U12-type spliceosomal introns of Insecta

Most of eukaryotic genes are interrupted by introns that need to be removed from pre-mRNAs before they can perform their function. This is done by complex machinery called spliceosome. Many eukaryotes possess two separate spliceosomal systems that process separate sets of introns. The major (U2) spliceosome removes majority of introns, while minute fraction of intron repertoire is processed by the minor (U12) spliceosome. These two populations of introns are called U2-type and U12-type, respectively. The latter fall into two subtypes based on the terminal dinucleotides. The minor spliceosomal system has been lost independently in some lineages, while in some others few U12-type introns persist. We investigated twenty insect genomes in order to better understand the evolutionary dynamics of U12-type introns. Our work confirms dramatic drop of U12-type introns in Diptera, leaving these genomes just with a handful cases. This is mostly the result of intron deletion, but in a number of dipteral cases, minor type introns were switched to a major type, as well. Insect genes that harbor U12-type introns belong to several functional categories among which proteins binding ions and nucleic acids are enriched and these few categories are also overrepresented among these genes that preserved minor type introns in Diptera.     

Whole-genome sequencing of a laboratory-evolved yeast strain

Background

Two categories of introns are known, a common U2 type and a rare U12 type. These two types of introns are removed by distinct spliceosomes. The phylogenetic distribution of spliceosomal RNAs that are characteristic of the U12 spliceosome, i.e. the U11, U12, U4atac and U6atac RNAs, suggest that U12 spliceosomes were lost in many phylogenetic groups. We have now examined the distribution of U2 and U12 introns in many of these groups.

Results

U2 and U12 introns were predicted by making use of available EST and genomic sequences. The results show that in species or branches where U12 spliceosomal components are missing, also U12 type of introns are lacking. Examples are the choanoflagellate Monosiga brevicollis, Entamoeba histolytica, green algae, diatoms, and the fungal lineage Basidiomycota. Furthermore, whereas U12 splicing does not occur in Caenorhabditis elegans, U12 introns as well as U12 snRNAs are present in Trichinella spiralis, which is deeply branching in the nematode tree. A comparison of homologous genes in T. spiralis and C. elegans revealed different mechanisms whereby U12 introns were lost.

Conclusions

The phylogenetic distribution of U12 introns and spliceosomal RNAs give further support to an early origin of U12 dependent splicing. In addition, this distribution identifies a large number of instances during eukaryotic evolution where such splicing was lost.     

Computational screen for spliceosomal RNA genes aids in defining the phylogenetic distribution of major and minor spliceosomal components

The RNA molecules of the spliceosome are critical for specificity and catalysis during splicing of eukaryotic pre-mRNA. In order to examine the evolution and phylogenetic distribution of these RNAs, we analyzed 149 eukaryotic genomes representing a broad range of phylogenetic groups. RNAs were predicted using high-sensitivity local alignment methods and profile HMMs in combination with covariance models. The results provide the most comprehensive view so far of the phylogenetic distribution of spliceosomal RNAs. RNAs were predicted in many phylogenetic groups where these RNA were not previously reported. Examples are RNAs of the major (U2-type) spliceosome in all fungal lineages, in lower metazoa and many protozoa. We also identified the minor (U12-type) spliceosomal U11 and U6atac RNAs in Acanthamoeba castellanii, where U12 spliceosomal RNA as well as minor introns were reported recently. In addition, minor-spliceosome-specific RNAs were identified in a number of phylogenetic groups where previously such RNAs were not observed, including the nematode Trichinella spiralis, the slime mold Physarum polycephalum and the fungal lineages Zygomycota and Chytridiomycota. The detailed map of the distribution of the U12-type RNA genes supports an early origin of the minor spliceosome and points to a number of occasions during evolution where it was lost.     

Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5'-cap structure

Few genes in the divergent eukaryote Trichomonas vaginalis have introns, despite the unusually large gene repertoire of this human-infective parasite. These introns are characterized by extended conserved regulatory motifs at the 5' and 3' boundaries, a feature shared with another divergent eukaryote, Giardia lamblia, but not with metazoan introns. This unusual characteristic of T. vaginalis introns led us to examine spliceosomal small nuclear RNAs (snRNAs) predicted to mediate splicing reactions via interaction with intron motifs. Here we identify T. vaginalis U1, U2, U4, U5, and U6 snRNAs, present predictions of their secondary structures, and provide evidence for interaction between the U2/U6 snRNA complex and a T. vaginalis intron. Structural models predict that T. vaginalis snRNAs contain conserved sequences and motifs similar to those found in other examined eukaryotes. These data indicate that mechanisms of intron recognition as well as coordination of the two catalytic steps of splicing have been conserved throughout eukaryotic evolution. Unexpectedly, we found that T. vaginalis spliceosomal snRNAs lack the 5' trimethylguanosine cap typical of snRNAs and appear to possess unmodified 5' ends. Despite the lack of a cap structure, U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III.     

Identification of the major spliceosomal RNAs in Dictyostelium discoideum reveals developmentally regulated U2 variants and polyadenylated snRNAs

Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5' cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5' end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm.     

Origin of Spliceosomal Introns and Alternative Splicing

In this work we review the current knowledge on the prehistory, origins, and evolution of spliceosomal introns. First, we briefly outline the major features of the different types of introns, with particular emphasis on the nonspliceosomal self-splicing group II introns, which are widely thought to be the ancestors of spliceosomal introns. Next, we discuss the main scenarios proposed for the origin and proliferation of spliceosomal introns, an event intimately linked to eukaryogenesis. We then summarize the evidence that suggests that the last eukaryotic common ancestor (LECA) had remarkably high intron densities and many associated characteristics resembling modern intron-rich genomes. From this intron-rich LECA, the different eukaryotic lineages have taken very distinct evolutionary paths leading to profoundly diverged modern genome structures. Finally, we discuss the origins of alternative splicing and the qualitative differences in alternative splicing forms and functions across lineages.     

pICln Inhibits snRNP Biogenesis by Binding Core Spliceosomal Proteins

The U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) form essential components of spliceosomes, the machinery that removes introns from pre-mRNAs in eukaryotic cells. A critical initial step in the complex process of snRNP biogenesis is the assembly of a group of common core proteins (Sm proteins) on spliceosomal snRNA. In this study we show by multiple independent methods that the protein pICln associates with Sm proteins in vivo and in vitro. The binding of pICln to Sm proteins interferes with Sm protein assembly on spliceosomal snRNAs and inhibits import of snRNAs into the nucleus. Furthermore, pICln prevents the interaction of Sm proteins with the survival of motor neurons (SMN) protein, an interaction that has been shown to be critical for snRNP biogenesis. These findings lead us to propose a model in which pICln participates in the regulation of snRNP biogenesis, at least in part by interfering with Sm protein interaction with SMN protein.     

The evolution of spliceosomal introns

Although the widespread proliferation of introns in eukaryotic protein-coding genes remains one of the most poorly understood aspects of genomic architecture, major advances have emerged recently from large-scale genome sequencing projects and functional analyses of mRNA-processing events. Evidence supports the idea that spliceosomal introns were not only present in the stem eukaryote but diverged into at least two distinct classes very early in eukaryotic evolution. Some rough estimates of intron turnover rates are provided, and a testable hypothesis for the origin of new introns is proposed. In light of recent findings on the molecular natural history of splicing, various aspects of the phylogenetic and physical distributions of introns can now be interpreted in a theoretical framework that jointly considers the population-genetic roles of mutation, random genetic drift, and natural selection.     

Evolutionary dynamics of U12-type spliceosomal introns

Background  

Many multicellular eukaryotes have two types of spliceosomes for the removal of introns from messenger RNA precursors. The major (U2) spliceosome processes the vast majority of introns, referred to as U2-type introns, while the minor (U12) spliceosome removes a small fraction (less than 0.5%) of introns, referred to as U12-type introns. U12-type introns have distinct sequence elements and usually occur together in genes with U2-type introns. A phylogenetic distribution of U12-type introns shows that the minor splicing pathway appeared very early in eukaryotic evolution and has been lost repeatedly.     

Association of intron phases with conservation at splice site sequences and evolution of spliceosomal introns.

How exon-intron structures of eukaryotic genes evolved under various evolutionary forces remains unknown. The phases of spliceosomal introns (the placement of introns with respect to reading frame) provide an opportunity to approach this question. When a large number of nuclear introns in protein-coding genes were analyzed, it was found that most introns were of phase 0, which keeps codons intact. We found that the phase distribution of spliceosomal introns is strongly correlated with the sequence conservation of splice signals in exons; the relatively underrepresented phase 2 introns are associated with the lowest conservation, the relatively overrepresented phase 0 introns display the highest conservation, and phase 1 introns are intermediate. Given the detrimental effect of mutations in exon sequences near splice sites as found in molecular experiments, the underrepresentation of phase 2 introns may be the result of deleterious-mutation-driven intron loss, suggesting a possible genetic mechanism for the evolution of intron-exon structures.     

Sm/Lsm Genes Provide a Glimpse into the Early Evolution of the Spliceosome

The spliceosome, a sophisticated molecular machine involved in the removal of intervening sequences from the coding sections of eukaryotic genes, appeared and subsequently evolved rapidly during the early stages of eukaryotic evolution. The last eukaryotic common ancestor (LECA) had both complex spliceosomal machinery and some spliceosomal introns, yet little is known about the early stages of evolution of the spliceosomal apparatus. The Sm/Lsm family of proteins has been suggested as one of the earliest components of the emerging spliceosome and hence provides a first in-depth glimpse into the evolving spliceosomal apparatus. An analysis of 335 Sm and Sm-like genes from 80 species across all three kingdoms of life reveals two significant observations. First, the eukaryotic Sm/Lsm family underwent two rapid waves of duplication with subsequent divergence resulting in 14 distinct genes. Each wave resulted in a more sophisticated spliceosome, reflecting a possible jump in the complexity of the evolving eukaryotic cell. Second, an unusually high degree of conservation in intron positions is observed within individual orthologous Sm/Lsm genes and between some of the Sm/Lsm paralogs. This suggests that functional spliceosomal introns existed before the emergence of the complete Sm/Lsm family of proteins; hence, spliceosomal machinery with considerably fewer components than today's spliceosome was already functional.     

Base pairing with U6atac snRNA is required for 5' splice site activation of U12-dependent introns in vivo.

The minor U12-dependent class of eukaryotic nuclear pre-mRNA introns is spliced by a distinct spliceosomal mechanism that requires the function of U11, U12, U5, U4atac, and U6atac snRNAs. Previous work has shown that U11 snRNA plays a role similar to U1 snRNA in the major class spliceosome by base pairing to the conserved 5'' splice site sequence. Here we show that U6atac snRNA also base pairs to the 5'' splice site in a manner analogous to that of U6 snRNA in the major class spliceosome. We show that splicing defective mutants of the 5'' splice site can be activated for splicing in vivo by the coexpression of compensatory U6atac snRNA mutants. In some cases, maximal restoration of splicing required the coexpression of compensatory U11 snRNA mutants. The allelic specificity of mutant phenotype suppression is consistent with Watson-Crick base pairing between the pre-mRNA and the snRNAs. These results provide support for a model of the RNA-RNA interactions at the core of the U12-dependent spliceosome that is strikingly similar to that of the major class U2-dependent spliceosome.     

Splicing of a divergent subclass of AT-AC introns requires the major spliceosomal snRNAs.

AT-AC introns constitute a minor class of eukaryotic pre-mRNA introns, characterized by 5''-AT and AC-3'' boundaries, in contrast to the 5''-GT and AG-3'' boundaries of the much more prevalent conventional introns. In addition to the AT-AC borders, most known AT-AC introns have highly conserved 5'' splice site and branch site sequence elements of 7-8 nt. Intron 6 of the nucleolar P120 gene and intron 2 of the SCN4A voltage-gated skeletal muscle sodium channel are AT-AC introns that have been shown recently to be processed via a unique splicing pathway involving several minor U snRNAs. Interestingly, intron 21 of the same SCN4A gene and the corresponding intron 25 of the SCN5A cardiac muscle sodium channel gene also have 5''-AT and AC-3'' boundaries, but they have divergent 5'' splice site and presumptive branch site sequences. Here, we report the accurate in vitro processing of these two divergent AT-AC introns and show that they belong to a functionally distinct subclass of AT-AC introns. Splicing of these introns does not require U12, U4atac, and U6atac snRNAs, but instead requires the major spliceosomal snRNAs U1, U2, U4, U5, and U6. Previous studies showed that G --> A mutation at the first position and G --> C mutation at the last position of a conventional yeast or mammalian GT-AG intron suppress each other in vivo, suggesting that the first and last bases participate in an essential non-Watson-Crick interaction. Our results show that such introns, hereafter termed AT-AC II introns, occur naturally and are spliced by a mechanism distinct from that responsible for processing of the apparently more common AT-AC I introns.     

Computational identification of four spliceosomal snRNAs from the deep-branching eukaryote Giardia intestinalis

RNAs processing other RNAs is very general in eukaryotes, but is not clear to what extent it is ancestral to eukaryotes. Here we focus on pre-mRNA splicing, one of the most important RNA-processing mechanisms in eukaryotes. In most eukaryotes splicing is predominantly catalysed by the major spliceosome complex, which consists of five uridine-rich small nuclear RNAs (U-snRNAs) and over 200 proteins in humans. Three major spliceosomal introns have been found experimentally in Giardia; one Giardia U-snRNA (U5) and a number of spliceosomal proteins have also been identified. However, because of the low sequence similarity between the Giardia ncRNAs and those of other eukaryotes, the other U-snRNAs of Giardia had not been found. Using two computational methods, candidates for Giardia U1, U2, U4 and U6 snRNAs were identified in this study and shown by RT-PCR to be expressed. We found that identifying a U2 candidate helped identify U6 and U4 based on interactions between them. Secondary structural modelling of the Giardia U-snRNA candidates revealed typical features of eukaryotic U-snRNAs. We demonstrate a successful approach to combine computational and experimental methods to identify expected ncRNAs in a highly divergent protist genome. Our findings reinforce the conclusion that spliceosomal small-nuclear RNAs existed in the last common ancestor of eukaryotes.     

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.     

Spliceosomal introns: new insights into their evolution

A new genome-wide analysis of spliceosomal introns indicates massive loss and gain of introns has taken place in many eukaryotic lineages. Only a small subset of the analyzed introns was present in the common ancestor of plants, fungi, animals and Plasmodium.