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The C-terminal RG dipeptide repeats of the spliceosomal Sm proteins D1 and D3 contain symmetrical dimethylarginines, which form a major B-cell epitope for anti-Sm autoantibodies.

The Sm proteins B/B', D1, D2, D3, E, F, and G are components of the small nuclear ribonucleoproteins U1, U2, U4/U6, and U5 that are essential for the splicing of pre-mRNAs in eukaryotes. D1 and D3 are among the most common antigens recognized by anti-Sm autoantibodies, an autoantibody population found exclusively in patients afflicted with systemic lupus erythematosus. Here we demonstrate by protein sequencing and mass spectrometry that all arginines in the C-terminal arginine-glycine (RG) dipeptide repeats of the human Sm proteins D1 and D3, isolated from HeLa small nuclear ribonucleoproteins, contain symmetrical dimethylarginines (sDMAs), a posttranslational modification thus far only identified in the myelin basic protein. The further finding that human D1 individually overexpressed in baculovirus-infected insect cells contains asymmetrical dimethylarginines suggests that the symmetrical dimethylation of the RG repeats in D1 and D3 is dependent on the assembly status of D1 and D3. In antibody binding studies, 10 of 11 anti-Sm patient sera tested, as well as the monoclonal antibody Y12, reacted with a chemically synthesized C-terminal peptide of D1 containing sDMA, but not with peptides containing asymmetrically modified or nonmodified arginines. These results thus demonstrate that the sDMA-modified C terminus of D1 forms a major linear epitope for anti-Sm autoantibodies and Y12 and further suggest that posttranslational modifications of Sm proteins play a role in the etiology of systemic lupus erythematosus.

Amino Acid Sequence↗

A 62,000 molecular weight spliceosome protein crosslinks to the intron polypyrimidine tract.

Incubation in HeLa nuclear extract of a 32P-labeled 61 nucleotide-long RNA corresponding to the lariat branch site/polypyrimidine tract/3' splice site of the first intron of human beta-globin pre-mRNA led to the crosslinking of a single protein of approximately 62,000 mol. wt. (p62). p62 corresponds to a polypyrimidine tract-binding protein recently described by Garcia-Blanco et al. (Genes & Dev. 3: 1874-1886, 1989). Crosslinking of p62 to the 61 nt RNA was highly sequence specific. No p62 crosslinking was observed with a 60 nt pGEM vector RNA, a 63 nt RNA antisense to the 61-mer or a 72 nt U2 RNA sequence. p62 crosslinking to the 61 nt RNA was competed by unlabeled 61 nt RNA, by beta-globin pre-mRNA containing intron 1, and by poly(U) and poly(C), but was competed to a lesser extent or not at all by pGEM RNA, a beta-globin RNA lacking intron 1, or poly(A). Experiments with mutated RNAs revealed that neither the lariat branch site adenosine nor the 3' splice site were required for p62 crosslinking to polypyrimidine tract-containing RNA. Elimination of the polypyrimidine tract reduced p62 crosslinking, as did mutation of a polypyrimidine tract UU dinucleotide to GA. However, replacement of a pyrimidine-rich tract immediately adjacent (3') to the lariat branch site with a 57% A + G pGEM vector RNA sequence also significantly reduced p62 crosslinking, indicating the involvement of both this pyrimidine-rich region and the classical polypyrimidine tract adjacent to the 3' splice site. The sites of protein interaction were further defined by RNase H protection experiments, the results of which confirmed the patterns of p62 crosslinking to mutant RNAs. p62 crosslinking was efficiently competed by a DNA oligonucleotide having the same sequence as the 61 nt RNA, showing that p62 requires neither ribose 2' OH groups nor uracil bases for its interaction with the polypyrimidine tract. p62 was not crosslinked to double-stranded 61 nt RNA. Q-Sepharose chromatography of HeLa nuclear extract yielded an unbound fraction (QU) in which p62 was the only polypyrimidine tract-crosslinkable protein and a bound fraction (QB) in which, surprisingly, several non-p62 proteins were crosslinkable to the polypyrimidine tract RNA. Yet, when the two Q-Sepharose fractions were combined, p62 strongly out-competed the otherwise-crosslinkable QB proteins for polypyrimidine tract RNA crosslinking. This indicates that p62 may have the highest affinity and/or crosslinking efficiency for the intron polypyrimidine tract of any HeLa nuclear protein.

Base Sequence↗

The spliceosomal snRNAs of Caenorhabditis elegans.

Nematodes are the only group of organisms in which both cis- and trans-splicing of nuclear mRNAs are known to occur. Most Caenorhabditis elegans introns are exceptionally short, often only 50 bases long. The consensus donor and acceptor splice site sequences found in other animals are used for both cis- and trans-splicing. In order to identify the machinery required for these splicing events, we have characterized the C. elegans snRNAs. They are similar in sequence and structure to those characterized in other organisms, and several sequence variations discovered in the nematode snRNAs provide support for previously proposed structure models. The C. elegans snRNAs are encoded by gene families. We report here the sequences of many of these genes. We find a highly conserved sequence, the proximal sequence element (PSE), about 65 bp upstream of all 21 snRNA genes thus far sequenced, including the SL RNA genes, which specify the snRNAs that provide the 5' exons in trans-splicing. The sequence of the C. elegans PSE is distinct from PSE's from other organisms.

Animals↗

Evolutionary conservation of the spliceosomal protein, U2B''.

U1 and U2snRNPs play key roles in pre-mRNA splicing. The interactions between the U1 and U2snRNP-specific proteins, U1A, U2A' and U2B'' and their respective UsnRNAs are of interest both to elucidate their roles in splicing, and as models to study RNA-protein interactions. We have cloned a full-length cDNA, encoding U2B'', from potato. This is the first report of a sequence for a plant UsnRNP protein. The plant U2B'' sequence exhibits extensive similarity with the human U2B'' protein at both the DNA and amino acid levels. The evolutionary conservation at the protein level, particularly in sequences implicated in determining specific binding to U2snRNA, suggests conservation of U2B'' function from plants to man. The significance of amino acid substitutions in the RNP-80 motif with respect to U2snRNA binding in plants is discussed.

Amino Acid Sequence↗

The 5' end domain of U2 snRNA is required to establish the interaction of U2 snRNP with U2 auxiliary factor(s) during mammalian spliceosome assembly.

Stable association of U2 snRNP with the branchpoint sequence of mammalian pre-mRNAs requires binding of a non-snRNP protein to the polypyrimidine tract. In order to determine how U2 snRNP contacts this protein, we have used an RNA containing the consensus 5' and the (Py)n-AG 3' splice sites but lacking the branchpoint sequence so as to prevent direct U2 snRNA base pairing to the branchpoint. Different approaches including electrophoretic separation of RNP complexes formed in nuclear extracts, RNase T1 protection immunoprecipitation assays with antibodies against snRNPs and UV cross-linking experiments coupled to immunoprecipitations allowed us to demonstrate that at least three splicing factors contact this RNA at 0 degree C without ATP. As expected, U1 snRNP interacts with the region comprising the 5' splice site. A protein of approximately 65,000 molecular weight recognizes the RNA specifically at the 5' boundary of the polypyrimidine tract. It could be either the U2 auxiliary factor (U2AF) (Zamore and Green (1989) PNAS 86, 9243-9247), the polypyrimidine tract binding protein (pPTB) (Garcia-Blanco et al. (1989) Genes and Dev. 3, 1874-1886) or a mixture of both. U2 snRNP also contacts the RNA in a way depending on p65 binding, thereby further arguing that the latter may correspond to the previously characterized U2AF and pPTB. Cleavage of U2 snRNA sequence by a complementary oligonucleotide and RNase H led us to conclude that the 5' terminus of U2 snRNA is required to ensure the contact between U2 snRNP and p65 bound to the RNA. More importantly, this conclusion can be extended to authentic pre-mRNAs. When we have used a human beta-globin pre-mRNA instead of the above artificial substrate, RNA bound p65 became precipitable by anti-(U2) RNP and anti-Sm antibodies except when the 5' end of U2 snRNA was selectively cleaved.

Animals↗

Mammalian polycomb-mediated repression of Hox genes requires the essential spliceosomal protein Sf3b1.

Polycomb group (PcG) proteins are responsible for the stable repression of homeotic (Hox) genes by forming multimeric protein complexes. We show (1) physical interaction between components of the U2 small nuclear ribonucleoprotein particle (U2 snRNP), including Sf3b1 and PcG proteins Zfp144 and Rnf2; and (2) that Sf3b1 heterozygous mice exhibit skeletal transformations concomitant with ectopic Hox expressions. These alterations are enhanced by Zfp144 mutation but repressed by Mll mutation (a trithorax-group gene). Importantly, the levels of Sf3b1 in PcG complexes were decreased in Sf3b1-heterozygous embryos. These findings suggest that Sf3b1-PcG protein interaction is essential for true PcG-mediated repression of Hox genes.

Animals↗

Domains of yeast U4 spliceosomal RNA required for PRP4 protein binding, snRNP-snRNP interactions, and pre-mRNA splicing in vivo.

U4 small nuclear RNA (snRNA) contains two intramolecular stem-loop structures, located near each end of the molecule. The 5' stem-loop is highly conserved in structure and separates two regions of U4 snRNA that base-pair with U6 snRNA in the U4/U6 small nuclear ribonucleoprotein particle (snRNP). The 3' stem-loop is highly divergent in structure among species and lies immediately upstream of the binding site for Sm proteins. To investigate the function of these two domains, mutants were constructed that delete the yeast U4 snRNA 5' stem-loop and that replace the yeast 3' stem-loop with that from trypanosome U4 snRNA. Both mutants fail to complement a null allele of the yeast U4 gene. The defects of the mutants have been examined in heterozygous strains by native gel electrophoresis, glycerol gradient centrifugation, and immunoprecipitation. The chimeric yeast-trypanosome RNA does not associate efficiently with U6 snRNA, suggesting that the 3' stem-loop of yeast U4 snRNA might be a binding site for a putative protein that facilitates assembly of the U4/U6 complex. In contrast, the 5' hairpin deletion mutant associates efficiently with U6 snRNA. However, it does not bind the U4/U6-specific protein PRP4 and does not assemble into a U4/U5/U6 snRNA. Thus, we propose that the role of the PRP4 protein is to promote interactions between the U4/U6 snRNP and the U5 snRNP.

Base Sequence↗

The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA.

The RNA helicase-like splicing factor PRP2 interacts only transiently with spliceosomes. To facilitate analysis of interactions of PRP2 with spliceosomal components, PRP2 protein was stalled in splicing complexes by two different methods. A dominant negative mutant form of PRP2 protein, which associates stably with spliceosomes, was found to interact directly with pre-mRNAs, as demonstrated by UV-crosslinking experiments. The use of various mutant and truncated pre-mRNAs revealed that this interaction requires a spliceable pre-mRNA and an assembled spliceosome; a 3' splice site is not required. To extend these observations to the wild-type PRP2 protein, spliceosomes were depleted of ATP; PRP2 protein interacts with pre-mRNA in these spliceosomes in an ATP-independent fashion. Comparison of RNA binding by PRP2 protein in the presence of ATP or gamma S-ATP showed that ATP hydrolysis rather than mere ATP binding is required to release PRP2 protein from pre-mRNA. As PRP2 is an RNA-stimulated ATPase, these experiments strongly suggest that the pre-mRNA is the native co-factor stimulating ATP hydrolysis by PRP2 protein in spliceosomes. Since PRP2 is a putative RNA helicase, we propose that the pre-mRNA is the target of RNA displacement activity of PRP2 protein, promoting the first step of splicing.

Adenosine Triphosphate↗

Arabidopsis TITAN-LIKE is required for U12-type intron splicing, especially of AT-AC subtypes.

Many eukaryotes possess two types of spliceosomes: the U2-dependent and U12-dependent spliceosomes. The U2-dependent spliceosome processes >99% of all introns, whereas the U12-dependent spliceosome acts on only ~0.3% of introns, one-third of which start with AT and end with AC, with the remainder having GT-AG termini. How the U12-dependent spliceosome splices two types of introns with different terminal sequences remains poorly understood. Human centrosomal AT-AC splicing factor (CENATAC) is a subunit of the U12-dependent spliceosome that is particularly required for the splicing of the AT-AC subtype. The Arabidopsis genome contains a single homolog, TITAN-LIKE (TTL), but its function in splicing remains unknown. Here, we generated ttl mutants and isolated two viable alleles, of which we analyzed one, designated ttl-142, to investigate TTL's function in splicing. ttl-142 carries a 42-nucleotide deletion that removes 14 amino acid residues from the predicted protein, and homozygous mutants exhibit morphological abnormalities. Most U12-dependent introns were less efficiently spliced in ttl-142 than in the wild type, with the splicing of AT-AC introns particularly suppressed. Splicing suppression in ttl-142 was more extensive than in a drol1 (defective repression of the OLE3:LUC1) mutant, which carries a mutation in a gene specifically required for AT-AC intron splicing. Conversely, fewer genes showed altered expression levels in ttl-142 than in drol1, and most differentially expressed genes differed between the two mutants. These results suggest that the phenotypes of ttl-142 and drol1 mutants may reflect the impairment of distinct spliceosomal functions.

Arabidopsis↗

Alterations of RNase H sensitivity of the 3' splice site region during the in vitro splicing reaction.

We have developed a splicing assay system with an immobilized pre-mRNA to study the mechanism of the splicing reaction after spliceosome assembly. Using this system, we have found that the second step of the splicing reaction could be dissected into two stages. After the 5' splice site reaction, at least two factors interact with the pre-formed spliceosome containing intermediate molecules in an ATP-independent manner to convert the spliceosome into a form competent for the 3' splice site reaction. Then, the 3' splice site reaction occurs on this spliceosome, if ATP is supplied to the reaction mixture. We have also investigated the dynamic state of the 3' splice site region in the spliceosomes during the splicing reaction by probing with RNase H sensitivity. Prior to the 5' splice site reaction, the 3' splice site region was protected from RNase H attack. The region became sensitive immediately after the 5' splice site reaction, and subsequently became resistant again as the spliceosome competent for the 3' splice site reaction was formed. These results suggest that the interaction of the 3' splice site region with some spliceosome components changes significantly during the splicing reaction.

Base Sequence↗

The origin of introns and their role in eukaryogenesis: a compromise solution to the introns-early versus introns-late debate?

BACKGROUND: Ever since the discovery of 'genes in pieces' and mRNA splicing in eukaryotes, origin and evolution of spliceosomal introns have been considered within the conceptual framework of the 'introns early' versus 'introns late' debate. The 'introns early' hypothesis, which is closely linked to the so-called exon theory of gene evolution, posits that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. Under this scenario, the absence of spliceosomal introns in prokaryotes is considered to be a result of "genome streamlining". The 'introns late' hypothesis counters that spliceosomal introns emerged only in eukaryotes, and moreover, have been inserted into protein-coding genes continuously throughout the evolution of eukaryotes. Beyond the formal dilemma, the more substantial side of this debate has to do with possible roles of introns in the evolution of eukaryotes. RESULTS: I argue that several lines of evidence now suggest a coherent solution to the introns-early versus introns-late debate, and the emerging picture of intron evolution integrates aspects of both views although, formally, there seems to be no support for the original version of introns-early. Firstly, there is growing evidence that spliceosomal introns evolved from group II self-splicing introns which are present, usually, in small numbers, in many bacteria, and probably, moved into the evolving eukaryotic genome from the alpha-proteobacterial progenitor of the mitochondria. Secondly, the concept of a primordial pool of 'virus-like' genetic elements implies that self-splicing introns are among the most ancient genetic entities. Thirdly, reconstructions of the ancestral state of eukaryotic genes suggest that the last common ancestor of extant eukaryotes had an intron-rich genome. Thus, it appears that ancestors of spliceosomal introns, indeed, have existed since the earliest stages of life's evolution, in a formal agreement with the introns-early scenario. However, there is no evidence that these ancient introns ever became widespread before the emergence of eukaryotes, hence, the central tenet of introns-early, the role of introns in early evolution of proteins, has no support. However, the demonstration that numerous introns invaded eukaryotic genes at the outset of eukaryotic evolution and that subsequent intron gain has been limited in many eukaryotic lineages implicates introns as an ancestral feature of eukaryotic genomes and refutes radical versions of introns-late. Perhaps, most importantly, I argue that the intron invasion triggered other pivotal events of eukaryogenesis, including the emergence of the spliceosome, the nucleus, the linear chromosomes, the telomerase, and the ubiquitin signaling system. This concept of eukaryogenesis, in a sense, revives some tenets of the exon hypothesis, by assigning to introns crucial roles in eukaryotic evolutionary innovation. CONCLUSION: The scenario of the origin and evolution of introns that is best compatible with the results of comparative genomics and theoretical considerations goes as follows: self-splicing introns since the earliest stages of life's evolution--numerous spliceosomal introns invading genes of the emerging eukaryote during eukaryogenesis--subsequent lineage-specific loss and gain of introns. The intron invasion, probably, spawned by the mitochondrial endosymbiont, might have critically contributed to the emergence of the principal features of the eukaryotic cell. This scenario combines aspects of the introns-early and introns-late views. REVIEWERS: this article was reviewed by W. Ford Doolittle, James Darnell (nominated by W. Ford Doolittle), William Martin, and Anthony Poole.

Journal Article↗

A supraspliceosome model for large nuclear ribonucleoprotein particles based on mass determinations by scanning transmission electron microscopy.

Pre-mRNA splicing is an important regulatory step in the expression of most eukaryotic genes. In vitro studies have shown splicing to occur within 50-60 S multi-component ribonucleoprotein (RNP) complexes termed spliceosomes. Studies of mammalian cell nuclei have revealed larger complexes that sediment at 200 S in sucrose gradients, termed large nuclear RNP (lnRNP) particles. These particles contain all factors required for pre-mRNA splicing, including the spliceosomal U snRNPs and protein splicing factors. Electron microscopy has shown them to consist of four apparently similar substructures. In this study, mass measurements by scanning transmission electron microscopy of freeze-dried mammalian lnRNP preparations, both confirm the similarity between the lnRNP particles and reveal the mass uniformity of their subunits. Thus, the tetrameric lnRNP particle has a mass of 21.1(+/-1.6) MDa, while each repeating subunit has a mass of 4.8(+/-0.5) MDa, which is close to the estimated mass of the fully assembled 60 S spliceosome. The 1.9 MDa discrepancy between the lnRNP particle's mass and the cumulative masses of its four subunits may be attributed to an additional domain frequently observed in the micrographs. Notably, strands and loops of RNA were often seen emanating from lnRNP particles positively stained with uranyl formate. Our results support the idea that the nuclear splicing machine is a supraspliceosome complex. For clarity, we define spliceosomes devoid of pre-mRNA as spliceosome cores, and propose that the supraspliceosome is constructed from one pre-mRNA, four spliceosome cores, each composed mainly of U snRNPs, and additional proteins. In this way a frame is provided to juxtapose exons about to be spliced.

Mass Spectrometry↗

Commitment to splice site pairing coincides with A complex formation.

Differential recognition of exons by the spliceosome regulates gene expression and exponentially increases the complexity of metazoan proteomes. After definition of the exons, the spliceosome is activated by a series of sequential structural rearrangements. Formation of the first ATP-independent spliceosomal complex commits the pre-mRNA to the general splicing pathway. However, the time at which a commitment to a specific splice site choice and pairing is made is unknown. Here, we demonstrate that alternative splicing patterns are irreversibly chosen at a kinetic step different from the ATP-independent commitment to splicing. Splice sites become committed at the first ATP-dependent spliceosomal complex when rearrangements lock U2 snRNP onto the pre-mRNA. Thus, commitment to the splicing pathway and commitment to splice site pairing are separate steps during spliceosomal assembly, and ATP hydrolysis drives the irreversible juxtaposition of exons within the spliceosome.

Animals↗

Human U4/U6.U5 and U4atac/U6atac.U5 tri-snRNPs exhibit similar protein compositions.

In the U12-dependent spliceosome, the U4atac/U6atac snRNP represents the functional analogue of the major U4/U6 snRNP. Little information is available presently regarding the protein composition of the former snRNP and its association with other snRNPs. In this report we show that human U4atac/U6atac di-snRNPs associate with U5 snRNPs to form a 25S U4atac/U6atac.U5 trimeric particle. Comparative analysis of minor and major tri-snRNPs by using immunoprecipitation experiments revealed that their protein compositions are very similar, if not identical. Not only U5-specific proteins but, surprisingly, all tested U4/U6- and major tri-snRNP-specific proteins were detected in the minor tri-snRNP complex. Significantly, the major tri-snRNP-specific proteins 65K and 110K, which are required for integration of the major tri-snRNP into the U2-dependent spliceosome, were among those proteins detected in the minor tri-snRNP, raising an interesting question as to how the specificity of addition of tri-snRNP to the corresponding spliceosome is maintained. Moreover, immunodepletion studies demonstrated that the U4/U6-specific 61K protein, which is involved in the formation of major tri-snRNPs, is essential for the association of the U4atac/U6atac di-snRNP with U5 to form the U4atac/U6atac.U5 tri-snRNP. Subsequent immunoprecipitation studies demonstrated that those proteins detected in the minor tri-snRNP complex are also incorporated into U12-dependent spliceosomes. This remarkable conservation of polypeptides between minor and major spliceosomes, coupled with the absence of significant sequence similarity between the functionally analogous snRNAs, supports an evolutionary model in which most major and minor spliceosomal proteins, but not snRNAs, are derived from a common ancestor.

Antibodies↗

Roles of PRP8 protein in the assembly of splicing complexes.

Three different approaches have been used to investigate the roles of the yeast U5 snRNP protein PRP8 in spliceosome assembly: genetic depletion of PRP8 protein in vivo, heat inactivation of temperature-sensitive prp8 protein in protoplasts and inhibition of PRP8 function with antibodies in vitro. In each case, U5 and U4/U6 snRNPs failed to assemble into the forming spliceosomes. In addition, extract prepared from PRP8-depleted cells and extract containing inactivated PRP8 protein had substantially reduced amounts of U4/U6.U5 triple snRNP complexes. Thus, functional PRP8 protein is required for the stable formation of U4/U6.U5 complexes without which spliceosomes fail to form. As spliceosome formation was also blocked by anti-PRP8 antibodies that apparently do not disrupt triple snRNPs, PRP8 protein may play a separate role in the assembly of triple snRNPs into spliceosomes. As a consequence of PRP8 depletion the levels of the U4, U5 and U6 snRNAs declined dramatically. We discuss this in the context of the known genetic interactions between PRP8 and putative RNA helicase (DEAD box protein) genes and propose that PRP8 protein plays a role in regulating dynamic RNA-RNA interactions in spliceosome assembly, possibly ensuring the correct directionality of these events.

Base Sequence↗