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PubMed1998

Modification of U6 spliceosomal RNA is guided by other small RNAs.

The vertebrate spliceosomal snRNAs are highly modified by pseudouridylation and 2'-O-methylation. We have identified novel conserved small RNAs that can direct addition of two methyl groups in U6 snRNA, at A47 and C77. These guide RNAs, mgU6-47 (methylation guide for U6 snRNA residue 47) and mgU6-77 contain boxes C, C', D, and D' and associate with fibrillarin. Each RNA can form a duplex with U6 snRNA positioning A47 and C77 for 2'-O-methylation. The antisense element of mgU6-77 can also position C2970 of 28S rRNA for 2'-O-methylation. Depletion of mgU6-77 from Xenopus oocytes prevents 2'-O-methylation of both C77 in U6 and C2970 in 28S; methylation can be restored by injecting in vitro transcribed mgU6-77. Thus, mgU6-77 appears to function in the 2'-O-methylation of two distinct classes of cellular RNA, snRNA, and rRNA.

Animals↗

PubMed2002

The spliceosome: no assembly required?

In this issue of Molecular Cell, Stevens et al. purify a large particle from yeast extracts that contains all five of the U snRNPs required for pre-mRNA splicing. The existence of this "penta-snRNP" suggests the provocative possibility that spliceosome assembly does not depend upon a pre-mRNA substrate.

RNA Precursors↗

PubMed1998

Probing of the spliceosome with site-specifically derivatized 5' splice site RNA oligonucleotides.

We have developed a site-specific chemical modification technique to incorporate a photoreactive azidophenacyl (APA) group at designated internal positions along the RNA phosphodiester backbone. Using this technique, we have analyzed interactions of the 5' splice site (5'SS) RNA within the spliceosome. Several crosslinked products can be detected within complex B using the derivatized 5'SS RNAs, including U6 snRNA, hPrp8p, and 114-, 90-, 70-, 54-, and 27-kDa proteins. The 5'SS RNAs derivatized at intron positions +4 to +8 crosslink to U6 snRNA, confirming the previously reported pairing interaction between these sequences. hPrp8p and p70 are crosslinked to the 5'SS RNA when the APA is placed within the 5' exon. Finally, a set of unidentified proteins, including p114, p54, and p27, is detected with the 5'SS RNA derivatized at intron positions +4 to +8. Introduction of the bulky APA group near the 5'SS junction (positions -2 to +3) strongly interferes with complex B formation and thus no APA crosslinks are observed at these positions. Together with our earlier observation that hPrp8p crosslinks to the GU dinucleotide at the 5' end of the intron, these results suggest that the inhibitory effect of APA results from steric hindrance of the hPrp8p:5'SS interaction. Unexpectedly, thio-modifications within the region of the 5'SS RNA that is involved in base pairing to U6 snRNA strongly stimulate complex B formation.

Allosteric Site↗

PubMed1998

Target discrimination by RNA-binding proteins: role of the ancillary protein U2A' and a critical leucine residue in differentiating the RNA-binding specificity of spliceosomal proteins U1A and U2B".

The spliceosomal proteins U1A and U2B" each use a homologous RRM domain to bind specifically to their respective snRNA targets, U1hpll and U2hpIV, two stem-loops that are similar yet distinct in sequence. Previous studies have shown that binding of U2B" to U2hpIV is facilitated by the ancillary protein U2A', whereas specific binding of U1A to U1hpll requires no cofactor. Here we report that U2A' enables U2B" to distinguish the loop sequence of U2hpIV from that of U1hpll but plays no role in stem sequence discrimination. Although U2A' can also promote heterospecific binding of U1A to U2hpIV, a much higher concentration of the ancillary protein is required due to the approximately 500-fold greater affinity of U2A' for U2B". Additional experiments have identified a single leucine residue in U1A(Leu-44) that is critical for the intrinsic specificity of this protein for the loop sequence of U1 hpll in preference to that of U2hpIV. Our data suggest that most of the difference in RNA-binding specificity between U1A and U2B" can be accounted for by this leucine residue and by the contribution of the ancillary protein U2A' to the specificity of U2B".

Amino Acid Sequence↗

PubMed2001

Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle.

In eukaryotic cells, freshly synthesized messenger RNA (pre-mRNA) contains stretches of non-coding RNA that must be excised before the RNA can be translated into protein. Their removal is catalysed by the spliceosome, a large complex formed when a number of small nuclear ribonucleoprotein particles (snRNPs) bind sequentially to the pre-mRNA. The first snRNP to bind is called U1; other snRNPs (U2, U4/U6 and U5) follow. Here we describe the three-dimensional structure of human U1 snRNP, determined by single-particle electron cryomicroscopy at 10 A resolution. The reconstruction reveals a doughnut-shaped central element that accommodates the seven Sm proteins common to all snRNPs, supporting a proposed model of circular Sm protein arrangement. By taking earlier biochemical results into account, we were able to assign the remaining density of the map to the other known components of U1 snRNP, deriving a structural model that describes the three-dimensional arrangement of proteins and RNA in U1 snRNP.

Cryoelectron Microscopy↗

PubMed2002

Partial correction of endogenous DeltaF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing.

Spliceosome-mediated RNA trans-splicing (SMaRT) was investigated as a means for functionally correcting endogenous DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) transcripts using in vitro human cystic fibrosis (CF) polarized airway epithelia and in vivo human CF bronchial xenografts. Recombinant adenovirus (Ad.CFTR-PTM) encoding a pre-therapeutic molecule (PTM) targeted to CFTR intron 9 corrected transepithelial cyclic AMP (cAMP)-sensitive short-circuit current (Isc) in DeltaF508 homozygous epithelia to a level 16% of that observed in normal human bronchial epithelia. Molecular analyses using RT-PCR and western blotting confirmed SMaRT-mediated partial correction of endogenous DeltaF508 messenger RNA (mRNA) transcripts and protein. In an in vivo model of DeltaF508 CF airway epithelia, human CF bronchial xenografts infected with Ad.CFTR-PTM also demonstrated partial correction of CFTR-mediated Cl- permeability at a level 22% of that seen in non-CF xenografts. These results provide functional evidence for SMaRT-mediated repair of mutant endogenous CFTR mRNA in intact polarized CF airway epithelial models.

Adenoviridae↗

PubMed2003

Phenotype correction of hemophilia A mice by spliceosome-mediated RNA trans-splicing.

Conventional gene therapy of hemophilia A relies on the transfer of factor VIII (FVIII; encoded by the F8 gene) cDNA. We carried out spliceosome-mediated RNA trans-splicing (SMaRT) to repair mutant FVIII mRNA. A pre-trans-splicing molecule (PTM) corrected endogenous FVIII mRNA in F8 knockout mice with the hemophilia A phenotype, producing sufficient functional FVIII to correct the hemophilia A phenotype. This is the first description of phenotypic correction of a genetic defect by RNA repair in a knockout animal model. Our results indicate the feasibility of using SMaRT to repair RNA for the treatment of genetic diseases.

Animals↗

PubMed2002

Sculpting of the spliceosomal branch site recognition motif by a conserved pseudouridine.

Pairing of a consensus sequence of the precursor (pre)-mRNA intron with a short region of the U2 small nuclear (sn)RNA during assembly of the eukaryotic spliceosome results in formation of a complementary helix of seven base pairs with a single unpaired adenosine residue. The 2' OH of this adenosine, called the branch site, brings about nucleophilic attack at the pre-mRNA 5' splice site in the first step of splicing. Another feature of this pairing is the phylogenetic conservation of a pseudouridine (psi) residue in U2 snRNA nearly opposite the branch site. We show that the presence of this psi in the pre-mRNA branch-site helix of Saccharomyces cerevisiae induces a dramatically altered architectural landscape compared with that of its unmodified counterpart. The psi-induced structure places the nucleophile in an accessible position for the first step of splicing.

Adenosine↗

PubMed2025

Alternative spliceosomal protein Eftud2 mediated Kif3a exon skipping promotes SHH-subgroup medulloblastoma progression.

Alternative splicing plays a pivotal role in various facets of organogenesis, immune response, and tumorigenesis. Medulloblastoma represents a prevalent childhood brain tumor, with approximately one-third classified as the Sonic Hedgehog (SHH) subgroup. Nevertheless, the contribution of alternative splicing to medulloblastoma oncogenesis remains elusive. This investigation delineated an upregulation of the spliceosomal protein Eftud2 in the SHH-subgroup medulloblastoma mouse model and human medulloblastoma patients. Targeted ablation of Eftud2 in granule precursor cells (GNPs) within the cerebellum prolonged the survival of SHH-subgroup medulloblastoma mice, indicating a putative association between Eftud2 expression and medulloblastoma prognosis. Functional assays unveiled that EFTUD2 depletion in human medulloblastoma cells significantly curtailed cellular proliferation by impeding the activation of the SHH signaling pathway. Through multi-omics sequencing analysis, it was discerned that Eftud2 influences exons 10-11 skipping of Kif3a, a kinesin motor critical for primary cilia formation. Notably, exons 10-11 skipping in Kif3a augmented human medulloblastoma cell proliferation by potentiating the transcriptional activity of Gli2. These findings underscore a robust correlation between Eftud2 and SHH-subgroup medulloblastoma, emphasizing its regulatory role in modulating downstream transcription factors through the alternative splicing of pivotal genes within the SHH signaling pathway, thereby propelling the aggressive proliferation of SHH-subgroup medulloblastoma.

Medulloblastoma↗

PubMed2005

Structural basis for m3G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1.

In higher eukaryotes the biogenesis of spliceosomal UsnRNPs involves a nucleocytoplasmic shuttling cycle. After the m7G-cap-dependent export of the snRNAs U1, U2, U4 and U5 to the cytoplasm, each of these snRNAs associates with seven Sm proteins. Subsequently, the m7G-cap is hypermethylated to the 2,2,7-trimethylguanosine (m3G)-cap. The import adaptor snurportin1 recognises the m3G-cap and facilitates the nuclear import of the UsnRNPs by binding to importin-beta. Here we report the crystal structure of the m3G-cap-binding domain of snurportin1 with bound m3GpppG at 2.4 A resolution, revealing a structural similarity to the mRNA-guanyly-transferase. Snurportin1 binds both the hypermethylated cap and the first nucleotide of the RNA in a stacked conformation. This binding mode differs significantly from that of the m7G-cap-binding proteins Cap-binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and viral protein 39 (VP39). The specificity of the m3G-cap recognition by snurportin1 was evaluated by fluorescence spectroscopy, demonstrating the importance of a highly solvent exposed tryptophan for the discrimination of m7G-capped RNAs. The critical role of this tryptophan and as well of a tryptophan continuing the RNA base stack was confirmed by nuclear import assays and cap-binding activity tests using several snurportin1 mutants.

Active Transport, Cell Nucleus↗

PubMed2005

Phosphorylation regulates the activity of the SMN complex during assembly of spliceosomal U snRNPs.

The assembly of spliceosomal U-rich small nuclear ribonucleoproteins (U snRNPs) is an ATP-dependent process mediated by the coordinated action of the SMN and the PRMT5 complex. Here, we provide evidence that the activity of this assembly machinery is regulated by means of post-translational modification. We show that two main components of the SMN/PRMT5 system, namely the survival motor neuron (SMN) protein (reduced levels thereof causing spinal muscular atrophy) and pICln, are phosphorylated in vivo. Both proteins share a previously unknown motif containing either one or two phosphoserines. Alteration of these residues in SMN (serines 28 and 31) significantly impairs the activity of the SMN complex. Despite the presence of SMN in both the nucleus and cytoplasm, we find that only the latter promotes efficient SMN-mediated U snRNP assembly activity. As cytoplasmic SMN is phosphorylated to a much larger extent, we hypothesize that this modification is a key activator of the SMN complex.

Amino Acid Sequence↗

PubMed1998

Structural/functional properties of a mammalian multi-component structure containing all major spliceosomal small nuclear ribonucleoprotein particles.

An approx. 40 S multi-component structure, consisting of all major spliceosomal small nuclear ribonucleoprotein particles (snRNP) (U1, U2, U4/U6 and U5) in stable association with a large number of polypeptides, mainly in the range 50-210 kDa, has been reported to exist within rat liver nuclear extracts [Guialis, Moraitou, Patrinou-Georgoula and Dangli (1991) Nucleic Acids Res. 19, 287-296]. Using a new polyclonal antibody recognizing a 63 kDa protein component of the complex, this multi-snRNP assembly was detected within rat liver nuclear extracts as efficiently as with the antibody for the U2 snRNP-specific B' polypeptide. The 63 kDa protein was found to correspond to the 66 kDa subunit of the splicing factor SF3a, a known integral component of the HeLa 17 S U2 snRNP. Anti-2,2,7-trimethylguanosine affinity chromatography was an easy and efficient way of purifying the multi-snRNP complex from rat liver 40 S heterogeneous nuclear ribonucleoprotein particle (hnRNP)-containing sucrose gradient fractions. By subsequent glycerol-gradient sedimentation, all known snRNP forms active in RNA splicing were identified among its constituents. A complex structurally similar to the rat multi-snRNP was also identified in HeLa nuclear extracts. Preservation of hnRNP-snRNP interactions was observed within HeLa 40 S fractions. Moreover, these fractions were capable of restoring splicing activity when applied in reconstitution studies to supplement a micrococcal nuclease-treated splicing extract.

Animals↗

PubMed2004

Roles of the U5 snRNP in spliceosome dynamics and catalysis.

Most protein-coding genes in eukaryotes are interrupted by non-coding intervening sequences (introns), which must be precisely removed from primary gene transcripts (pre-mRNAs) before translation of the message into protein. Intron removal by pre-mRNA splicing occurs in the nucleus and is catalysed by complex ribonucleoprotein machines called spliceosomes. These molecular machines consist of several small nuclear RNA molecules and their associated proteins [together termed snRNP (small nuclear ribonucleoprotein) particles], plus multiple accessory factors. Of particular interest are the U2, U5 and U6 snRNPs, which play crucial roles in the catalytic steps of splicing. In the present review, we summarize our current understanding of the role played by the protein components of the U5 snRNP in pre-mRNA splicing, which include some of the largest and most highly conserved nuclear proteins.

Catalysis↗

PubMed2000

The spliceosomal intron of the rolA gene of agrobacterium rhizogenes is a prokaryotic promoter.

Agrobacterium rhizogenes transfers DNA (T-DNA) from its Ri plasmid to plant cells. All T-DNA genes are expressed in plant cells. The rolA gene is the only T-DNA gene that contains an intron in the untranslated leader region of its mRNA. This paper shows that (i) the rolA gene is also transcribed in bacteria; (ii) the 85 bp corresponding to the spliceosomal intron drives prokaryotic gene expression in agrobacteria, in free-living rhizobia and in bacteroids within root nodules; and (iii) promoter activity is abolished by the deletion of 63 bp from its 5' end and is reduced by mutations changing its sequence near the putative -10 region. The expression pattern of a chimeric reporter gene shows that, in free-living bacteria, gene expression takes place during the exponential phase of growth and increases at the onset of the stationary phase. Within root nodules, reporter gene expression occurs in the invasion, nitrogen fixing and senescent zones.

Bacterial Proteins↗

PubMedDate not supplied

Key sequences involved in the spreading of the systemic autoimmune response to spliceosomal proteins.

Immune spreading to multiple intracellular antigens is likely to be of primary importance in organ-specific and systemic autoimmune diseases. A number of mechanisms by which immune spreading may occur from only a single autoreactive epitope have been proposed. Search for an initiator or early epitope thus represents an important area of investigation. For example, many studies have focused on the identification of epitopes recognized by the antibodies from both patients with systemic lupus erythematosus (SLE) and lupus-prone mice. Recently, an autoepitope present in the 70K U1 ribonucleo protein (RNP) and recognized by CD4+ T cells from lupus mice has also been identified. Here, we analyze the results of B- and T-cell-epitope mapping studies of several RNPs present in the spliceosome and propose a model of epitope spreading. In this model, a consensus sequence (the RNP motif) conserved in many nuclear, nucleolar and cytoplasmic antigens, might play a role as 'driver' epitope. This hypothesis is based on the observation that this sequence is recognized by CD4+ T cells from lupus mice and is often targeted by autoantibodies, very early during the course of the disease. Targeting this region that is repeated in different self-antigens, might represent an interesting strategy to interfere with the continuous T-cell stimulation and exposure to specific antigens.

Animals↗

PubMed2003

U4 small nuclear RNA can function in both the major and minor spliceosomes.

U4 small nuclear RNA (snRNA) and U6 snRNA form a base-paired di-snRNP complex that is essential for pre-mRNA splicing of the major class of metazoan nuclear introns. The functionally analogous but highly diverged U4atac and U6atac snRNAs form a similar complex that is involved in splicing of the minor class of introns. Previous results with mutants of U6atac in which a substructure was replaced by the analogous structure from U6 snRNA suggested that wild-type U4 snRNA might be able to interact productively with the mutant U6atac snRNA. Here we show that a mutant U4 snRNA designed to base pair with a mutant U6atac snRNA can activate U12-dependent splicing when coexpressed in an in vivo genetic suppression assay. This genetic interaction could also be demonstrated in an in vitro crosslinking assay. These results show that a U4/U6atac di-snRNP can correctly splice a U12-dependent intron and suggest that the specificity for spliceosome type resides in the U6 and U6atac small nuclear ribonucleoproteins. Further experiments suggest that expression of a mutant U4 snRNA that can bind to wild-type U6atac snRNA alters the specificity of some splice sites, providing an evolutionary rationale for maintaining two U4-like snRNAs.

Arabidopsis↗

PubMed2004

Molecular imaging of gene expression in living subjects by spliceosome-mediated RNA trans-splicing.

Spliceosome-mediated RNA trans-splicing (SMaRT) provides an effective means to reprogram mRNAs and the proteins they encode. SMaRT technology has a broad range of applications, including RNA repair and molecular imaging, each governed by the nature of the sequences delivered by the pre-trans-splicing molecule. Here, we show the ability of SMaRT to optically image the expression of an exogenous gene at the level of pre-mRNA splicing in cells and living animals. Because of the modular design of pre-trans-splicing molecules, there is great potential to employ SMaRT to image the expression of any arbitrary gene of interest in living subjects. In this report, we describe a model system that demonstrates the feasibility of imaging gene expression by transsplicing in small animals. This represents a previously undescribed approach to molecular imaging of mRNA levels in living subjects.

Animals↗

PubMed2005

Spliceosomal introns in the deep-branching eukaryote Trichomonas vaginalis.

Eukaryotes have evolved elaborate splicing mechanisms to remove introns that would otherwise destroy the protein-coding capacity of genes. Nuclear premRNA splicing requires sequence motifs in the intron and is mediated by a ribonucleoprotein complex, the spliceosome. Here we demonstrate the presence of a splicing apparatus in the protist Trichomonas vaginalis and show that RNA motifs found in yeast and metazoan introns are required for splicing. We also describe the first introns in this deep-branching lineage. The positions of these introns are often conserved in orthologous genes, indicating they were present in a common ancestor of trichomonads, yeast, and metazoa. All examined T. vaginalis introns have a highly conserved 12-nt 3' splice-site motif that encompasses the branch point and is necessary for splicing. This motif is also found in the only described intron in a gene from another deep-branching eukaryote, Giardia intestinalis. These studies demonstrate the conservation of intron splicing signals across large evolutionary distances, reveal unexpected motif conservation in deep-branching lineages that suggest a simplified mechanism of splicing in primitive unicellular eukaryotes, and support the presence of introns in the earliest eukaryote.

Animals↗