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Lineage-specific loss and divergence of functionally linked genes in eukaryotes.

By comparing 4,344 protein sequences from fission yeast Schizosaccharomyces pombe with all available eukaryotic sequences, we identified those genes that are conserved in S. pombe and nonfungal eukaryotes but are missing or highly diverged in the baker's yeast Saccharomyces cerevisiae. Since the radiation from the common ancestor with S. pombe, S. cerevisiae appears to have lost about 300 genes, and about 300 more genes have diverged by far beyond expectation. The most notable feature of the set of genes lost in S. cerevisiae is the coelimination of functionally connected groups of proteins, such as the signalosome and the spliceosome components. We predict similar coelimination of the components of the posttranscriptional gene-silencing system that includes the recently identified RNA-dependent RNA polymerase. Because one of the functions of posttranscriptional silencing appears to be "taming" of retrotransposons, the loss of this system in yeast could have triggered massive retrotransposition, resulting in elimination of introns and subsequent loss of spliceosome components that become dispensable. As the genome database grows, systematic analysis of coordinated gene loss may become a general approach for predicting new components of functional systems or even defining previously unknown functional complexes.

Biological Evolution↗

Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor.

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA branch site is an early step in spliceosome assembly and appears to commit a pre-mRNA to the splicing pathway. We have shown previously that this ATP-dependent binding requires a non-rnRNP factor, U2 snRNP auxiliary factor (U2AF), in addition to U2 snRNP. In this report we have identified U2AF, purified it to homogeneity, and characterized its biochemical properties. Purified U2AF comprises roughly equimolar quantities of two polypeptides, approximately 65 kDa and approximately 35 kDa, which appear to be associated. Measured by ultraviolet crosslinking, the 65-kDa polypeptide binds specifically to the polypyrimidine tract/3' splice site region. U2AF binds rapidly at 4 degrees C in the absence of ATP and remains associated with the pre-mRNA following U2 snRNP binding. Thus, the simple binding of U2AF initiates mammalian spliceosome assembly by facilitating the ATP-dependent binding of U2 snRNP.

Adenosine Triphosphate↗

Seven newly discovered intron positions in the triose-phosphate isomerase gene: evidence for the introns-late theory.

The gene encoding the glycolytic enzyme triose-phosphate isomerase (TPI; EC 5.3.1.1) has been central to the long-standing controversy on the origin and evolutionary significance of spliceosomal introns by virtue of its pivotal support for the introns-early view, or exon theory of genes. Putative correlations between intron positions and TPI protein structure have led to the conjecture that the gene was assembled by exon shuffling, and five TPI intron positions are old by the criterion of being conserved between animals and plants. We have sequenced TPI genes from three diverse eukaryotes--the basidiomycete Coprinus cinereus, the nematode Caenorhabditis elegans, and the insect Heliothis virescens--and have found introns at seven novel positions that disrupt previously recognized gene/protein structure correlations. The set of 21 TPI introns now known is consistent with a random model of intron insertion. Twelve of the 21 TPI introns appear to be of recent origin since each is present in but a single examined species. These results, together with their implication that as more TPI genes are sequenced more intron positions will be found, render TPI untenable as a paradigm for the introns-early theory and, instead, support the introns-late view that spliceosomal introns have been inserted into preexisting genes during eukaryotic evolution.

Amino Acid Sequence↗

In vitro trans-splicing in Saccharomyces cerevisiae.

The interactions established at the 5'-splice site during spliceosome assembly are likely to be important for both precise recognition of the upstream intron boundary and for positioning this site in the active center of the spliceosome. Definition of the RNA-RNA and the RNA-protein interactions at the 5' splice site would be facilitated by the use of a small substrate amenable to modification during chemical synthesis. We describe a trans-splicing reaction performed in Saccharomyces cerevisiae extracts in which the 5' splice site and the 3' splice site are on separate molecules. The RNA contributing the 5' splice site is only 20 nucleotides long and was synthesized chemically. The trans-splicing reaction is accurate and has the same sequence, ATP, and Mg2+ requirements as cis-splicing. We also report how deoxy substitutions around the 5'-splice site affect trans-splicing efficiency.

Actins↗

Association of the mammalian helicase MAH with the pre-mRNA splicing complex.

Conversion of pre-mRNAs into mature mRNAs includes several consecutive enzymatic modification steps that are carried out in the spliceosomes. Helicases have been shown to contribute to these catalytic processes both in yeast and in mammalian cells. Our results identify the mammalian protein MAH (matrix-associated helicase) as a new helicase present in the spliceosome complex. Sequence comparison describes MAH as the first higher eukaryotic member of the helicase superfamily I, with demonstrated enzymatic activity. Because MAH does not bind small nuclear ribonucleoproteins (snRNPs), it appears to be a non-snRNP binding factor of the splicing complex. In conclusion, our data suggest the involvement of MAH in processing of pre-mRNAs in mammalian cells.

Animals↗

Cloning of mDEAH9, a putative RNA helicase and mammalian homologue of Saccharomyces cerevisiae splicing factor Prp43.

Yeast splicing factor Prp43, a DEAH box protein of the putative RNA helicase/RNA-dependent NTPase family, is a splicing factor that functions late in the pre-mRNA splicing pathway to facilitate spliceosome disassembly. In this paper we report cDNA cloning and characterization of mDEAH9, an apparent mammalian homologue of Prp43. Amino acid sequence comparison revealed that the two proteins are approximately 65% identical over a 500-aa region spanning the central helicase domain and the C-terminal region. Expression of mDEAH9 in S. cerevisiae bearing a temperature-sensitive mutation in prp43 was sufficient to restore growth at the nonpermissive temperature. This functional complementation was specific, as mouse mDEAH9 failed to complement mutations in related splicing factor genes prp16 or prp22. Finally, double label immunofluorescence experiments performed with mammalian cells revealed colocalization of mDEAH9 and splicing factor SC35 in punctate nuclear speckles. Thus, the hypothesis that mDEAH9 represents the mammalian homologue of yeast Prp43 is supported by its high sequence homology, functional complementation, and colocalization with a known splicing factor in the nucleus. Our results provide additional support for the hypothesis that the spliceosomal machinery that mediates regulated, dynamic changes in conformation of pre-mRNA and snRNP RNAs has been highly conserved through evolution.

Amino Acid Sequence↗

Serine-arginine (SR)-rich splicing factors have an exon-independent function in pre-mRNA splicing.

Two distinct functions have been proposed for the serine-arginine (SR)-rich family of splicing factors. First, SR proteins are essential splicing factors and are thought to function by mediating protein-protein interactions within the intron during spliceosome assembly. Second, SR proteins bind to exonic enhancer sequences and recruit spliceosome components to adjacent introns. The latter activity is required for splice-site recognition and alternative splicing. Until now it has not been possible to determine whether the requirement for SR proteins in the basic splicing reaction is a secondary consequence of their exon-dependent recruitment function. Here we show that RNA substrates containing only 1 nt of exon sequence can undergo the first step of the splicing reaction in vitro and that this activity requires SR proteins. Thus, we provide direct evidence that SR proteins have both exon-independent and exon-dependent functions in pre-mRNA splicing.

Exons↗

Disruption of base-paired U4.U6 small nuclear RNAs induced by mammalian heterogeneous nuclear ribonucleoprotein C protein.

Due to 3' end modifications, mammalian U6 small nuclear RNA (snRNA) is heterogeneous in size. The major form terminates with five U residues and a 2',3'-cyclic phosphate, but multiple RNAs containing up to 12 U residues have a 3'-OH end. They are labeled in the presence of [alpha-32P]UTP by the terminal uridylyl transferase activity present in HeLa cell nuclear extracts. That these forms all enter the U6 snRNA-containing particles, U4.U6, U4.U5.U6, and the spliceosome, has been demonstrated previously. Here, we report an interaction between the heterogeneous nuclear ribonucleoprotein (hnRNP) C protein, an abundant nuclear pre-mRNA binding protein, and the U6 snRNAs that have the longest uridylate stretches. This U6 snRNA subset is free of any one of the other snRNPs, since anti-Sm antibodies failed to immunoprecipitate hnRNP C protein. Furthermore, isolated U4.U6 snRNPs containing U6 snRNAs with long oligouridylate stretches are disrupted upon binding of hnRNP C protein either purified from HeLa cells or produced as recombinant protein from Escherichia coli. In view of these data and our previous proposal that the U6 snRNA active in splicing has 3'-OH end, we discuss a model where the hnRNP C protein has a decisive function in the catalytic activation of the spliceosome by allowing the release of U4 snRNP.

Base Composition↗

Role of the constitutive splicing factors U2AF65 and SAP49 in suboptimal RNA splicing of novel retroviral mutants.

Retroviruses display a unique form of alternative splicing in which both spliced and unspliced RNAs accumulate in the cytoplasm. Simple retroviruses, such as avian sarcoma virus, do not encode regulatory proteins that affect splicing; this process is controlled solely through interactions between the viral RNA and the host cell splicing machinery. Previously, we described the selection and characterization of novel avian sarcoma virus mutants. These viruses were separated into two classes based upon analysis of splicing intermediates produced in infected cells and in a cell-free system. One class, which included mutants with altered polypyrimidine tract or branch point sequences, showed significant accumulation of intermediates, suggesting that splicing was regulated in step 2. The other class, which included mutants with deletions of exonic enhancer sequences, did not accumulate splicing intermediates, suggesting that splicing was regulated before step 1 of the splicing reaction. In this report, we show that a mutant blocked at step 1 fails to form a stable spliceosomal complex, whereas one blocked at step 2 shows a defect in its ability to transit through the last spliceosomal complex. Using UV cross-linking methods, we show that regulation at each step is associated with specific changes in the binding of cellular splicing factors. Regulation at step 1 is correlated with decreased cross-linking of the factor U2AF65, whereas regulation at step 2 is correlated with enhanced cross-linking of the factor SAP49. Because these mutations were isolated by selection for replication-competent viruses, we conclude that retroviral splicing may be regulated in vivo through altered binding of constitutive splicing factors.

Alternative Splicing↗

Purification of the spliced leader ribonucleoprotein particle from Leptomonas collosoma revealed the existence of an Sm protein in trypanosomes. Cloning the SmE homologue.

Trans-splicing in trypanosomes involves the addition of a common spliced leader (SL) sequence, which is derived from a small RNA, the SL RNA, to all mRNA precursors. The SL RNA is present in the cell in the form of a ribonucleoprotein, the SL RNP. Using conventional chromatography and affinity selection with 2'-O-methylated RNA oligonucleotides at high ionic strength, five proteins of 70, 16, 13, 12, and 8 kDa were co-selected with the SL RNA from Leptomonas collosoma, representing the SL RNP core particle. Under conditions of lower ionic strength, additional proteins of 28 and 20 kDa were revealed. On the basis of peptide sequences, the gene coding for a protein with a predicted molecular weight of 11.9 kDa was cloned and identified as homologue of the cis-spliceosomal SmE. The protein carries the Sm motifs 1 and 2 characteristic of Sm antigens that bind to all known cis-spliceosomal uridylic acid-rich small nuclear RNAs (U snRNAs), suggesting the existence of Sm proteins in trypanosomes. This finding is of special interest because trypanosome snRNPs are the only snRNPs examined to date that are not recognized by anti-Sm antibodies. Because of the early divergence of trypanosomes from the eukaryotic lineage, the trypanosome SmE protein represents one of the primordial Sm proteins in nature.

Amino Acid Sequence↗

ICln is essential for cellular and early embryonic viability.

pICln is a 26-kDa protein that is ubiquitously expressed and highly conserved from Xenopus laevis to Homo sapiens. The physiological functions of pICln remain to be established. To address this question, we disrupted the ICln gene in embryonic stem cells. We found that murine embryos lacking ICln die early in gestation (between stages E3.5 and E7.5). Furthermore, we found that ICln is essential for embryonic stem cell viability. Previously, we showed that pICln interacts directly with a homolog of a yeast protein that binds a PAK-like kinase and participates in the regulation of cell morphology and cell cycling. pICln also forms a complex with several core spliceosomal proteins, and this interaction may play a role in the regulation of spliceosomal biogenesis. Collectively, these data strongly suggest that pICln participates in critical cellular pathways, including regulation of the cell cycle and RNA processing.

3T3 Cells↗

New roles for the Snp1 and Exo84 proteins in yeast pre-mRNA splicing.

The mammalian 70K protein, a component of the U1 small nuclear ribonucleoprotein involved in pre-mRNA splicing, interacts with a number of proteins important for regulating constitutive and alternative splicing. Similar proteins that interact with the yeast homolog of the 70K protein, Snp1p, have yet to be identified. We used the two-hybrid system to find four U1-Snp1 associating (Usa) proteins. Two of these proteins physically associate with Snp1p as assayed by coimmunoprecipitation. One is Prp8p, a known, essential spliceosomal component. This interaction suggests some novel functions for Snp1p and the U1 small nuclear ribonucleoprotein late in spliceosome development. The other, Exo84p, is a conserved subunit of the exocyst, an eight-protein complex functioning in secretion. We show here that Exo84p is also involved in pre-mRNA splicing. A temperature-sensitive exo84 mutation caused increased ratios of pre-mRNA to mRNA for the Rpl30 and actin transcripts in cells incubated at the non-permissive temperature. The mutation also led to a defect in splicing and prespliceosome formation in vitro; an indication that Exo84p has a direct role in splicing. The results elucidate a surprising link between splicing and secretion.

Actins↗

Identification and characterization of Gemin7, a novel component of the survival of motor neuron complex.

The survival of motor neurons (SMN) protein is the product of the gene mutated or deleted in the neurodegenerative disease, spinal muscular atrophy. SMN is part of a large macromolecular complex that also contains Gemin2, Gemin3, Gemin4, Gemin5, and Gemin6. The SMN complex functions in the assembly of spliceosomal small nuclear ribonucleoproteins and probably other ribonucleoprotein particles. We have identified a novel protein component of the SMN complex termed Gemin7 using native purified SMN complexes and peptide sequencing by mass spectrometry. Coimmunoprecipitation and immunolocalization experiments demonstrate that Gemin7 is a component of the SMN complex and colocalizes with SMN in the cytoplasm and in gems. Binding experiments show that Gemin7 interacts directly with SMN and Gemin6 and mediates the association of Gemin6 with the SMN complex. The amino acid sequence of Gemin7 does not contain any recognizable motifs with the exception of several arginine and glycine repeats that are necessary for its interaction with SMN. Moreover, Gemin7 interacts with several Sm proteins of spliceosomal small nuclear ribonucleoproteins, in particular, with SmE. With the identification of Gemin7, the inventory of the core components of the SMN complex appears essentially complete.

Amino Acid Sequence↗

Network of interactions of a novel plant-specific Arg/Ser-rich protein, atRSZ33, with atSC35-like splicing factors.

Arg/Ser-rich (RS) proteins play a crucial role in splicing and are implicated in splice site selection in metazoa. In plants, intron recognition seems to differ from the one in animals due to specific factor requirements. Here we describe a new plant-specific RS-rich protein, atRSZ33, with a unique domain structure consisting of an RNA recognition motif (RRM), two zinc knuckles embedded in a basic RS region, and an acidic C-terminal domain. atRSZ33 was found to be a phosphoprotein that concentrates in nuclear speckles and is predominantly present in roots and flowers. In a yeast two-hybrid screen, atRSZ33 interacted with splicing factors atSRp34/SR1, an Arabidopsis ortholog of human SF2/ASF; atRSZp21 and atRSZp22, which are similar to the human 9G8; and three novel SC35-like splicing factors termed atSCL28, atSCL30, and atSCL33/SR33. Two further members of the SCL family, namely SCL30a and the ortholog of mammalian SC35, atSC35, were also found to interact with atRSZ33. These interactions were verified by in vitro binding assays; furthermore, the transcriptional activity of atRSZ33 was found to overlap with the ones of its interacting partners. These specific interactions coupled with the many similarities of atRSZ33 to SR proteins suggest that its main activity is in spliceosome assembly. Mapping of regions necessary for protein-protein interaction between atRSZ33 and atSCL33/SR33 revealed that both zinc knuckles together with a small part of the RS and the RRM domain are required for efficient binding. However, the interacting domain is relatively small, allowing binding of additional proteins, a feature that is consistent with the proposed role of atRSZ33 in spliceosome assembly.

Amino Acid Sequence↗

Homomeric ring assemblies of eukaryotic Sm proteins have affinity for both RNA and DNA. Crystal structure of an oligomeric complex of yeast SmF.

Sm and Sm-like proteins are key components of small ribonucleoproteins involved in many RNA and DNA processing pathways. In eukaryotes, these complexes contain seven unique Sm or Sm-like (Lsm) proteins assembled as hetero-heptameric rings, whereas in Archaea and bacteria six or seven-membered rings are made from only a single polypeptide chain. Here we show that single Sm and Lsm proteins from yeast also have the capacity to assemble into homo-oligomeric rings. Formation of homo-oligomers by the spliceosomal small nuclear ribonucleoprotein components SmE and SmF preclude hetero-interactions vital to formation of functional small nuclear RNP complexes in vivo. To better understand these unusual complexes, we have determined the crystal structure of the homomeric assembly of the spliceosomal protein SmF. Like its archaeal/bacterial homologs, the SmF complex forms a homomeric ring but in an entirely novel arrangement whereby two heptameric rings form a co-axially stacked dimer via interactions mediated by the variable loops of the individual SmF protein chains. Furthermore, we demonstrate that the homomeric assemblies of yeast Sm and Lsm proteins are capable of binding not only to oligo(U) RNA but, in the case of SmF, also to oligo(dT) single-stranded DNA.

Binding Sites↗

Nuclear coactivator-62 kDa/Ski-interacting protein is a nuclear matrix-associated coactivator that may couple vitamin D receptor-mediated transcription and RNA splicing.

Nuclear coactivator-62 kDa/Ski-interacting protein (NCoA62/SKIP) is a putative vitamin D receptor (VDR) and nuclear receptor coactivator protein that is unrelated to other VDR coactivators such as those in the steroid receptor coactivator (SRC) family. The mechanism through which NCoA62/SKIP functions in VDR-activated transcription is unknown. In the present study, we identified a nuclear localization sequence in the COOH terminus of NCoA62/SKIP and showed that NCoA62/SKIP was targeted to nuclear matrix subdomains. Chromatin immunoprecipitation studies revealed that endogenous NCoA62/SKIP associated in a 1,25-dihydroxyvitamin D3-dependent manner with VDR target genes in ROS17/2.8 osteosarcoma cells. A cyclic pattern of promoter occupancy by VDR, SRC-1, and NCoA62/SKIP was observed, with NCoA62/SKIP entering these promoter complexes after SRC-1. These studies provide strong support for the proposed role of NCoA62/SKIP as a VDR transcriptional coactivator, and they indicate that key mechanistic differences probably exist between NCoA62/SKIP and SRC coactivators. To explore potential mechanisms, NCoA62/SKIP-interacting proteins were purified from HeLa cell nuclear extracts and identified by mass spectrometry. The identified proteins represent components of the spliceosome as well as other nuclear matrix-associated proteins. Here, we show that a dominant negative inhibitor of NCoA62/SKIP (dnNCoA62/SKIP) interfered with appropriate splicing of transcripts derived from 1,25-dihydroxyvitamin D3-induced expression of a growth hormone minigene cassette. Taken together, these data show that NCoA62/SKIP has properties that are consistent with those of nuclear receptor coactivators and with RNA spliceosome components, thus suggesting a potential role for NCoA62/SKIP in coupling VDR-mediated transcription to RNA splicing.

Animals↗

Motifs IV and V in the DEAH box splicing factor Prp22 are important for RNA unwinding, and helicase-defective Prp22 mutants are suppressed by Prp8.

The yeast pre-mRNA splicing factor Prp22 is a member of the DEAH box family of nucleic acid-stimulated ATPases and RNA helicases. Here we report a mutational analysis of 16 conserved residues in motifs Ia ((534)TQPRRVAA(541)), IV ((695)LVFLTG(700)), and V ((757)TNIAETSIT(765)). Mutants T757A, I764A, and T765A were lethal, and F697A cells did not grow at < or =30 degrees C. The mutant proteins failed to catalyze mRNA release from the spliceosome in vitro, and they were deficient for RNA unwinding. The F697A, I764A, and T765A proteins were active for ATP hydrolysis in the presence of RNA cofactor. The T757A mutant retained basal ATPase activity but was not stimulated by RNA, whereas ATP hydrolysis by T765A was strictly dependent on the RNA cofactor. Thus Thr-757 and Thr-765 in motif V link ATP hydrolysis to the RNA cofactor. To illuminate the mechanism of Prp22-catalyzed mRNA release, we performed a genetic screen to identify extragenic suppressors of the cold-sensitive growth defect of a helicase/release-defective Prp22 mutant. We identified one of the suppressors as a missense mutation of PRP8 (R1753K), a protein component of the U5 small nuclear ribonucleoprotein. We show that PRP8-R1753K suppressed multiple helicase-deficient prp22 mutations, including the lethal I764A mutation. Replacing Arg-1753 of Prp8 by either Lys, Ala, Gln, or Glu resulted in suppression of helicase-defective Prp22 mutants. Prp8-Arg1753 mutations by themselves caused temperature-sensitive growth defects in a PRP22 strain. These findings suggest a model whereby Prp22 disrupts an RNA/protein or RNA/RNA interaction in the spliceosome that is normally stabilized by Prp8.

Amino Acid Sequence↗

Tudor domains bind symmetrical dimethylated arginines.

The Tudor domain is an approximately 60-amino acid structure motif in search of a function. Herein we show that the Tudor domains of the spinal muscular atrophy gene product SMN, the splicing factor 30 kDa (SPF30), and the Tudor domain-containing 3 (TDRD3) proteins interacted with arginine-glycine-rich motifs in a methylarginine-dependent manner. The Tudor domains also associated with methylarginine-containing cellular proteins, providing evidence that methylated arginines represent physiological ligands for this protein module. In addition, we report that spliceosomal small nuclear ribonucleoprotein particles core Sm proteins accumulated in the cytoplasm when arginine methylation was inhibited with adenosine dialdehyde or in the presence of an excessive amount of unmethylated arginine-glycine-rich peptides. These data provide in vivo evidence in support of a role for arginine methylation in the proper assembly and localization of spliceosomal Sm proteins.

Amino Acid Sequence↗