The splicing regulatory element, UGCAUG, is phylogenetically and spatially conserved in introns that flank tissue-specific alternative exons

Previous studies have identified UGCAUG as an intron splicing enhancer that is frequently located adjacent to tissue-specific alternative exons in the human genome. Here, we show that UGCAUG is phylogenetically and spatially conserved in introns that flank brain-enriched alternative exons from fish to man. Analysis of sequence from the mouse, rat, dog, chicken and pufferfish genomes revealed a strongly statistically significant association of UGCAUG with the proximal intron region downstream of brain-enriched alternative exons. The number, position and sequence context of intronic UGCAUG elements were highly conserved among mammals and in chicken, but more divergent in fish. Control datasets, including constitutive exons and non-tissue-specific alternative exons, exhibited a much lower incidence of closely linked UGCAUG elements. We propose that the high sequence specificity of the UGCAUG element, and its unique association with tissue-specific alternative exons, mark it as a critical component of splicing switch mechanism(s) designed to activate a limited repertoire of splicing events in cell type-specific patterns. We further speculate that highly conserved UGCAUG-binding protein(s) related to the recently described Fox-1 splicing factor play a critical role in mediating this specificity.     

A computational scan for U12-dependent introns in the human genome sequence

U12-dependent introns are found in small numbers in most eukaryotic genomes, but their scarcity makes accurate characterisation of their properties challenging. A computational search for U12-dependent introns was performed using the draft version of the human genome sequence. Human expressed sequences confirmed 404 U12-dependent introns within the human genome, a 6-fold increase over the total number of non-redundant U12-dependent introns previously identified in all genomes. Although most of these introns had AT-AC or GT-AG terminal dinucleotides, small numbers of introns with a surprising diversity of termini were found, suggesting that many of the non-canonical introns found in the human genome may be variants of U12-dependent introns and, thus, spliced by the minor spliceosome. Comparisons with U2-dependent introns revealed that the U12-dependent intron set lacks the ‘short intron’ peak characteristic of U2-dependent introns. Analysis of this U12-dependent intron set confirmed reports of a biased distribution of U12-dependent introns in the genome and allowed the identification of several alternative splicing events as well as a surprising number of apparent splicing errors. This new larger reference set of U12-dependent introns will serve as a resource for future studies of both the properties and evolution of the U12 spliceosome.     

Strong population structure characterizes weediness gene evolution in the invasive grass species Brachypodium distachyon

Mediterranean annual grasses have invaded California and have replaced vast areas of native grassland. One of these invasive grasses is Brachypodium distachyon , a new model species for the grasses with extensive genomic resources and a nearly completed genome sequence. This study shows that the level of genetic variation in invaded California grasslands is lower compared to the native range in Eurasia. The invaded regions are characterized by highly differentiated populations of B. distachyon isolated by distance, most likely as a result of founder effects and a dearth of outcrossing events. EXP6 and EXP10 encoding α-expansins responsible for rapid growth, and AGL11 and AGL13 encoding proteins involved in vegetative phase regulation, appear to be under purifying selection with no evidence for local adaptation. Our data show that B. distachyon has diverged only recently from related Brachypodium species and that tetraploidization might have been as recent as a few thousand years ago. Observed low genetic variation in EXP10 and AGL13 appears to have been present in Eurasia before tetraploidization, potentially as a result of strong selective pressures on advantageous mutations, which are most likely responsible for its fast growth and rapid completion of its life cycle.     

A ribonuclease III domain protein functions in group II intron splicing in maize chloroplasts

Chloroplast genomes in land plants harbor approximately 20 group II introns. Genetic approaches have identified proteins involved in the splicing of many of these introns, but the proteins identified to date cannot account for the large size of intron ribonucleoprotein complexes and are not sufficient to reconstitute splicing in vitro. Here, we describe an additional protein that promotes chloroplast group II intron splicing in vivo. This protein, RNC1, was identified by mass spectrometry analysis of maize (Zea mays) proteins that coimmunoprecipitate with two previously identified chloroplast splicing factors, CAF1 and CAF2. RNC1 is a plant-specific protein that contains two ribonuclease III (RNase III) domains, the domain that harbors the active site of RNase III and Dicer enzymes. However, several amino acids that are essential for catalysis by RNase III and Dicer are missing from the RNase III domains in RNC1. RNC1 is found in complexes with a subset of chloroplast group II introns that includes but is not limited to CAF1- and CAF2-dependent introns. The splicing of many of the introns with which it associates is disrupted in maize rnc1 insertion mutants, indicating that RNC1 facilitates splicing in vivo. Recombinant RNC1 binds both single-stranded and double-stranded RNA with no discernible sequence specificity and lacks endonuclease activity. These results suggest that RNC1 is recruited to specific introns via protein-protein interactions and that its role in splicing involves RNA binding but not RNA cleavage activity.     

Identification of common genetic variation that modulates alternative splicing

Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron–exon boundary, although the distance between these SNPs and the intron–exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.     

Intronic alternative splicing regulators identified by comparative genomics in nematodes

Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high- scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (T)GCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis, (T)CTATC, is important for alternative splicing regulation of the unc-52 gene.     

Trans-splicing group II introns in plant mitochondria: the complete set of cis-arranged homologs in ferns, fern allies, and a hornwort.

The fragmentation of group II introns without concomitant loss of splicing competence is illustrated by extraordinary gene arrangements in plant mitochondrial genomes. The mitochondrial genes nad1, nad2, and nad5, all encoding subunits of the NADH dehydrogenase, require trans-splicing for functional assembly of their mRNAs in flowering plants. Tracing the origins of trans-splicing group II introns shows that they have evolved from formerly cis-arranged homologs whose descendants can still be identified in lineages of early branching land plants. In this contribution we present the full set of ancestor introns for all five conserved mitochondrial trans-splicing positions. These introns are strikingly small in the quillwort Isoetes lacustris, the continuous nad2 gene intron in this species representing the smallest (389 nt) land plant group II intron yet identified. cDNA analysis shows correct splicing of the introns in vivo and also identifies frequent RNA editing events in the flanking nad gene exons. Other representatives of the ancestral cis-arranged introns are identified in the fern Osmunda regalis, the horsetail Equisetum telmateia, and the hornwort Anthoceros crispulus. Only the now identified intron in Osmunda carries significant traces of a former maturase reading frame. The identification of a continuous homolog in Anthoceros demonstrates that intron invasion into the affected genes in some cases predated the split of vascular and nonvascular plants more than 400 million years ago. As an alternative to disruption after size increase, the respective introns can get secondarily lost in certain lineages.     

Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish

Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated Cʹ-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish.