in the plant gene expression field, and because of its rather ‘friendly’ length one never loses interest before reaching the end of a chapter – thus it succeeds in keeping the reader's attention throughout. Because of its specific focus on plants, the book is a very warmly welcomed contribution to the somewhat limited plant molecular literature. The book is also very well structured. The first chapter deals with fundamental aspects of gene expression, providing context and a nice overview of the topic. The next three chapters then cover specific types of regulatory mechanism (i.e. multiple transcript initiation, alternative transcript processing, and messenger RNA 3′-end formation and polyadenylation). There then follows a chapter focused on regulation by small RNAs, which also covers the use of RNA interference as a molecular tool for the manipulation of gene expression. Finally, the closing chapter deals with the control of gene expression by the transport, localization and turnover of messenger RNA. The chapters are very informative and not only describe the regulatory mechanisms per se, but also link them with real examples of how they affect plant growth and development. Moreover, where applicable, comparisons with the mechanisms that operate in animals are given, providing a very useful indication of the aspects of gene expression that have diverged in the different kingdoms. The information provided is very much state-of-the art; the book not only summarizes current knowledge within each of the relevant fields, but also mentions uncertainties and debates about unresolved issues. Shortcomings are few and far between. We noted that discussion of the unique regulatory mechanisms that operate within chloroplasts and mitochondria is restricted to short sections in the opening chapters. This is obviously a reflection of the authors' intention to focus on the nucleocytosolic system. Nonetheless, in view of the volume's stated focus on plants, a little more information on the plant-specific, endosymbiotic organelle would have been welcome. Our only other criticism pertains to the absence of colour figures from the locations in which they are actually discussed (i.e. within each chapter). Instead, grey-scale figures are included in the chapters, and those needing colour for better explanation are condensed into a separate colour plates section. While this is not optimal, it is nonetheless quite common in such volumes and is presumably borne of cost considerations. This issue aside, we found the illustrations to be very clear and informative. Without doubt, this is a book that can be recommended to scientists working within the field of plant gene expression, including cell biologists, molecular biologists, geneticists and biochemists. Moreover, those interested in animal gene expression will find it useful, since numerous excellent examples of cross-kingdom similarity and dissimilarity are provided throughout the text. The book is particularly well suited for use in graduate student education because, as already mentioned, each chapter begins with a fundamental overview before entering into more detailed analysis: thus complexity increases gradually within each chapter, making the work quite accessible. Libraries should be encouraged to acquire the book as it has the potential to become a cornerstone within the subject for some years. If one is interested in how plant growth and development are regulated by gene expression, and by RNA in particular, then this is a key piece of literature. Regulation of gene expression in plants is an excellent guide to the fantastic world of RNA molecules in plants.

Functional significance of the alternative transcript processing of the Arabidopsis floral promoter FCA.

The Arabidopsis gene FCA encodes an RNA binding protein that functions to promote the floral transition. The FCA transcript is alternatively processed to yield four transcripts, the most abundant of which is polyadenylated within intron 3. We have analyzed the role of the alternative processing on the floral transition. The introduction of FCA intronless transgenes resulted in increased FCA protein levels and accelerated flowering, but no role in flowering was found for products of the shorter transcripts. The consequences of the alternative processing on the FCA expression pattern were determined using a series of translational FCA-beta-glucuronidase fusions. The inclusion of FCA genomic sequence containing the alternatively processed intron 3 restricted the expression of the transgene predominantly to shoot and root apices and young flower buds. Expression of this fusion also was delayed developmentally. Therefore, the alternative processing of the FCA transcript limits, both spatially and temporally, the amount of functional FCA protein. Expression in roots prompted an analysis of root development, which indicated that FCA functions more generally than in the control of the floral transition.