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Significant advances have been made in elucidating the biogenesis pathway and three-dimensional structure of the UsnRNPs, the building blocks of the spliceosome. U2 and U4/U6*U5 tri-snRNPs functionally associate with the pre-mRNA at an earlier stage of spliceosome assembly than previously thought, and additional evidence supporting UsnRNA-mediated catalysis of pre-mRNA splicing has been presented.
A novel form of spliceosome, containing the minor snRNPs U11 and U12, splices a class of pre-mRNA introns with non-consensus splice sites. This unexpected spliceosome diversity has interesting implications for the evolution and expression of eukaryotic genes.
A minor class of pre-mRNA introns whose excision requires a spliceosome containing U11, U12, U4atac/U6atac, and U5 snRNPs has been identified in plants, insects, and vertebrates. We have characterized single loci that specify the U6atac and U12 snRNAs of Drosophila melanogaster. P element-mediated disruptions of the U6atac and U12 genes cause lethality during the third instar larval and embryonic stages, respectively, and are rescued by U6atac and U12 transgenes. The P element disruption of U6atac results in excision defects of U12-type introns from several transcripts including an alternative U12-dependent spliced isoform of prospero, a homeodomain protein required for CNS development. Thus, we demonstrate the requirement for the U12 spliceosome in the development of a metazoan organism.
Conformational change within the spliceosome is required between the first catalytic step of pre-mRNA splicing, when the branch site (BS) attacks the 5' splice site, and the second step, when the 5' exon attacks the 3' splice site, yielding mRNA and lariat-intron products. A genetic screen for suppressors of BS A-to-G mutants, which stall between the two steps, identified Prp8, the highly conserved spliceosomal factor. prp8 suppressors facilitate the second step for multiple intron mutants and interact functionally with first step suppressors, alleles of PRP16 and U6 snRNA. Genetic interactions among prp8, prp16, and U6 alleles suggest that these factors control a common stage in first-to-second step transition. We propose that mutant substrates are utilized by alteration of the equilibrium between first/second step conformations, resembling tRNA miscoding caused by altered equilibrium between open/closed ribosomal conformations. This mechanistic commonality suggests that alteration of rearrangements represents an evolutionarily convenient way of modulating substrate selectivity.
Gene therapy of large genes (e.g. plectin and collagen genes) is hampered by size limitations for insertions of the currently used viral vectors. To reduce the size of these insertions spliceosome-mediated RNA trans-splicing (SMaRT), which provides intron-specific gene-correction at the pre-RNA level, can be an alternative approach. To test its applicability in skin gene therapy, SMaRT was used in the context of the 4003delTC mutation in the collagen XVII gene (COL17A1) causing generalized atrophic benign junctional epidermolysis bullosa. A beta-galactosidase (beta-gal) trans-splicing assay system was established using intron 51 of COL17A1 as the target for trans-splicing. In this system, intron 51 is flanked by the 5'exon and the 3'exon of the beta-gal gene, the latter containing two in-frame stop codons. Cotransfection of a pre-trans-splicing molecule consisting of the binding domain of intron 51 and the 3'exon of beta-gal without the stop codons resulted in a 300-fold increase of beta-gal activity compared to controls. A 2-3-fold increase in efficiency was obtained through an elongation of the binding domains. Replacement of the complete 3'end of the COL17A1 gene was shown using a collagen XVII mini-gene construct. The beta-gal assay was used in human keratinocytes to evaluate the influence of a keratinocyte-specific spliceosome background. Reverse transcription polymerase chain reaction and beta-gal activity assay showed functional correction of the stop-codons in cultured human keratinocytes and in an immortalized GABEB cell line harbouring the 4003delTC mutation. These results demonstrate that SMaRT is feasible in a keratinocyte-specific context and therefore may be applied in skin gene therapy.
Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors--the spliceosome--is a large multi-protein complex. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.
We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-mRNA encoding the beta-subunit of human chorionic gonadotropin gene 6 and pre-trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis- and trans-splicing demonstrated that cis-splicing can be inhibited by trans-splicing. RNA repair in a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significant beta-galactosidase activity. These observations suggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy.
The spliceosomal snRNPs U1, U2, U4 and U5 contain a common RNP structure termed the Sm core that is formed by the binding of Sm proteins onto the U snRNA. Although isolated Sm proteins assemble spontaneously onto U snRNAs in vitro, there is increasing evidence that SMN and its interactor Gemin2 are involved in this process in vivo. Here, we describe a cell-free assay system for the assembly of U snRNPs that closely reproduces in vivo conditions. Using this system, we show that assembly of U1 snRNP depends on ATP. Immunodepletion of SMN-Gemin2 from the extract abolished assembly even though the extract contained high levels of Sm proteins. An affinity-purified macromolecular SMN complex consisting of 16 components including all Sm proteins restored assembly in the immunodepleted extract. These data provide the first direct evidence that a complex containing SMN and Gemin2 mediates the active assembly of spliceosomal U snRNPs.
The origins and importance of spliceosomal introns comprise one of the longest-abiding mysteries of molecular evolution. Considerable debate remains over several aspects of the evolution of spliceosomal introns, including the timing of intron origin and proliferation, the mechanisms by which introns are lost and gained, and the forces that have shaped intron evolution. Recent important progress has been made in each of these areas. Patterns of intron-position correspondence between widely diverged eukaryotic species have provided insights into the origins of the vast differences in intron number between eukaryotic species, and studies of specific cases of intron loss and gain have led to progress in understanding the underlying molecular mechanisms and the forces that control intron evolution.
The small nuclear RNA U4atac is a core component of the minor spliceosome. In humans, homozygous or compound heterozygous point mutations in U4atac cause rare developmental disorders, such as Roifman syndrome, characterized by growth restriction, brain anomalies, and immune deficiency. To better define the pathophysiological role of U4atac mutations, we here establish a model of minor spliceosome dysfunction by generating a Drosophila melanogaster CRISPR/Cas9-induced U4atac mutant in the highly conserved stem II region. U4atac homozygous mutants exhibit growth and neurodevelopmental defects, immunodeficiency, and gastrointestinal symptoms. Using bulk RNA-sequencing and functional assays, we reveal that mutations in U4atac affect the splicing of a large set of transcripts involved in innate immunity, hematopoiesis, and intestinal cell functions, including the Drosophila Janus kinase (JAK) homolog hopscotch (hop). Importantly, U4atac deficiency reduces Hop expression and causes Hop-related hematopoietic defects at the embryonic and larval stages. Notably, we also observe reduced expression of Jak1 and attenuated activation of downstream signaling in patients with Roifman syndrome. Thus, our work identifies alterations of Jak signaling as part of the pathogenesis of RNU4atac-opathy.
The spliceosome is a macromolecular machine that carries out the excision of introns from eukaryotic pre-mRNAs and splicing together of exons. Four large RNA-protein complexes, called the U1, U2, U4/U6 and U5 small nuclear ribonucleoprotein particles (snRNPs), and some non-snRNP proteins assemble around three short conserved sequences within the intron in an ordered manner to form the active spliceosome. We aim to provide insight into the molecular details of the mechanism of pre-mRNA splicing through crystallographic studies of the snRNPs. We have solved the X-ray crystal structure of some snRNP proteins as part of either protein-protein complexes or RNA-protein complexes. These structures have provided an important insight into the overall architecture of the U1 and U2 snRNPs and the mechanisms of RNA-protein and protein-protein recognition.
Spliceosome-associated proteins (SAPs) 61, 62, and 114 can be UV-crosslinked to pre-mRNA in purified spliceosomal complexes and are associated with U2 small nuclear ribonucleoproteins (snRNP). These proteins also compose the essential heterotrimeric splicing factor SF3a, and products of yeast pre-mRNA processing genes PRP9, PRP11, and PRP21 are their likely yeast counterparts. We report the isolation of a cDNA encoding SAP 61 and find that it is 30% identical in amino acid sequence to PRP9. A C-terminal Cys2His2 zinc-finger-like motif, which could be involved in the pre-mRNA binding, is the most highly conserved region of the protein. We also demonstrate specific protein-protein interactions between SAPs 61 and 114 and show that the N terminus of SAP 61 is required for this interaction. Significantly, the corresponding proteins are also known to interact in yeast: PRP9 interacts with PRP21, and the N-terminal portion of PRP9 is required. Previous work showed that direct interactions also occur between SAPs 62 and 114 and between the corresponding PRPs 11 and 21. These observations indicate that the specific protein-protein interactions that occur between the three prespliceosomal factors have been conserved between yeast and mammals.
Chlorarachniophyte algae contain a complex, multi-membraned chloroplast derived from the endosymbiosis of a eukaryotic alga. The vestigial nucleus of the endosymbiont, called the nucleomorph, contains only three small linear chromosomes with a haploid genome size of 380 kb and is the smallest known eukaryotic genome. Nucleotide sequence data from a subtelomeric fragment of chromosome III were analyzed as a preliminary investigation of the coding capacity of this vestigial genome. Several housekeeping genes including U6 small nuclear RNA (snRNA), ribosomal proteins S4 and S13, a core protein of the spliceosome [small nuclear ribonucleoprotein (snRNP) E], and a cip-like protease (clpP) were identified. Expression of these genes was confirmed by combinations of Northern blot analysis, in situ hybridization, immunocytochemistry, and cDNA analysis. The protein-encoding genes are typically eukaryotic in overall structure and their messenger RNAs are polyadenylylated. A novel feature is the abundance of 18-, 19-, or 20-nucleotide introns; the smallest spliceosomal introns known. Two of the genes, U6 and S13, overlap while another two genes, snRNP E and clpP, are cotranscribed in a single mRNA. The overall gene organization is extraordinarily compact, making the nucleomorph a unique model for eukaryotic genomics.
The Saccharomyces cerevisiae genes PRP2, PRP16, and PRP22 encode pre-mRNA splicing factors that belong to the highly conserved "DEAH" family of putative RNA helicases. We previously identified two additional members of this family, JA1 and JA2. To investigate its biological function, we cloned the JA1 gene and generated alleles carrying mutations identical to those found in highly conserved regions of other members of the DEAH family. A ja1 allele carrying a mutation identical to that in the temperature-sensitive (ts) prp22-1 gene conferred ts phenotype when integrated into the genome of a wild-type strain by gene replacement. Northern analysis of RNA obtained from the ts strain shifted to a nonpermissive temperature revealed accumulation of unspliced pre-mRNAs and excised intron lariats. Furthermore, analysis of splicing complexes showed that intron lariats accumulated in spliceosomes. The results presented indicate that JA1 encodes a pre-mRNA processing factor (Prp) involved in disassembly of spliceosomes after the release of mature mRNA. We have therefore renamed this gene PRP43.
The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately downstream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3' splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3' splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind to the ESS showed that the U-rich 5' region binds U2AF65 and polypyrimidine tract binding protein, the C-rich central part binds 35- and 54-55-kDa serine/arginine-rich (SR) proteins, and the AG-rich 3' end binds alternative splicing factor/splicing factor 2. Mutational and functional studies indicated that the most critical region of the ESS maps to the central C-rich core (GGCUCCCCC). This core sequence, along with additional nonspecific downstream nucleotides, is sufficient for partial suppression of spliceosome assembly and splicing of BPV-1 pre-mRNAs. The inhibition of splicing by the ESS can be partially relieved by excess purified HeLa SR proteins, suggesting that the ESS suppresses pre-mRNA splicing by interfering with normal bridging and recruitment activities of SR proteins.
The evolutionary origin of spliceosomal introns has been the subject of much controversy. Introns are proposed to have been both lost and gained during evolution. If the gain or loss of introns are unique events in evolution, they can serve as markers for phylogenetic analysis. We have made an extensive survey of the phylogenetic distribution of seven spliceosomal introns that are present in Fugu genes, but not in their mammalian homologues; we show that these introns were acquired by actinopterygian (ray-finned) fishes at various stages of evolution. We have also investigated the intron pattern of the rhodopsin gene in fishes, and show that the four introns found in the ancestral chordate rhodopsin gene were simultaneously lost in a common ancestor of ray-finned fishes. These changes in introns serve as excellent markers for phylogenetic analysis because they reliably define clades. Our intron-based cladogram establishes the difficult-to-ascertain phylogenetic relationships of some ray-finned fishes. For example, it shows that bichirs (Polypterus) are the sister group of all other extant ray-finned fishes.
We have analyzed the sequence requirements of the human U5 RNA during small nuclear ribonucleoprotein (snRNP) and spliceosome assembly. A collection of mutant derivatives of the human U5 RNA gene was constructed in a U1 expression vector and transiently transfected in mammalian cells. Using immunoprecipitation and affinity selection assays, the cap trimethylation, the binding of Sm proteins and of the U5 snRNP-specific protein p220, as well as the assembly of the U4/U5/U6 triple snRNP and of spliceosomes were determined. By mutational analysis we were able to assign distinct functions to several structural elements of the human U5 RNA. Efficient binding of the Sm proteins requires the 3' stem-loop. Both the Sm protein-binding site and the 3' stem-loop are necessary for the formation of the trimethyl guanosine cap, consistent with Sm protein binding being a prerequisite for cap trimethylation. Specific elements of the U5 RNA 5' stem-loop contribute to efficient p220 association, in particular stem Ib. Interestingly, the highly conserved loop I appears to be a multifunctional element; in addition to its function in splice-site selection the 5' loop is involved in binding of p220 and in the assembly of the U4/U5/U6 triple snRNP. In sum, this mutational analysis has identified four functional domains of the human U5 RNA.
Two sequences important for pre-mRNA splicing precede the 3' end of introns in higher eukaryotes, the branch point (BP) and the polypyrimidine (Py) tract. Initial recognition of these signals involves cooperative binding of the splicing factor SF1/mammalian branch point binding protein (mBBP) to the BP and of U2AF(65) to the Py tract. Both factors are required for recruitment of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) to the BP in reactions reconstituted from purified components. In contrast, extensive depletion of ST1/BBP in Saccharomyces cerevisiae does not compromise spliceosome assembly or splicing significantly. As BP sequences are less conserved in mammals, these discrepancies could reflect more stringent requirements for SF1/BBP in this system. We report here that extensive depletion of SF1/mBBP from nuclear extracts of HeLa cells results in only modest reduction of their activity in spliceosome assembly and splicing. Some of these effects reflect differences in the kinetics of U2 snRNP binding. Although U2AF(65) binding was reduced in the depleted extracts, the defects caused by SF1/mBBP depletion could not be fully restored by an increase in occupancy of the Py tract by exogenously added U2AF(65), arguing for a role of SF1/mBBP in U2 snRNP recruitment distinct from promoting U2AF(65) binding.