Pre-mRNA splicing and its cotranscriptional connections.

Transcription of eukaryotic genes by RNA polymerase II (Pol II) yields RNA precursors containing introns that must be spliced out and the flanking exons ligated together. Splicing is catalyzed by a dynamic ribonucleoprotein complex called the spliceosome. Recent evidence has shown that a large fraction of splicing occurs cotranscriptionally as the RNA chain is extruded from Pol II at speeds of up to 5 kb/minute. Splicing is more efficient when it is tethered to the transcription elongation complex, and this linkage permits functional coupling of splicing with transcription. We discuss recent progress that has uncovered a network of connections that link splicing to transcript elongation and other cotranscriptional RNA processing events.

Splice site proximity influences alternative exon definition.

Alternative splicing enables higher eukaryotes to expand mRNA diversity from a finite number of genes through highly combinatorial splice site selection mechanisms that are influenced by the sequence of competing splice sites, cis-regulatory elements binding trans-acting factors, the length of exons and introns harbouring alternative splice sites and RNA secondary structures at putative splice junctions. To test the hypothesis that the intron definition or exon definition modes of splice site recognition direct the selection of alternative splice patterns, we created a database of alternative splice site usage (ALTssDB). When alternative splice sites are embedded within short introns (intron definition), the 5' and 3' splice sites closest to each other across the intron preferentially pair, consistent with previous observations. However, when alternative splice sites are embedded within large flanking introns (exon definition), the 5' and 3' splice sites closest to each other across the exon are preferentially selected. Thus, alternative splicing decisions are influenced by the intron and exon definition modes of splice site recognition. The results demonstrate that the spliceosome pairs splice sites that are closest in proximity within the unit of initial splice site selection.

Allosteric regulation of U1 snRNP by splicing regulatory proteins controls spliceosomal assembly.

Alternative splicing is responsible for much of the transcriptomic and proteomic diversity observed in eukaryotes and involves combinatorial regulation by many cis-acting elements and trans-acting factors. SR and hnRNP splicing regulatory proteins often have opposing effects on splicing efficiency depending on where they bind the pre-mRNA relative to the splice site. Position-dependent splicing repression occurs at spliceosomal E-complex, suggesting that U1 snRNP binds but cannot facilitate higher order spliceosomal assembly. To test the hypothesis that the structure of U1 snRNA changes during activation or repression, we developed a method to structure-probe native U1 snRNP in enriched conformations that mimic activated or repressed spliceosomal E-complexes. While the core of U1 snRNA is highly structured, the 5' end of U1 snRNA shows different SHAPE reactivities and psoralen crosslinking efficiencies depending on where splicing regulatory elements are located relative to the 5' splice site. A motif within the 5' splice site binding region of U1 snRNA is more reactive toward SHAPE electrophiles when repressors are bound, suggesting U1 snRNA is bound, but less base-paired. These observations demonstrate that splicing regulators modulate splice site selection allosterically.

The Ideal Cardiac Mapping System.

Cardiac mapping has witnessed significant and unprecedented progress over more than a century. At present, several mapping/imaging technologies are commercially available, alone or in combination. This article briefly discusses the advantages and limitations (disadvantages) of each technique.

Combinatorial regulation of alternative splicing.

The generation of protein coding mRNAs from pre-mRNA is a fundamental biological process that is required for gene expression. Alternative pre-mRNA splicing is responsible for much of the transcriptomic and proteomic diversity observed in higher order eukaryotes. Aberrations that disrupt regular alternative splicing patterns are known to cause human diseases, including various cancers. Alternative splicing is a combinatorial process, meaning many factors affect which two splice sites are ligated together. The features that dictate exon inclusion are comprised of splice site strength, intron-exon architecture, RNA secondary structure, splicing regulatory elements, promoter use and transcription speed by RNA polymerase and the presence of post-transcriptional nucleotide modifications. A comprehensive view of all of the factors that influence alternative splicing decisions is necessary to predict splicing outcomes and to understand the molecular basis of disease. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.

Inappropriate ICD shock due to hot tub-induced external electrical interference.

BACKGROUND: A 72-year-old white male with a history of rapid nonsustained ventricular tachycardia, hypertrophic cardiomyopathy, and intermittent Brugada-type ECG had a single-lead implantable cardioverter-defibrillator (ICD) implantation and received a sudden ICD shock while in the hot tub. To the best of our knowledge this is the first case report of hot tub jet-induced inappropriate ICD shock. METHODS: ICD interrogation and analysis of intracardiac electrograms and event markers. RESULTS: ICD interrogation revealed inappropriate ICD shocks due to electrical interference of hot tub engine; 60-cycle electrical artifact mimicking fast ventricular fibrillation erroneously detected by the device. The device then delivered a 34.8 joules shock while the patient was actually in sinus rhythm. CONCLUSIONS: Electrical interference due to external sources such as hot tub engines may occur and produce an inappropriate detection and ICD shock. Precaution and patient education is warranted.

Preparation of Splicing Competent Nuclear Extract from Mammalian Cells and In Vitro Pre-mRNA Splicing Assay.

The ability to perform in vitro splicing assays has paved the way for in-depth studies of the mechanisms and machinery involved in the process of splicing. The in vitro splicing assay is a valuable experimental approach that combines the complexity of the spliceosome and regulatory systems with the flexibility of performing endless splicing and alternative splicing reactions. Through the use of crude nuclear extract and radiolabeled pre-mRNA, spliced mRNAs can be visualized using autoradiography for downstream analysis. This chapter describes the necessary steps to perform an in vitro splicing reaction, including the generation of the key components necessary for the splicing reaction; nuclear extract.

Hypertension, left ventricular hypertrophy, and sudden cardiac death.

Hypertension (HTN) is the most common cause of hypertensive heart disease, which comprises of left ventricular hypertrophy (LVH), left atrial enlargement, diastolic dysfunction, functional mitral regurgitation and neurohormonal changes. All of these lead to significant arrhythmias such as atrial fibrillation (AF) as well as ventricular arrhythmias, and are known risk factors for sudden cardiac death (SCD). The association between LVH and SCD is well established, especially in the presence of myocardial ischemia, fibrosis and scar tissue, and AF. Inflammation, fibrosis and oxidative stress, as well as ischemia play a significant role and are the leading pathways to remodeling, arrhythmias, and SCD. Aggressive HTN control may lead, at least in part, to regression of LVH and thus lower the risk of AF and SCD. Therefore, LVH is a powerful, independent predictor of AF, ventricular arrhythmias and SCD, and is significantly underrecognized. Further investigation of the relationship and management of diastolic dysfunction, LVH and genetic factors and their association with SCD is certainly warranted.

Ranolazine: Electrophysiologic Effect, Efficacy, and Safety in Patients with Cardiac Arrhythmias.

Ranolazine is currently approved as an antianginal agent in patients with chronic angina (class IIA). Ranolazine exhibits antiarrhythmic effects that are related to its multichannel blocking effect, predominantly inhibition of late sodium (late INa) current and the rapid potassium rectifier current (IKr), as well as ICa, late ICa, and INa-Ca. It also suppresses the early and delayed after depolarizations. Ranolazine is effective in the suppression of atrial and ventricular arrhythmias (off-label use) without significant proarrhythmic effect. Currently, ongoing trials are evaluating the efficacy and safety of ranolazine in patients with cardiac arrhythmias; preliminary results suggest that ranolazine, when used alone or in combination with dronedarone, is safe and effective in reducing atrial fibrillation. Ranolazine is not currently approved by the US Food and Drug Administration as an antiarrhythmic agent.

Left ventricular hypertrophy and arrhythmogenesis.

Left ventricular hypertrophy (LVH) poses an independent risk of increased morbidity and mortality, including atrial arrhythmias, ventricular arrhythmias, and sudden cardiac death. The most common causes of LVH are hypertension and valvular heart disease. Electrocardiography and echocardiography are the first steps in the diagnosis and evaluation of therapy in patients with LVH. Cardiac MRI is the gold standard in diagnosis and assessment of response to therapy. Management of LVH should be based on etiology, evidence, and guideline adherence. Timely and optimal management of the underlying cause of LVH results in improvement (regression) of LVH and its related complications.

Generation of oligonucleotides under hydrothermal conditions by non-enzymatic polymerization.

We previously reported that 5'-mononucleotides organized within a multilamellar lipid matrix can produce oligomers in the anhydrous phase of hydration-dehydration (HD) cycles. However, hydrolysis of oligomers can occur during hydration, and it is important to better understand the steady state in which ester bond synthesis is balanced by hydrolysis. In order to study condensation products of mononucleotides and hydrolysis of their polymers, we established a simulation of HD cycles that would occur on the early Earth when volcanic land masses emerged from the ocean over 4 billion years ago. At this stage on early Earth, precipitation produced hydrothermal fields characterized by small aqueous pools undergoing evaporation and refilling at elevated temperatures. Here, we confirm that under these conditions, the chemical potential made available by cycles of hydration and dehydration is sufficient to drive synthesis of ester bonds. If 5'-mononucleotides are in solution at millimolar concentrations, then oligomers resembling RNA are synthesized and exist in a steady state with their monomers. Furthermore, if the mononucleotides can form complementary base pairs, then some of the products have properties suggesting that secondary structures are present, including duplex species stabilized by hydrogen bonds.