Stoichiometry of a regulatory splicing complex revealed by single-molecule analyses

Splicing is regulated by complex interactions of numerous RNA-binding proteins. The molecular mechanisms involved remain elusive, in large part because of ignorance regarding the numbers of proteins in regulatory complexes. Polypyrimidine tract-binding protein (PTB), which regulates tissue-specific splicing, represses exon 3 of α-tropomyosin through distant pyrimidine-rich tracts in the flanking introns. Current models for repression involve either PTB-mediated looping or the propagation of complexes between tracts. To test these models, we used single-molecule approaches to count the number of bound PTB molecules both by counting the number of bleaching steps of GFP molecules linked to PTB within complexes and by analysing their total emissions. Both approaches showed that five or six PTB molecules assemble. Given the domain structures, this suggests that the molecules occupy primarily multiple overlapping potential sites in the polypyrimidine tracts, excluding propagation models. As an alternative to direct looping, we propose that repression involves a multistep process in which PTB binding forms small local loops, creating a platform for recruitment of other proteins that bring these loops into close proximity

Interplay between Exonic Splicing Enhancers, mRNA Processing, and mRNA Surveillance in the Dystrophic Mdx Mouse

BACKGROUND: Pre-mRNA splicing, the removal of introns from RNA, takes place within the spliceosome, a macromolecular complex composed of five small nuclear RNAs and a large number of associated proteins. Spliceosome assembly is modulated by the 5′ and 3′ splice site consensus sequences situated at the ends of each intron, as well as by exonic and intronic splicing enhancers/silencers recognized by SR and hnRNP proteins. Nonsense mutations introducing a premature termination codon (PTC) often result in the activation of cellular quality control systems that reduce mRNA levels or alter the mRNA splicing pattern. The mdx mouse, a commonly used genetic model for Duchenne muscular dystrophy (DMD), lacks dystrophin by virtue of a premature termination codon (PTC) in exon 23 that also severely reduces the level of dystrophin mRNA. However, the effect of the mutation on dystrophin RNA processing has not yet been described. METHODOLOGY/PRINCIPAL FINDING: Using combinations of different biochemical and cellular assays, we found that the mdx mutation partially disrupts a multisite exonic splicing enhancer (ESE) that is recognized by a 40 kDa SR protein. In spite of the presence of an inefficient intron 22 3′ splice site containing the rare GAG triplet, the mdx mutation does not activate nonsense-associated altered splicing (NAS), but induces exclusively nonsense-mediated mRNA decay (NMD). Functional binding sites for SR proteins were also identified in exon 22 and 24, and in vitro experiments show that SR proteins can mediate direct association between exon 22, 23, and 24. CONCLUSIONS/SIGNIFICANCE: Our findings highlight the complex crosstalk between trans-acting factors, cis-elements and the RNA surveillance machinery occurring during dystrophin mRNA processing. Moreover, they suggest that dystrophin exon–exon interactions could play an important role in preventing mdx exon 23 skipping, as well as in facilitating the pairing of committed splice sites

Local Dimensionality Reduction for Non-Parametric Regression

Locally-weighted regression is a computationally-efficient technique for non-linear regression. However, for high-dimensional data, this technique becomes numerically brittle and computationally too expensive if many local models need to be maintained simultaneously. Thus, local linear dimensionality reduction combined with locally-weighted regression seems to be a promising solution. In this context, we review linear dimensionalityreduction methods, compare their performance on non-parametric locally-linear regression, and discuss their ability to extend to incremental learning. The considered methods belong to the following three groups: (1) reducing dimensionality only on the input data, (2) modeling the joint input-output data distribution, and (3) optimizing the correlation between projection directions and output data. Group 1 contains principal component regression (PCR); group 2 contains principal component analysis (PCA) in joint input and output space, factor analysis, and probabilistic PCA; and group 3 contains reduced rank regression (RRR) and partial least squares (PLS) regression. Among the tested methods, only group 3 managed to achieve robust performance even for a non-optimal number of components (factors or projection directions). In contrast, group 1 and 2 failed for fewer components since these methods rely on the correct estimate of the true intrinsic dimensionality. In group 3, PLS is the only method for which a computationally-efficient incremental implementation exists

A Compact Representation of Drawing Movements with Sequences of Parabolic Primitives

Some studies suggest that complex arm movements in humans and monkeys may optimize several objective functions, while others claim that arm movements satisfy geometric constraints and are composed of elementary components. However, the ability to unify different constraints has remained an open question. The criterion for a maximally smooth (minimizing jerk) motion is satisfied for parabolic trajectories having constant equi-affine speed, which thus comply with the geometric constraint known as the two-thirds power law. Here we empirically test the hypothesis that parabolic segments provide a compact representation of spontaneous drawing movements. Monkey scribblings performed during a period of practice were recorded. Practiced hand paths could be approximated well by relatively long parabolic segments. Following practice, the orientations and spatial locations of the fitted parabolic segments could be drawn from only 2–4 clusters, and there was less discrepancy between the fitted parabolic segments and the executed paths. This enabled us to show that well-practiced spontaneous scribbling movements can be represented as sequences (“words”) of a small number of elementary parabolic primitives (“letters”). A movement primitive can be defined as a movement entity that cannot be intentionally stopped before its completion. We found that in a well-trained monkey a movement was usually decelerated after receiving a reward, but it stopped only after the completion of a sequence composed of several parabolic segments. Piece-wise parabolic segments can be generated by applying affine geometric transformations to a single parabolic template. Thus, complex movements might be constructed by applying sequences of suitable geometric transformations to a few templates. Our findings therefore suggest that the motor system aims at achieving more parsimonious internal representations through practice, that parabolas serve as geometric primitives and that non-Euclidean variables are employed in internal movement representations (due to the special role of parabolas in equi-affine geometry)

Chloroplast DNA Microsatellites Reveal Contrasting Phylogeographic Structure in Mahogany (Swietenia macrophylla King, Meliaceae) from Amazonia and Central America

Big-leaf mahogany (Swietenia macrophylla) is one of the most valuable and overharvested timber trees of tropical America. A description of the organization of genetic variation across its broad range would be useful for management of genetic diversity and for understanding its demographic history. Here we report on a phylogeographic analysis of mahogany based on six polymorphic cpDNA simple sequence repeat loci (cpSSRs) genotyped in 16 populations distributed across the Brazilian Amazon and Mesoamerica (N = 245 individuals). Of the 31 cpDNA haplotypes identified, 15 occurred in Amazonia and 16 in Mesoamerica with no single haplotype shared between the two regions. The populations from Central America showed moderate differentiation (FST = 0.36) while within population genetic diversity was generally high (mean Nei's HE = 0.639). In contrast, the Amazonian populations were strongly differentiated (FST = 0.95) and contained low haplotype diversity (mean HE = 0.176), with the exception of the highly diverse Marajoara population from the Eastern Amazon (HE = 0.925). SAMOVA identified a single Mesoamerican phylogroup and four Amazonian phylogroups, indicating stronger phylogeographic structure within Amazonia. The results demonstrate high levels of cpDNA variation and differentiation of regional S. macrophylla populations, and provide the first evidence of a major phylogeographic break between Mesoamerican and South American mahogany populations

A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes

Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations(1-4). In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear(1,2,5,6). By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms(7) (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene