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PubMed2001

The 65 and 110 kDa SR-related proteins of the U4/U6.U5 tri-snRNP are essential for the assembly of mature spliceosomes.

The association of the U4/U6.U5 tri-snRNP with pre-spliceosomes is a poorly understood step in the spliceosome assembly pathway. We have identified two human tri-snRNP proteins (of 65 and 110 kDa) that play an essential role in this process. Characterization by cDNA cloning of the 65 and 110 kDa proteins revealed that they are likely orthologues of the yeast spliceosomal proteins Sad1p and Snu66p, respectively. Immunodepletion of either protein from the HeLa cell nuclear extracts inhibited pre-mRNA splicing due to a block in the formation of mature spliceosomes, but had no effect on the integrity of the U4/U6.U5 tri-snRNP. Spliceosome assembly and splicing catalysis could be restored to the respective depleted extract by the addition of recombinant 65 or 110 kDa protein. Our data demonstrate that both proteins are essential for the recruitment of the tri-snRNP to the pre-spliceosome but not for the maintenance of the tri-snRNP stability. Moreover, since both proteins contain an N-terminal RS domain, they could mediate the association of the tri-snRNP with pre-spliceosomes by interaction with members of the SR protein family.

Amino Acid Sequence↗

PubMed2005

Spliceosome disassembly catalyzed by Prp43 and its associated components Ntr1 and Ntr2.

Two novel yeast splicing factors required for spliceosome disassembly have been identified. Ntr1 and Ntr2 (NineTeen complex-Related proteins) were identified for their weak association with components of the Prp19-associated complex. Unlike other Prp19-associated components, these two proteins were primarily associated with the intron-containing spliceosome during the splicing reaction. Extracts depleted of Ntr1 or Ntr2 exhibited full splicing activity, but accumulated large amounts of lariat-intron in the spliceosome after splicing, indicating that the normal function of the Prp19-associated complex in spliceosome activation was not affected, but spliceosome disassembly was hindered. Immunoprecipitation analysis revealed that Ntr1 and Ntr2 formed a stable complex with DExD/H-box RNA helicase Prp43 in the splicing extract. Ntr1 interacted with Prp43 through the N-terminal G-patch domain, with Ntr2 through a middle region, and with itself through the carboxyl half of the protein. The affinity-purified Ntr1-Ntr2-Prp43 complex could catalyze disassembly of the spliceosome in an ATP-dependent manner, separating U2, U5, U6, NTC (NineTeen Complex), and lariat-intron. This is the first demonstration of physical disassembly of the spliceosome, catalyzed by a complex containing a DExD/H-box RNA helicase and two accessory factors, which might function in targeting the helicase to the correct substrate.

Adenosine Triphosphate↗

PubMed2006

Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP.

Two different models currently exist for the assembly pathway of the spliceosome, namely, the traditional model, in which spliceosomal snRNPs associate in a stepwise, ordered manner with the pre-mRNA, and the holospliceosome model, in which all spliceosomal snRNPs preassemble into a penta-snRNP complex. Here we have tested whether the spliceosomal A complex, which contains solely U1 and U2 snRNPs bound to pre-mRNA, is a functional, bona fide assembly intermediate. Significantly, A complexes affinity-purified from nuclear extract depleted of U4/U6 snRNPs (and thus unable to form a penta-snRNP) supported pre-mRNA splicing in nuclear extract depleted of U2 snRNPs, whereas naked pre-mRNA did not. Mixing experiments with purified A complexes and naked pre-mRNA additionally confirmed that under these conditions, A complexes do not form de novo. Thus, our studies demonstrate that holospliceosome formation is not a prerequisite for generating catalytically active spliceosomes and that, at least in vitro, the U1 and U2 snRNPs can functionally associate with the pre-mRNA, prior to and independent of the tri-snRNP. The ability to isolate functional spliceosomal A complexes paves the way to study in detail subsequent spliceosome assembly steps using purified components.

HeLa Cells↗

PubMed2006

Functional links between the Prp19-associated complex, U4/U6 biogenesis, and spliceosome recycling.

The Prp19-associated complex, consisting of at least eight protein components, is involved in spliceosome activation by specifying the interaction of U5 and U6 with pre-mRNA for their stable association with the spliceosome after U4 dissociation. We show here that yeast cells depleted of one or two of the Prp19-associated components, accumulate the free form of U4. In NTC25-deleted cells, the level of U6 was also reduced. Extracts prepared from NTC25-deleted cells contained neither free U4 nor U6 and were ineffective in spliceosome recycling in the in vitro splicing reaction. Overexpression of U6 partially rescued the temperature-sensitive growth defect and decreased the relative amount of free U4 in NTC25-deleted cells, indicating that the accumulation of free U4 was a consequence of insufficient amounts of U6 snRNA. Extracts prepared from U6-overproducing NTC25-deleted cells containing free-form U6 were capable of spliceosome recycling, suggesting a role of free U6 RNP in spliceosome recycling. Our results demonstrate that in addition to direct participation in spliceosome activation, the Prp19-associated complex has an indirect role in spliceosome recycling through affecting the biogenesis of U4/U6 snRNP in the in vivo splicing reaction.

Blotting, Northern↗

PubMed1987

Splicing of yeast nuclear pre-mRNA in vitro requires a functional 40S spliceosome and several extrinsic factors.

We have previously shown that extracts prepared from most of the yeast temperature-sensitive rna mutants are heat sensitive for pre-mRNA splicing in vitro, and that the products of the corresponding RNA genes are essential for the early stages of the splicing region. In this report, we demonstrate that most heat-inactivated mutant extracts do not form the spliceosome, suggesting that their gene products are likely to be involved in spliceosome formation. Heat-inactivated rna2 extracts, on the other hand, do form a splicing-dependent 40S complex containing uncleaved pre-mRNA exclusively. The pre-mRNA in the 40S complex can be converted to the splicing products in the presence of ATP and complementing extracts. These results demonstrate that: (1) the 40S complex formed in heat-inactivated rna2 extracts is a spliceosome (termed the rna2 delta spliceosome), (2) the spliceosome is a functional intermediate in the splicing pathway, and (3) the splicing process can be dissected into two steps, spliceosome formation and cleavage-ligation reactions. Additional results indicate that at least two extrinsic factors, as well as the RNA2 gene product, are required for complementation of the rna2 delta spliceosome. A three-step mechanism for nuclear pre-mRNA splicing in yeast is proposed.

Genes, Fungal↗

PubMed1996

Splicing of precursors to mRNA in higher plants: mechanism, regulation and sub-nuclear organisation of the spliceosomal machinery.

The removal of introns from pre-mRNA transcripts and the concomitant ligation of exons is known as pre-mRNA splicing. It is a fundamental aspect of constitutive eukaryotic gene expression and an important level at which gene expression is regulated. The process is governed by multiple cis-acting elements of limited sequence content and particular spatial constraints, and is executed by a dynamic ribonucleoprotein complex termed the spliceosome. The mechanism and regulation of pre-mRNA splicing, and the sub-nuclear organisation of the spliceosomal machinery in higher plants is reviewed here. Heterologous introns are often not processed in higher plants indicating that, although highly conserved, the process of pre-mRNA splicing in plants exhibits significant differences that distinguish it from splicing in yeast and mammals. A fundamental distinguishing feature is the presence of and requirement for AU or U-rich intron sequence in higher-plant pre-mRNA splicing. In this review we document the properties of higher-plant introns and trans-acting spliceosomal components and discuss the means by which these elements combine to determine the accuracy and efficiency of pre-mRNA processing. We also detail examples of how introns can effect regulated gene expression by affecting the nature and abundance of mRNA in plants and list the effects of environmental stresses on splicing. Spliceosomal components exhibit a distinct pattern of organisation in higher-plant nuclei. Effective probes that reveal this pattern have only recently become available, but the domains in which spliceosomal components concentrate were identified in plant nuclei as enigmatic structures some sixty years ago. The organisation of spliceosomal components in plant nuclei is reviewed and these recent observations are unified with previous cytochemical and ultrastructural studies of plant ribonuleoprotein domains.

Cell Nucleus↗

PubMed1999

Transient interaction of BBP/ScSF1 and Mud2 with the splicing machinery affects the kinetics of spliceosome assembly.

Removal of introns from pre-mRNA is an essential step of gene expression. The splicing reaction is catalyzed in a large complex termed the spliceosome. Introns are recognized during the early steps of spliceosome assembly with the formation of commitment complexes. Intron recognition is mediated by the interaction of splicing factors with conserved sequences present in the pre-mRNA. BBP/SF1 participates in this recognition by interacting with the pre-mRNA branch point in both yeast and mammals. This protein, which is essential in yeast, also interacts with the U2AF65/Mud2 splicing factor. However, its precise role in splicing complex formation is still unclear. We have now analyzed the presence of BBP and Mud2 in yeast splicing complexes using supershift and coprecipitation assays. We found that BBP is present together with Mud2 in commitment complex 2 (CC2), but is not detectable in commitment complex 1 (CC1). Furthermore, genetic and biochemical depletion of BBP demonstrated that it is required for CC2 formation. In addition we observed that BBP and Mud2 are not detectable in pre-spliceosomes. These are the first commitment complex components that are shown to be released during or immediately after pre-spliceosome formation. Interestingly, depletion of BBP or disruption of MUD2 had no significant effect on pre-spliceosome formation and splicing in vitro but led to a transient accumulation of CC1. These observations support a model in which BBP and Mud2 are recycled during transition from CC2 to pre-spliceosome.

DNA-Binding Proteins↗

PubMed2002

Early organization of pre-mRNA during spliceosome assembly.

Intron excision from precursor mRNAs (pre-mRNAs) in eukaryotes requires juxtaposition of reactive functionalities within the substrate at the heart of the spliceosome where the two chemical steps of splicing occur. Although a series of interactions between pre-mRNAs, pre-spliceosomal and spliceosomal factors is well established, the molecular mechanisms of splicing machinery assembly, as well as the temporal basis for organization of the substrate for splicing, remain poorly understood. Here we have used a directed hydroxyl radical probe tethered to pre-mRNA substrates to map the structure of the pre-mRNA substrate during the spliceosome assembly process. These studies indicate an early organization and proximation of conserved pre-mRNA sequences during spliceosome assembly/recruitment and suggest a mechanism for the formation of the final active site of the mature spliceosome.

Adenosine Triphosphate↗

PubMed2006

Proximity of conserved U6 and U2 snRNA elements to the 5' splice site region in activated spliceosomes.

Major structural changes occur in the spliceosome during its catalytic activation, which immediately precedes the splicing of pre-mRNA. Whereas changes in snRNA conformation are well documented at the level of secondary RNA-RNA interactions, little is known about the tertiary structure of this RNA-RNA network, which comprises the spliceosome's catalytic core. Here, we have used the hydroxyl-radical probe Fe-BABE, tethered to the tenth nucleotide (U(+10)) of the 5' end of a pre-mRNA intron, to map RNA-RNA proximities in spliceosomes. These studies revealed that several conserved snRNA regions are close to U(+10) in activated spliceosomes, namely (i) the U6 snRNA ACAGAG-box region, (ii) portions of the U6 intramolecular stem-loop (U6-ISL) including a nucleotide implicated in the first catalytic step (U74), and (iii) the region of U2 that interacts with the branch point. These data constrain the relative orientation of these structural elements with respect to U(+10) in the activated spliceosome. Upon conversion of the activated spliceosome to complex C, the accessibility of U6-ISL to hydroxyl-radical cleavage is altered, suggesting rearrangements after the first catalytic step.

Base Sequence↗

PubMed2006

Crystal structure of a core spliceosomal protein interface.

The precise excision of introns from precursor mRNAs (pre-mRNAs) in eukaryotes is accomplished by the spliceosome, a complex assembly containing five small nuclear ribonucleoprotein (snRNP) particles. Human p14, a component of the spliceosomal U2 and U11/U12 snRNPs, has been shown to associate directly with the pre-mRNA branch adenosine early in spliceosome assembly and within the fully assembled spliceosome. Here we report the 2.5-A crystal structure of a complex containing p14 and a peptide derived from the p14-associated U2 snRNP component SF3b155. p14 contains an RNA recognition motif (RRM), the surface of which is largely occluded by a C-terminal alpha-helix and a portion of the SF3b155 peptide. An analysis of RNA.protein crosslinking to wild-type and mutant p14 shows that the branch adenosine directly interacts with a conserved aromatic within a pocket on the surface of the complex. This result, combined with a comparison of the structure with known RRMs and pseudoRRMs as well as model-building by using the electron cryomicroscopy structure of a spliceosomal U11/U12 di-snRNP, suggests that p14.SF3b155 presents a noncanonical surface for RNA recognition at the heart of the mammalian spliceosome.

Adenosine↗

PubMed2006

Inhibition of a spliceosome turnover pathway suppresses splicing defects.

Defects in assembly are suggested to signal the dissociation of faulty splicing complexes. A yeast genetic screen was performed to identify components of the putative discard pathway. Weak mutant alleles of SPP382 (also called NTR1) were found to suppress defects in two proteins required for spliceosome activation, Prp38p and Prp8p. Spp382p is shown necessary for cellular splicing, with premRNA and, for some alleles, excised intron, accumulating after inactivation. Like spp382-1, a mutant allele of AAR2 was identified in this suppressor screen. Like Spp382p, Aar2p has a reported role in spliceosome recycling and is found with Spp382p in a complex recovered with a mutant version of the spliceosomal core protein Prp8p. Possible insight into to the spp382 suppressor phenotype is provided by the observation that defective splicing complexes lacking the 5' exon cleavage intermediate are recovered by a tandem affinity purification-tagged Spp382 derivative. Stringent proteomic and two-hybrid analyses show that Spp382p also interacts with Cwc23p, a DNA J-like protein present in the spliceosome and copurified with the Prp43p DExD/H-box ATPase. Spp382p binds Prp43p and Prp43p requires Spp382p for intron release from the spliceosome. Consistent with a related function in the removal of defective complexes, three prp43 mutants are also shown to suppress splicing defects, with efficiencies inversely proportionate to the measured ATPase activities. These and related genetic data support the existence of a Spp382p-dependent turnover pathway acting on defective spliceosomes.

Adenosine Triphosphatases↗

PubMed2002

Purification and electron microscopic visualization of functional human spliceosomes.

Pre-mRNA splicing takes place in a large and highly dynamic complex known as the spliceosome. Here we report the optimization of a maltose-binding protein (MBP) affinity-purification method to isolate functional spliceosomes for electron microscopic analysis. Visualization of the spliceosome preparations revealed distinct 40-60 nm particles. Immunogold-conjugated antibodies to spliceosome components specifically label these particles, which are eliminated by treatment with either RNase or protease. Moreover, spliceosomes assembled on two different pre-mRNAs are indistinguishable. This first visualization of purified functional spliceosomes assembled in vitro reveals striking structural features, including one or more central cavities and multiple elongate lobes.

Base Sequence↗

PubMed2005

Complex spliceosomal organization ancestral to extant eukaryotes.

In higher eukaryotes, introns are spliced out of protein-coding mRNAs by the spliceosome, a massive complex comprising five non-coding RNAs (ncRNAs) and about 200 proteins. By comparing the differences between spliceosomal proteins from many basal eukaryotic lineages, it is possible to infer properties of the splicing system in the last common ancestor of extant eukaryotes, the eukaryotic ancestor. We begin with the hypothesis that, similar to intron length (that appears to have increased in multicellular eukaryotes), the spliceosome has increased in complexity throughout eukaryotic evolution. However, examination of the distribution of spliceosomal components indicates that not only was a spliceosome present in the eukaryotic ancestor but it also contained most of the key components found in today's eukaryotes. All the small nuclear ribonucleoproteins (snRNPs) protein components are likely to have been present, as well as many splicing-related proteins. Both major and trans-splicing are likely to have been present, and the spliceosome had already formed links with other cellular processes such as transcription and capping. However, there is no evidence as yet to suggest that minor (U12-dependent) splicing was present in the eukaryotic ancestor. Although the last common ancestor of extant eukaryotes appears to show much of the molecular complexity seen today, we do not, from this work, infer anything of the properties of the earlier "first eukaryote."

Eukaryotic Cells↗

PubMed2000

Widespread occurrence of spliceosomal introns in the rDNA genes of ascomycetes.

Spliceosomal (pre-mRNA) introns have previously been found in eukaryotic protein-coding genes, in the small nuclear RNAs of some fungi, and in the small- and large-subunit ribosomal DNA genes of a limited number of ascomycetes. How the majority of these introns originate remains an open question because few proven cases of recent and pervasive intron origin have been documented. We report here the widespread occurrence of spliceosomal introns (69 introns at 27 different sites) in the small- and large-subunit nuclear-encoded rDNA of lichen-forming and free-living members of the Ascomycota. Our analyses suggest that these spliceosomal introns are of relatively recent origin, i.e., within the Euascomycetes, and have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into rRNAs. The spliceosome itself, and not an external agent (e.g., transposable elements, group II introns), may have given rise to these introns. A nonrandom sequence pattern was found at sites flanking the rRNA spliceosomal introns. This pattern (AG-intron-G) closely resembles the proto-splice site (MAG-intron-R) postulated for intron insertions in pre-mRNA genes. The clustered positions of spliceosomal introns on secondary structures suggest that particular rRNA regions are preferred sites for insertion through reverse-splicing.

Ascomycota↗

PubMed1996

A human RNA helicase-like protein, HRH1, facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome.

Because the nuclear export of mRNA occurs only after the splicing reaction is completed, intron-containing pre-mRNA does not normally appear in the cytoplasm. As a mechanism to secure this, intron-containing RNA is retained in the nucleus via formation of the spliceosome. Therefore, the process of releasing spliced mRNA from the spliceosome after completion of splicing is an essential step for triggering the nuclear export of the spliced mRNA. In budding yeast, RNA helicase-like protein Prp22 is implicated in this process. Here we demonstrate the function of HRH1, a human protein homologous to Prp22, in mammalian cells using dominant-negative HRH1++ mutants (dn-HRH1). dn-HRH1 protein stalls on the spliceosome and prevents release of the spliced RNA from the spliceosome in vitro. Expression of dn-HRH1 in mammalian cells leads to inhibition of splicing and to extensive nuclear export of unspliced pre-mRNA, probably because of the incapability of recycling spliceosome components that normally retain the pre-mRNA in the nucleus. The arginine/serine-rich domain (RS domain) of HRH1, which is missing in Prp22, confers a nuclear localization signal, and appears to facilitate the interaction of HRH1 with the spliceosome. This is the first report on a bona fide mammalian homolog of yeast Prp splicing factor, and also on a mammalian RNA helicase-like splicing factor.

Amino Acid Sequence↗

PubMed1999

Human step II splicing factor hSlu7 functions in restructuring the spliceosome between the catalytic steps of splicing.

The spliceosome catalyzes pre-mRNA splicing in two steps. After catalytic step I, a major remodeling of the spliceosome occurs to establish the active site for step II. Here, we report the isolation of a cDNA encoding hSlu7, the human homolog of the yeast second step splicing factor Slu7. We show that hSlu7 associates with the spliceosome late in the splicing pathway, but at a stage prior to recognition of the 3' splice site for step II. In the absence of hSlu7, splicing is stalled between the catalytic steps in a novel complex, the CDeltahSlu7 complex. We provide evidence that this complex differs significantly in structure from the known spliceosomal complexes, yet is a functional intermediate between the catalytic steps of splicing. Together, our observations indicate that hSlu7 is required for a structural alteration of the spliceosome prior to the establishment of the catalytically active spliceosome for step II.

Adenoviridae↗

PubMed1992

Protein components specifically associated with prespliceosome and spliceosome complexes.

We have carried out a systematic analysis of the protein composition of highly purified mammalian spliceosomes. We show that > 30 distinct proteins, including 20 previously unidentified components [designated spliceosome-associated proteins (SAPs)], are specifically associated with the spliceosome in a salt-resistant complex. In contrast to these spliceosome-specific proteins, we show that hnRNP proteins are not tightly associated with purified prespliceosome and spliceosome complexes. The splicing factor U2AF65, U1 snRNP-specific proteins, and several SAPs are present in the earliest prespliceosome complex (E). A set of 10 proteins is then added to the first ATP-dependent prespliceosome complex (A), and concomitantly, a significant decrease in the level of U2AF65 is observed. The fully assembled spliceosome is formed by the addition of 12 proteins in a reaction that requires ATP and both the 5' and 3' splice sites.

Adenosine Triphosphate↗

PubMed1999

Identification of both shared and distinct proteins in the major and minor spliceosomes.

In metazoans, two distinct spliceosomes catalyzing pre-messenger RNA splicing have been identified. Here, the human U11/U12 small nuclear ribonucleoprotein (snRNP), a subunit of the minor (U12-dependent) spliceosome, was isolated. Twenty U11/U12 proteins were identified, including subsets unique to the minor spliceosome or common to both spliceosomes. Common proteins include four U2 snRNP polypeptides that constitute the essential splicing factor SF3b. A 35-kilodalton U11-associated protein homologous to the U1 snRNP 70K protein was also identified. These data provide fundamental information about proteins of the minor spliceosome and shed light on its evolutionary relationship to the major spliceosome.

Amino Acid Sequence↗