Deletion of many yeast introns reveals a minority of genes that require splicing for function

Splicing regulates gene expression and contributes to proteomic diversity in higher eukaryotes. However, in yeast only 283 of the 6000 genes contain introns and their impact on cell function is not clear. To assess the contribution of introns to cell function, we initiated large-scale intron deletions in yeast with the ultimate goal of creating an intron-free model eukaryote. We show that about one-third of yeast introns are not essential for growth. Only three intron deletions caused severe growth defects, but normal growth was restored in all cases by expressing the intronless mRNA from a heterologous promoter. Twenty percent of the intron deletions caused minor phenotypes under different growth conditions. Strikingly, the combined deletion of all introns from the 15 cytoskeleton-related genes did not affect growth or strain fitness. Together, our results show that although the presence of introns may optimize gene expression and provide benefit under stress, a majority of introns could be removed with minor consequences on growth under laboratory conditions, supporting the view that many introns could be phased out of Saccharomyces cerevisiae without blocking cell growth.     

In silico screening of archaeal tRNA-encoding genes having multiple introns with bulge-helix-bulge splicing motifs

In archaeal species, several transfer RNA genes have been reported to contain endogenous introns. Although most of the introns are located at anticodon loop regions between nucleotide positions 37 and 38, a number of introns at noncanonical sites and six cases of tRNA genes containing two introns have also been documented. However, these tRNA genes are often missed by tRNAscan-SE, the software most widely used for the annotation of tRNA genes. We previously developed SPLITS, a computational tool to identify tRNA genes containing one intron at a noncanonical position on the basis of its discriminative splicing motif, but the software was limited in the detection of tRNA genes with multiple introns at noncanonical sites. In this study, we initially updated the system as SPLITSX in order to correctly predict known tRNA genes as well as novel ones with multiple introns. By a comprehensive search for tRNA genes in 29 archaeal genomes using SPLITSX, we listed 43 novel candidates that contain introns at noncanonical sites. As a result, 15 contained two introns and three contained three introns within the respective putative tRNA genes. Moreover, the candidates completely complemented all the codons of two archaeal species of uncultured methanogenic archaeon, RC-I and Thermofilum pendens Hrk 5, with novel candidates that were not detectable by tRNAscan-SE alone.     

RNAmotifs: prediction of multivalent RNA motifs that control alternative splicing

RNA-binding proteins (RBPs) regulate splicing according to position-dependent principles, which can be exploited for analysis of regulatory motifs. Here we present RNAmotifs, a method that evaluates the sequence around differentially regulated alternative exons to identify clusters of short and degenerate sequences, referred to as multivalent RNA motifs. We show that diverse RBPs share basic positional principles, but differ in their propensity to enhance or repress exon inclusion. We assess exons differentially spliced between brain and heart, identifying known and new regulatory motifs, and predict the expression pattern of RBPs that bind these motifs. RNAmotifs is available at https://bitbucket.org/rogrro/rna_motifs.     

Tissue-specific splicing of disordered segments that embed binding motifs rewires protein interaction networks

Alternative inclusion of exons increases the functional diversity of proteins. Among alternatively spliced exons, tissue-specific exons play a critical role in maintaining tissue identity. This raises the question of how tissue-specific protein-coding exons influence protein function. Here we investigate the structural, functional, interaction, and evolutionary properties of constitutive, tissue-specific, and other alternative exons in human. We find that tissue-specific protein segments often contain disordered regions, are enriched in posttranslational modification sites, and frequently embed conserved binding motifs. Furthermore, genes containing tissue-specific exons tend to occupy central positions in interaction networks and display distinct interaction partners in the respective tissues, and are enriched in signaling, development, and disease genes. Based on these findings, we propose that tissue-specific inclusion of disordered segments that contain binding motifs rewires interaction networks and signaling pathways. In this way, tissue-specific splicing may contribute to functional versatility of proteins and increases the diversity of interaction networks across tissues.     

Identification of BHB splicing motifs in intron-containing tRNAs from 18 archaea: evolutionary implications

Most introns of archaeal tRNA genes (tDNAs) are located in the anticodon loop, between nucleotides 37 and 38, the unique location of their eukaryotic counterparts. However, in several Archaea, mostly in Crenarchaeota, introns have been found at many other positions of the tDNAs. In the present work, we revisit and extend all previous findings concerning the identification, exact location, size, and possible fit to the proposed bulge-helix-bulge structural motif (BHB, now renamed hBHBh') of the sequences spanning intron-exon junctions in intron-containing tRNAs of 18 archaea. A total of 103 introns were found located at the usual position 37/38 and 33 introns at 14 other different positions, that is, in the anticodon stem and loop, in the D-and T-loops, in the V-arm, or in the amino acid arm. For introns located at 37/38 and elsewhere in the pre-tRNA, canonical hBHBh' motifs were not always found. Instead, a relaxed hBH or HBh' motif including the constant central 4-bp helix H flanked by one helix (h or h') on either side generating only one bulge could be disclosed. Also, for introns located elsewhere than at position 37/38, the hBHBh' (or HBh') structure competes with the three-dimensional structure of the mature tRNA, attesting to important structural rearrangements during the complex multistep maturation-splicing processes. A homotetramer-type of splicing endonuclease (like in all Crenarchaeota) instead of a homodimeric-type of enzyme (as in most Euryarchaeota) appears to best fit the requirement for splicing introns at relaxed hBH or HBh' motifs, and may represent the most primitive form of this enzyme.     

Intron definition in splicing of small Drosophila introns.

Approximately half of the introns in Drosophila melanogaster are too small to function in a vertebrate and often lack the pyrimidine tract associated with vertebrate 3' splice sites. Here, we report the splicing and spliceosome assembly properties of two such introns: one with a pyrimidine-poor 3' splice site and one with a pyrimidine-rich 3' splice site. The pyrimidine-poor intron was absolutely dependent on its small size for in vivo and in vitro splicing and assembly. As such, it had properties reminiscent of those of yeast introns. The pyrimidine-rich intron had properties intermediate between those of yeasts and vertebrates. This 3' splice site directed assembly of ATP-dependent complexes when present as either an intron or exon and supported low levels of in vivo splicing of a moderate-length intron. We propose that splice sites can be recognized as pairs across either exons or introns, depending on which distance is shorter, and that a pyrimidine-rich region upstream of the 3' splice site facilitates the exon mode.     

Identification of splicing signals in introns of yeast mitochondrial split genes: mutational alterations in intron bI1 and secondary structures in related introns.

Four mitochondrial mutations are known to block excision of intron I1 of the cob gene in S.cerevisiae. The nucleotide sequence alteration of one of them, M4873, has been determined. It is a deletion of 1 bp in a run of five G's at a distance of 30 to 34 bp upstream to the 3' splice point. Reversion is found to occur by restoration of the run of five G's either by insertion of 1 G (wild type reversion) or by transition A leads to G next to this run of G's (pseudo-wild type reversion). The effect of mutation and reversion on RNA splicing indicates that the run of five G's is of critical importance for intron I1 excision, possibly in participating in the formation of a splice signal with a helical structure. This presumption is confirmed by the observation that this sequence is part of a larger sequence of some 80 bp next to the 3' splice point which is conserved to some extend in the four mitochondrial introns (bI1, aI1, aI2, aI5) that survive after excision as circular RNAs. Most striking is the conservation of this sequence at the level of secondary structure.     

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.     

Branchpoint selection in the splicing of U12-dependent introns in vitro

In metazoans, splicing of introns from pre-mRNAs can occur by two pathways: the major U2-dependent or the minor U12-dependent pathways. Whereas the U2-dependent pathway has been well characterized, much about the U12-dependent pathway remains to be discovered. Most of the information regarding U12-type introns has come from in vitro studies of a very few known introns of this class. To expand our understanding of U12-type splicing, especially to test the hypothesis that the simple base-pairing mechanism between the intron and U12 snRNA defines the branchpoint of U12-dependent introns, additional in vitro splicing substrates were created from three putative U12-type introns: the third intron of the Xenopus RPL1 a gene (XRP), the sixth intron of the Xenopus TFIIS.oA gene (XTF), and the first intron of the human Sm E gene (SME). In vitro splicing in HeLa nuclear extract confirmed U12-dependent splicing of each of these introns. Surprisingly, branchpoint mapping of the XRP splicing intermediate shows use of the upstream rather than the downstream of two consecutive adenosines within the branchpoint sequence (BPS), contrary to the prediction based on alignment with the sixth intron of human P120, a U12-dependent intron whose branch site was previously determined. Also, in the SME intron, the position of the branchpoint A residue within the region base paired with U12 differs from that in P120 and XTF. Analysis of these three additional introns therefore rules out simple models for branchpoint selection by the U12-type spliceosome.     

Analysis of the role of Caenorhabditis elegans GC-AG introns in regulated splicing

GC-AG introns represent 0.7% of total human pre-mRNA introns. To study the function of GC-AG introns in splicing regulation, 196 cDNA-confirmed GC-AG introns were identified in Caenorhabditis elegans. These represent 0.6% of the cDNA- confirmed intron data set for this organism. Eleven of these GC-AG introns are involved in alternative splicing. In a comparison of the genomic sequences of homologous genes between C.elegans and Caenorhabditis briggsae for 26 GC-AG introns, the C at the +2 position is conserved in only five of these introns. A system to experimentally test the function of GC-AG introns in alternative splicing was developed. Results from these experiments indicate that the conserved C at the +2 position of the tenth intron of the let-2 gene is essential for developmentally regulated alternative splicing. This C allows the splice donor to function as a very weak splice site that works in balance with an alternative GT splice donor. A weak GT splice donor can functionally replace the GC splice donor and allow for splicing regulation. These results indicate that while the majority of GC-AG introns appear to be constitutively spliced and have no evolutionary constraints to prevent them from being GT-AG introns, a subset of GC-AG introns is involved in alternative splicing and the C at the +2 position of these introns can have an important role in splicing regulation.     

Nuclear genes that promote splicing of group I introns in the chloroplast 23S rRNA and psbA genes in Chlamydomonas reinhardtii

Defence against pathogens in Arabidopsis is orchestrated by at least three signalling molecules: salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). The hrl1 (hypersensitive response-like lesions 1) mutant of Arabidopsis is characterized by spontaneous necrotic lesions, accumulation of reactive oxygen species, constitutive expression of SA- and ET/JA-responsive defence genes, and enhanced resistance to virulent bacterial and oomycete pathogens. Epistasis analyses of hrl1 with npr1, etr1, coi1 and SA-depleted nahG plants revealed novel interactions between SA and ET/JA signalling pathways in regulating defence gene expression and cell death. RNA gel-blot analysis of RNA isolated separately from the lesion+ and the lesion- leaves of double mutants of hrl1 revealed different signalling requirements for the expression of defence genes in these tissues. Expression of the ET/JA-responsive PDF1.2 gene was markedly reduced in hrl1 npr1 and in SA-depleted hrl1 nahG plants. In hrl1 nahG plants, expression of PDF1.2 was regulated by benzathiadiazole in a concentration-dependent manner: induced at low concentration and suppressed at high concentration. The hrl1 etr1 plants lacked systemic PR-1 expression, and exhibited compromised resistance to virulent Pseudomonas syringae and Peronospora parasitica. Inhibiting JA responses in hrl1 coi1 plants lead to exaggerated cell death and severe stunting of plants. Finally, the hrl1 mutation lead to elevated expression of AtrbohD, which encodes a major subunit of the NADPH oxidase complex. Our results indicate that defence gene expression and resistance against pathogens in hrl1 is regulated synergistically by SA and ET/JA defence pathways.     

Interactions between introns via exon definition in plant pre-mRNA splicing

The barley gene Mlo encodes a prototype of a novel class of plant proteins. In mlo mutants, absence of the 60 kDa wild-type Mlo protein results in broad-spectrum resistance to the powdery mildew fungus, Erysiphe graminis f. sp. hordei . To directly assess its function, Mlo was transiently expressed with a marker gene encoding a modified green fluorescent protein (GFP) in leaf epidermal cells of mlo resistant barley lines. Fungal inoculation of epidermal cells transfected with wild-type Mlo led to haustorium formation and abundant sporulation. Therefore, expression of the wild-type Mlo gene, in mlo resistant genotypes, is both necessary and sufficient to restore susceptibility to fungal attack. Complementation of mlo resistance alleles was restricted to single host cells, indicating a cell-autonomous function for the wild-type Mlo protein. We discuss our findings with respect to source–sink relationships of plants and biotrophic fungi and the potentially wide-ranging use of the transient complementation assay to analyse host compatibility and defence in response to powdery mildew attack.     

Violating the splicing rules: TG dinucleotides function as alternative 3' splice sites in U2-dependent introns

Background  

Despite some degeneracy of sequence signals that govern splicing of eukaryotic pre-mRNAs, it is an accepted rule that U2-dependent introns exhibit the 3' terminal dinucleotide AG. Intrigued by anecdotal evidence for functional non-AG 3' splice sites, we carried out a human genome-wide screen.     

Identification of structural motifs and amino acids within the structure of human heparan sulfate 3-O-sulfotransferase that mediate enzymatic function.

In an accompanying paper [J. R. Myette, Z. Shriver, J. Liu, G. Venkataraman, and R. Sasisekharan (2002) Biochem. Biophys. Res. Commun. 290, 1206-1213], we described the purification and biochemical characterization of a soluble, recombinantly expressed form of the human heparan sulfate 3-O-sulfotransferase (3-OST-1). Such an important first step enables detailed structure-function studies for this class of enzymes. Herein, we describe a complimentary, structure-based homology modeling approach for predicting 3-OST-1 structure. This approach employs a variety of structural analysis and molecular modeling tools used in conjunction with protein crystallographic studies of related enzymes. In this manner, we describe important motifs within the predicted three-dimensional structure of the enzyme and identify specific amino acids that are likely important for enzymatic function.     

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.