Apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro

Trans-splicing, the in vivo joining of two RNA molecules, is well characterized in several groups of simple organisms but was long thought absent from fungi, plants and mammals. However, recent bioinformatic analyses of expressed sequence tag (EST) databases suggested widespread trans-splicing in mammals^1-2^. Splicing, including the characterised trans-splicing systems, involves conserved sequences at the splice junctions. Our analysis of a yeast non-coding RNA revealed that around 30% of the products of reverse transcription lacked an internal region of 117 nt, suggesting that the RNA was spliced. The junction sequences lacked canonical splice-sites but were flanked by direct repeats, and further analyses indicated that the apparent splicing actually arose because reverse transcriptase can switch templates during transcription^3^. Many newly identified, apparently trans-spliced, RNAs lacked canonical splice sites but were flanked by short regions of homology, leading us to question their authenticity. Here we report that all reported categories of non-canonical splicing could be replicated using an in vitro reverse transcription system with highly purified RNA substrates. We observed the reproducible occurrence of ostensible trans-splicing, exon shuffling and sense-antisense fusions. The latter generate apparent antisense non-coding RNAs, which are also reported to be abundant in humans^4^. Different reverse transcriptases can generate different products of template switching, providing a simple diagnostic. Many reported examples of splicing in the absence of canonical splicing signals may be artefacts of cDNA preparation

Differential Sperm Motility Mediates the Sex Ratio Drive Shaping Mouse Sex Chromosome Evolution

The search for morphological or physiological differences between X- and Y-bearing mammalian sperm has provoked controversy for decades. Many potential differences have been proposed, but none validated, while accumulating understanding of syncytial sperm development has cast doubt on whether such differences are possible even in principle. We present the first ever mammalian experimental model to trace a direct link from a measurable physiological difference between X- and Y-bearing sperm to the resulting skewed sex ratio. We show that in mice with deletions on chromosome Yq, birth sex ratio distortion is due to a relatively greater motility of X-bearing sperm, and not to any aspect of sperm/egg interaction. Moreover, the morphological distortion caused by Yq deletion is more severe in Y-bearing sperm, providing a potential hydrodynamic basis for the altered motility. This reinforces a growing body of work indicating that sperm haploid selection is an important and underappreciated evolutionary force

Deficiency in the Multicopy Sycp3-Like X-Linked Genes Slx and Slxl1 Causes Major Defects in Spermatid Differentiation

Slx and Slxl1 are genes present in multiple copies on the mouse X chromosome. Using transgenically-delivered small interfering RNAs to disrupt their function, we show that Slx and Slxl1 are important for normal sperm differentiation and male fertility

Avian W and mammalian Y chromosomes convergently retained dosage-sensitive regulators

After birds diverged from mammals, different ancestral autosomes evolved into sex chromosomes in each lineage. In birds, females are ZW and males are ZZ, but in mammals females are XX and males are XY. We sequenced the chicken W chromosome, compared its gene content with our reconstruction of the ancestral autosomes, and followed the evolutionary trajectory of ancestral W-linked genes across birds. Avian W chromosomes evolved in parallel with mammalian Y chromosomes, preserving ancestral genes through selection to maintain the dosage of broadly expressed regulators of key cellular processes. We propose that, like the human Y chromosome, the chicken W chromosome is essential for embryonic viability of the heterogametic sex. Unlike other sequenced sex chromosomes, the chicken W chromosome did not acquire and amplify genes specifically expressed in reproductive tissues. We speculate that the pressures that drive the acquisition of reproduction-related genes on sex chromosomes may be specific to the male germ line

Spatial Proximity and Similarity of the Epigenetic State of Genome Domains

Recent studies demonstrate that the organization of the chromatin within the nuclear space might play a crucial role in the regulation of gene expression. The ongoing progress in determination of the 3D structure of the nuclear chromatin allows one to study correlations between spatial proximity of genome domains and their epigenetic state. We combined the data on three-dimensional architecture of the whole human genome with results of high-throughput studies of the chromatin functional state and observed that fragments of different chromosomes that are spatially close tend to have similar patterns of histone modifications, methylation state, DNAse sensitivity, expression level, and chromatin states in general. Moreover, clustering of genome regions by spatial proximity produced compact clusters characterized by the high level of histone modifications and DNAse sensitivity and low methylation level, and loose clusters with the opposite characteristics. We also associated the spatial proximity data with previously detected chimeric transcripts and the results of RNA-seq experiments and observed that the frequency of formation of chimeric transcripts from fragments of two different chromosomes is higher among spatially proximal genome domains. A fair fraction of these chimeric transcripts seems to arise post-transcriptionally via trans-splicing

Evolutionary Convergence on Highly-Conserved 3′ Intron Structures in Intron-Poor Eukaryotes and Insights into the Ancestral Eukaryotic Genome

The presence of spliceosomal introns in eukaryotes raises a range of questions about genomic evolution. Along with the fundamental mysteries of introns' initial proliferation and persistence, the evolutionary forces acting on intron sequences remain largely mysterious. Intron number varies across species from a few introns per genome to several introns per gene, and the elements of intron sequences directly implicated in splicing vary from degenerate to strict consensus motifs. We report a 50-species comparative genomic study of intron sequences across most eukaryotic groups. We find two broad and striking patterns. First, we find that some highly intron-poor lineages have undergone evolutionary convergence to strong 3′ consensus intron structures. This finding holds for both branch point sequence and distance between the branch point and the 3′ splice site. Interestingly, this difference appears to exist within the genomes of green alga of the genus Ostreococcus, which exhibit highly constrained intron sequences through most of the intron-poor genome, but not in one much more intron-dense genomic region. Second, we find evidence that ancestral genomes contained highly variable branch point sequences, similar to more complex modern intron-rich eukaryotic lineages. In addition, ancestral structures are likely to have included polyT tails similar to those in metazoans and plants, which we found in a variety of protist lineages. Intriguingly, intron structure evolution appears to be quite different across lineages experiencing different types of genome reduction: whereas lineages with very few introns tend towards highly regular intronic sequences, lineages with very short introns tend towards highly degenerate sequences. Together, these results attest to the complex nature of ancestral eukaryotic splicing, the qualitatively different evolutionary forces acting on intron structures across modern lineages, and the impressive evolutionary malleability of eukaryotic gene structures

Alternative Splicing of the Cardiac Sodium Channel Creates Multiple Variants of Mutant T1620K Channels

Alternative splicing creates several Nav1.5 transcripts in the mammalian myocardium and in various other tissues including brain, dorsal root ganglia, breast cancer cells as well as neuronal stem cell lines. In total nine Nav1.5 splice variants have been discovered. Four of them, namely Nav1.5a, Nav1.5c, Nav1.5d, and Nav1.5e, generate functional channels in heterologous expression systems. The significance of alternatively spliced transcripts for cardiac excitation, in particular their role in SCN5A channelopathies, is less well understood. In the present study, we systematically investigated electrophysiological properties of mutant T1620K channels in the background of all known functional Nav1.5 splice variants in HEK293 cells. This mutation has been previously associated with two distinct cardiac excitation disorders: with long QT syndrome type 3 (LQT3) and isolated cardiac conduction disease (CCD). When investigating the effect of the T1620K mutation, we noticed similar channel defects in the background of hNav1.5, hNav1.5a, and hNav1.5c. In contrast, the hNav1.5d background produced differential effects: In the mutant channel, some gain-of-function features did not emerge, whereas loss-of-function became more pronounced. In case of hNav1.5e, the neonatal variant of hNav1.5, both the splice variant itself as well as the corresponding mutant channel showed electrophysiological properties that were distinct from the wild-type and mutant reference channels, hNav1.5 and T1620K, respectively. In conclusion, our data show that alternative splicing is a mechanism capable of generating a variety of functionally distinct wild-type and mutant hNav1.5 channels. Thus, the cellular splicing machinery is a potential player affecting genotype-phenotype correlations in SCN5A channelopathies