HiFENS: High-throughput FISH detection of endogenous pre-mRNA splicing isoforms (original) (raw)
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Molecular and Cellular Biology, 2010
Bioactive compounds have been invaluable for dissecting the mechanisms, regulation, and functions of cellular processes. However, very few such reagents have been described for pre-mRNA splicing. To facilitate their systematic discovery, we developed a high-throughput cell-based assay that measures pre-mRNA splicing by utilizing a quantitative reporter system with advantageous features. The reporter, consisting of a destabilized, intron-containing luciferase expressed from a short-lived mRNA, allows rapid screens (<4 h), thereby obviating the potential toxicity of splicing inhibitors. We describe three inhibitors (out of >23,000 screened), all pharmacologically active: clotrimazole, flunarizine, and chlorhexidine. Interestingly, none was a general splicing inhibitor. Rather, each caused distinct splicing changes of numerous genes. We further discovered the target of action of chlorhexidine and show that it is a selective inhibitor of specific Cdc2-like kinases (Clks) that phos...
High-throughput quantification of splicing isoforms
RNA, 2010
Most human messenger RNAs (mRNAs) are alternatively spliced and many exhibit disease-specific splicing patterns. However, the contribution of most splicing events to the development and maintenance of human diseases remains unclear. As the contribution of alternative splicing events to diagnosis and prognosis is becoming increasingly recognized, it becomes important to develop precise methods to quantify the abundance of these isoforms in clinical samples. Here we present a pipeline for realtime PCR annotation of splicing events (RASE) that allows accurate identification of a large number of splicing isoforms in human tissues. The RASE automatically designed specific primer pairs for 81% of all alternative splicing events in the NCBI build 36 database. Experimentally, the majority of the RASE designed primers resulted in isoform-specific amplification suitable for quantification in human cell lines or in formalin-fixed, paraffin-embedded (FFPE) RNA extract. Using this pipeline it is now possible to rapidly identify splicing isoform signatures in different types of human tissues or to validate complete sets of data generated by microarray expression profiling and deep sequencing techniques.
Analyzing mechanisms of alternative pre-mRNA splicing using in vitro splicing assays
Methods, 2005
The development of in vitro assays to analyze pre-mRNA splicing resulted in the discovery of many fundamental features characterizing splicing signals and the machinery that completes this process. Because in vitro assays can be manipulated by various biochemical approaches, the versatility of investigating alternative pre-mRNA splicing in the test tube appears endless. Importantly, modifications in reaction conditions can lead to the accumulation, isolation, and characterization of reaction intermediates, a prerequisite for gaining mechanistic insights into how the spliceosome carries out intron removal, and how regulatory elements assist the general splicing machinery in defining splice sites and alternative exons. These considerable experimental advantages have made the in vitro splicing system a standard assay, even though this approach is independent from RNA transcription and other RNA processing events, and in some respects deviates from the natural process of mRNA biogenesis. Here, we describe the tools and techniques necessary to carry out in vitro splicing assays. Analyses of various experimental designs are presented to highlight the approaches taken to gain insights into the mechanisms by which splice site recognition and activation are communicated with the general splicing machinery. Methods to measure the kinetics of splicing, to observe the formation of the pre-spliceosomal complexes, and to manipulate and modify the in vitro system to resolve the regulatory influences in alternative splicing are presented.
Embracing the complexity of pre-mRNA splicing
Cell Research, 2010
Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome, which catalyzes the removal of non-coding intronic sequences to assemble exons into mature mRNAs prior to export and translation. Defects in splicing lead to many human genetic diseases , and splicing mutations in a number of genes involved in growth control have been implicated in multiple types of cancer . Given the complexity of higher eukaryotic genes and the relatively low level of splice-site conservation, the precision of the splicing machinery in recognizing and pairing splice sites is remarkable. Introns ranging in size from less than 100 up to 100 000 bases are removed efficiently. At the same time, a large number of alternative splicing events are observed between different cell types, developmental stages, and during other biological processes. Of the approximately 25 000 genes encoded by the human genome [3], more than 90% are believed to produce transcripts that are alternatively spliced . Thus, alternative splicing of pre-mRNAs can lead to the production of multiple protein isoforms from a single pre-mRNA, significantly enriching the proteomic diversity of higher eukaryotic organisms . Because regulation of this process can determine the timing and location in which a particular protein isoform is produced, changes in alternative splicing patterns modulate many cellular activities. This extensive alternative splicing implies a significant flexibility of the spliceosome to identify and process exons within a given pre-mRNA.
Genome-Wide Approaches to Monitor Pre-mRNA Splicing
Elsevier eBooks, 2010
Pre-mRNA processing is an essential control-point in the gene expression pathway of eukaryotic organisms. The budding yeast Saccharomyces cerevisiae offers a powerful opportunity to examine the regulation of this pathway. In this chapter, we will describe methods that have been developed in our lab and others to examine pre-mRNA splicing from a genome-wide perspective in yeast. Our goal is to provide all of the necessary information-from microarray design to experimental setup to data analysis-to facilitate the widespread use of this technology.
Signals, pathways and splicing regulation
The International Journal of Biochemistry & Cell Biology, 2007
Alternative splicing of messenger RNA precursors is an extraordinary source of protein diversity and the regulation of this process is crucial for diverse cellular functions in both physiological and pathological situations. For many years, several signaling pathways have been implicated in alternative splicing regulation. Recent work has begun to unravel the molecular mechanisms by which extracellular stimuli activate signaling cascades that modulate the activity of the splicing machinery and therefore the splicing pattern of many different target messenger RNA precursors. These experiments are revealing unexpected aspects of the mechanism that control splicing and the consequences of the regulated splicing events.
Alternative splicing as a biomarker and potential target for drug discovery
Acta pharmacologica Sinica, 2015
Alternative splicing is a key process of multi-exonic gene expression during pre-mRNA maturation. In this process, particular exons of a gene will be included within or excluded from the final matured mRNA, and the resulting transcripts generate diverse protein isoforms. Recent evidence demonstrates that approximately 95% of human genes with multiple exons undergo alternative splicing during pre-mRNA maturation. Thus, alternative splicing plays a critical role in physiological processes and cell development programs, and.dysregulation of alternative splicing is highly associated with human diseases, such as cancer, diabetes and neurodegenerative diseases. In this review, we discuss the regulation of alternative splicing, examine the relationship between alternative splicing and human diseases, and describe several approaches that modify alternative splicing, which could aid in human disease diagnosis and therapy.
Alternative splicing: decoding an expansive regulatory layer
2012
Alternative splicing (AS) is the process by which splice sites in precursor (pre)-mRNA are differentially selected to produce multiple mRNA and protein isoforms. During the past few years the application of genome-wide profiling technologies coupled with bioinformatic approaches has transformed our understanding of AS complexity and regulation. These studies are further driving research directed at elucidating the functions of networks of regulated AS events in the context of normal physiology and disease. Major strides have also been made in understanding how AS is functionally integrated with-and coupled to-gene regulation at the level of chromatin and transcription. Particularly intriguing is the discovery of new AS 'switches' that control transcriptional networks required for animal development and behavior.
From single splicing events to thousands: the ambiguous step forward in splicing research
2013
Since the discovery of RNA splicing in 1977 the knowledge of this important biological process has increased steadily following the identification of many of the mechanistic features of splicing: from the basic cis-acting splicing signals, through the detail composition and dynamics of the spliceosome, to the role played by accessory splicing factors and their interactions. Moreover, the realization that most genes undergo alternative splicing has had a strong impact in the overall cell proteome and metabolism research fields and also in better appraising the fundamental role played by splicing defects in human disease. This robust growth of knowledge is due in particular to the development of new powerful technical tools that range from methodologies useful to focus on single events in extreme detail to microarray and high-throughput RNA sequencing approaches that aim at providing a global vision of splicing changes. Here, we will discuss how these techniques relate to each other in terms of their respective strengths and weaknesses. In particular, we will focus on their value for evaluating the biological significance of splicing events. Finally, we provide some views on how these methodologies should move forward to improve our basic and applied knowledge of RNA splicing.