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PubMed1994

Crystal structure at 1.92 A resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin.

The crystal structure of the RNA-binding domain of the small nuclear ribonucleoprotein U1A bound to a 21-nucleotide RNA hairpin has been determined at 1.92 A resolution. The ten-nucleotide RNA loop binds to the surface of the beta-sheet as an open structure, and the AUUGCAC sequence of the loop interacts extensively with the conserved RNP1 and RNP2 motifs and the C-terminal extension of the RNP domain. These interactions include stacking of RNA bases with aromatic side chains of proteins and many direct and water-mediated hydrogen bonds. The structure reveals the stereochemical basis for sequence-specific RNA recognition by the RNP domain.

Amino Acid Sequence↗
PubMed1995

The distribution of a spliceosome protein in cereal (Triticeae) interphase nuclei from cells with different metabolic activities and through the cell cycle.

A monoclonal antibody (mAb) KSm2 and three human Sm sera, which detect the 'D' polypeptide in mammals and is associated with the U1, U2, U4/U6, U5, U7, U9-U12 small nuclear RNAs, have been used in Western blotting to show that the antigen is conserved in wheat (Triticum aestivum cv. Beaver). Immunocytochemistry to wheat and a barley (Hordeum vulgare) suspension culture using the mAb KSm2 has shown that the 'D' polypeptide occurs in interphase nuclei as speckles and foci outside the nucleoli and as tracks. Nucleoli usually had at least one focus at their periphery. Immunocytochemistry at the electron microscope level of resolution has shown that the signal occurs between chromatin axes and predominantly in the outer domain of the nucleus. Analysis of the barley suspension culture demonstrated that there was a significant increase in antigen through G1, S to G2. In the wheat meristematic cells at prophase only the foci remained, and at metaphase the distribution of foci was asymmetrical with foci occurring either at one pole or on the metaphase plate. At telophase the foci appeared to decrease in size and were incorporated non-uniformly into the daughter nuclei because of their asymmetric distribution at metaphase. When wheat was grown at a range of temperatures, the root tip meristematic nuclei showed a number of different organization patterns. The average number of nucleoli increased and their size decreased. At the same time there was an increase in the average total area of foci as temperature increased from 4 degrees, 10 degrees, 25 degrees, 34 degrees, to 37 degrees C. Thus the 'D' polypeptide distribution changes with metabolic activity and through the cell cycle.

Antibodies, Monoclonal↗
PubMed2003

A new Drosophila spliceosomal intron position is common in plants.

The 25-year-old debate about the origin of introns between proponents of "introns early" and "introns late" has yielded significant advances, yet important questions remain to be ascertained. One question concerns the density of introns in the last common ancestor of the three multicellular kingdoms. Approaches to this issue thus far have relied on counts of the numbers of identical intron positions across present-day taxa on the assumption that the introns at those sites are orthologous. However, dismissing parallel intron gain for those sites may be unwarranted, because various factors can potentially constrain the site of intron insertion. Demonstrating parallel intron gain is severely handicapped, because intron sequences often evolve exceedingly fast and intron phylogenetic distributions are usually ambiguous, such that alternative loss and gain scenarios cannot be clearly distinguished. We have identified an intron position that was gained independently in animals and plants in the xanthine dehydrogenase gene. The extremely disjointed phylogenetic distribution of the intron argues strongly for separate gain rather than recurrent loss. If the observed phylogenetic pattern had resulted from recurrent loss, all observational support previously gathered for the introns-late theory of intron origins based on the phylogenetic distribution of introns would be invalidated.

Animals↗
PubMed2000

The spliceosomal snRNP core complex of Trypanosoma brucei: cloning and functional analysis reveals seven Sm protein constituents.

Each of the trypanosome small nuclear ribonucleoproteins (snRNPs) U2, U4/U6, and U5, as well as the spliced leader (SL) RNP, contains a core of common proteins, which we have previously identified. This core is unusual because it is not recognized by anti-Sm Abs and it associates with an Sm-related sequence in the trypanosome small nuclear RNAs (snRNAs). Using peptide sequences derived from affinity-purified U2 snRNP proteins, we have cloned cDNAs for five common proteins of 8.5, 10, 12.5, 14, and 15 kDa of Trypanosoma brucei and identified them as Sm proteins SmF (8.5 kDa), -E (10 kDa), -D1 (12.5 kDa), -G (14 kDa), and -D2 (15 kDa), respectively. Furthermore, we found the trypanosome SmB (T. brucei) and SmD3 (Trypanosoma cruzi) homologues through database searches, thus completing a set of seven canonical Sm proteins. Sequence comparisons of the trypanosome proteins revealed several deviations in highly conserved positions from the Sm consensus motif. We have identified a network of specific heterodimeric and -trimeric Sm protein interactions in vitro. These results are summarized in a model of the trypanosome Sm core, which argues for a strong conservation of the Sm particle structure. The conservation extends also to the functional level, because at least one trypanosome Sm protein, SmG, was able to specifically complement a corresponding mutation in yeast.

Amino Acid Sequence↗
PubMed1996

Heteronuclear ribonucleoproteins C1 and C2, components of the spliceosome, are specific targets of interleukin 1beta-converting enzyme-like proteases in apoptosis.

Apoptosis induced by a variety of agents results in the proteolytic cleavage of a number of cellular substrates by enzymes related to interleukin 1beta-converting enzyme (ICE). A small number of substrates for these enzymes have been identified to date, including enzymes involved in DNA repair processes: poly(ADP-ribose) polymerase and DNA-dependent protein kinase. We describe here for the first time the specific cleavage of the heteronuclear ribonucleoproteins (hnRNPs) C1 and C2 in apoptotic cells induced to undergo apoptosis by a variety of stimuli, including ionizing radiation, etoposide, and ceramide. No cleavage was observed in cells that are resistant to apoptosis induced by ionizing radiation. Protease inhibitor data implicate the involvement of an ICE-like protease in the cleavage of hnRNP C. Using recombinant ICE-like proteases and purified hnRNP C proteins in vitro, we show that the C proteins are cleaved by Mch3alpha and CPP32 and, to a lesser extent, by Mch2alpha, but not by ICE, Nedd2, Tx, or the cytotoxic T-cell protease granzyme B. The results described here demonstrate that the hnRNP C proteins, abundant nuclear proteins thought to be involved in RNA splicing, belong to a critical set of protein substrates that are cleaved by ICE-like proteases during apoptosis.

Apoptosis↗
PubMed2000

The trans-spliceosomal U4 RNA from the monogenetic trypanosomatid Leptomonas collosoma. Cloning and identification of a transcribed trna-like element that controls its expression.

U4 small nuclear RNA is essential for trans-splicing. Here we report the cloning of U4 snRNA gene from Leptomonas collosoma and analysis of elements controlling its expression. The trypanosome U4 RNA is the smallest known, it carries an Sm-like site, and has the potential for extensive intermolecular base pairing with the U6 RNA. Sequence analysis of the U4 locus indicates the presence of a tRNA-like element 86 base pairs upstream of the gene that is divergently transcribed to yield a stable small tRNA-like RNA. Two additional tRNA genes, tRNA(Pro) and tRNA(Gly), were found upstream of this element. By stable expression of a tagged U4 RNA, we demonstrate that the tRNA-like gene, but not the upstream tRNA genes, is essential for U4 expression and that the B box but not the A Box of the tRNA-like gene is crucial for expression in vivo. Mapping the 2'-O-methyl groups on U4 and U6 small nuclear RNAs suggests the presence of modifications in canonical positions. However, the number of modified nucleotides is fewer than in mammalian homologues. The U4 genomic organization including both tRNA-like and tRNA genes may represent a relic whereby trypanosomatids "hired" tRNA genes to provide extragenic promoter elements. The close proximity of tRNA genes to the tRNA-like molecule in the U4 locus further suggests that the tRNA-like gene may have evolved from a tRNA member of this cluster.

Animals↗
PubMed2000

A general approach for identification of RNA-protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs). Analysis of RNA-protein contacts in native U1 and U4/U6.U5 snRNPs.

We describe a novel approach to identify RNA-protein cross-linking sites within native small nuclear ribonucleoprotein (snRNP) particles from HeLa cells. It combines immunoprecipitation of the UV-irradiated particles under semi-denaturing conditions with primer extension analysis of the cross-linked RNA moiety. In a feasibility study, we initially identified the exact cross-linking sites of the U1 70-kDa (70K) protein in stem-loop I of U1 small nuclear RNA (snRNA) within purified U1 snRNPs and then confirmed the results by a large-scale preparation that allowed N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry of purified cross-linked peptide-oligonucleotide complexes. We identified Tyr(112) and Leu(175) within the RNA-binding domain of the U1 70K protein to be cross-linked to G(28) and U(30) in stem-loop I, respectively. We further applied our immunoprecipitation approach to HeLa U5 snRNP, as part of purified 25 S U4/U6.U5 tri-snRNPs. Cross-linking sites between the U5-specific 220-kDa protein (human homologue of Prp8p) and the U5 snRNA were located at multiple nucleotides within the highly conserved loop 1 and at one site in internal loop 1 of U5 snRNA. The cross-linking of four adjacent nucleotides indicates an extended interaction surface between loop 1 and the 220-kDa protein. In summary, our approach provides a rapid method for identification of RNA-protein contact sites within native snRNP particles as well as other ribonucleoprotein particles.

Amino Acid Sequence↗
PubMed2006

RNA structural requirements for the association of the spliceosomal hPrp31 protein with the U4 and U4atac small nuclear ribonucleoproteins.

The kink-turn, a stem I-internal loop-stem II structure of the 5 ' stem-loop of U4 and U4atac small nuclear (sn) RNAs bound by 15.5K protein is required for binding of human Prp31 protein (hPrp31) during U4 and U4atac snRNP assembly. In box C/D snoRNPs a similar kink-turn with bound 15.5K protein is required for selective binding of proteins NOP56 and NOP58. Here we analyzed RNA structural requirements for association of hPrp31 with U4 snRNP in vitro by hydroxyl radical footprinting. hPrp31 induced protection of the terminal penta-loop, as well as of stems I and II flanking the kink-turn. Similar protection was found with U4/U6 snRNA duplex prebound with 15.5K protein. A detailed mutational analysis of the U4 snRNA elements by electrophoretic mobility shift analysis revealed that stem I could not be shortened, although it tolerated sequence alterations. However, introduction of a third Watson-Crick base pair into stem II significantly reduced hPrp31 binding. While stem I of U4atac snRNA showed relaxed binding requirements, its stem II requirements were likewise restricted to two base pairs. In contrast, as shown previously, stem II of the kink-turn motif in box C/D snoRNAs is comprised of three base pairs, and NOP56 and NOP58 require a G-C pair at the central position. This indicates that hPrp31 binding specificity is achieved by the recognition of the two base pair long stem II of the U4 and U4atac snRNAs and suggests how discrimination is achieved by RNA structural elements during assembly of U4/U6 and U4atac/U6atac snRNPs and box C/D snoRNPs.

Eye Proteins↗
PubMed2000

An ATP-dependent, Ran-independent mechanism for nuclear import of the U1A and U2B" spliceosome proteins.

Nuclear import of the two uracil-rich small nuclear ribonucleoprotein (U snRNP) components U1A and U2B" is mediated by unusually long and complex nuclear localization signals (NLSs). Here we investigate nuclear import of U1A and U2B" in vitro and demonstrate that it occurs by an active, saturable process. Several lines of evidence suggest that import of the two proteins occurs by an import mechanism different to those characterized previously. No cross competition is seen with a variety of previously studied NLSs. In contrast to import mediated by members of the importin-beta family of nucleocytoplasmic transport receptors, U1A/U2B" import is not inhibited by either nonhydrolyzable guanosine triphosphate (GTP) analogues or by a mutant of the GTPase Ran that is incapable of GTP hydrolysis. Adenosine triphosphate is capable of supporting U1A and U2B" import, whereas neither nonhydrolyzable adenosine triphosphate analogues nor GTP can do so. U1A and U2B" import in vitro does not require the addition of soluble cytosolic proteins, but a factor or factors required for U1A and U2B" import remains tightly associated with the nuclear fraction of conventionally permeabilized cells. This activity can be solubilized in the presence of elevated MgCl(2). These data suggest that U1A and U2B" import into the nucleus occurs by a hitherto uncharacterized mechanism.

Adenosine Triphosphate↗
PubMed2001

Promiscuity of pre-mRNA spliceosome-mediated trans splicing: a problem for gene therapy?

Trans splicing of messenger RNA has been used in experimental settings to replace mutant RNA sequences. We investigated the feasibility of utilizing trans splicing to replace a mutant RET protooncogene sequence known to inappropriately activate this tyrosine kinase receptor. We constructed a pre-trans-splicing molecule (PTM) consisting of a binding domain complementary to the target intron, the 3' splicing signal sequence (3'ss), derived from adenovirus major late transcript intron 1 and a molecular tag sequence. Accurately targeted trans splicing between the human RET exons and the PTM was demonstrated in NIH 3T3 cells cotransfected with the human RET minigene and the PTM. The efficiency of specific trans splicing was estimated to be no more than 15% in the cotransfection experiment. However, in addition to the targeted trans splicing, nontargeted trans splicing to RET exons was observed. Furthermore, the rapid amplification of 5' cDNA ends (5' RACE) analysis demonstrated that nontargeted trans splicing occurred with endogenously expressed pre-mRNAs in TT cells and that specific trans splicing to RET was a rare event. Attempts to reduce nonspecificity by the addition of a stem-loop to the trans-splicing construct designed to suppress nonspecific splicing failed to have the desired effect. These observations suggest that overexpression of a trans-splicing construct containing a 3'ss results in promiscuous trans splicing and raise significant questions about the specificity and usefulness of currently used trans-splicing approaches. In addition, these findings raise the possibility that nonspecific spliced products may be produced by a variety of gene therapy constructs.

3T3 Cells↗
PubMed1998

Structural similarities between hammerhead ribozymes and the spliceosomal RNAs could be responsible for lack of ribozyme cleavage in yeast.

Hammerhead ribozymes have been proposed as potential therapeutic agents for the treatment of viral and other diseases. However, a clear understanding of the cleavage reaction in vivo is not available at present. In these studies, we chose the yeast Saccharomyces cerevisiae as a model system to study the effects of hammerhead cleavage on gene expression in vivo. Several reporter genes were employed to monitor the self-cleaving characteristics of three different ribozymes. We show that these ribozymes decrease expression of some reporter genes by interfering with splice site selection or translation initiation and not by in vivo cleavage of the RNA transcripts. In fact, it appears that although these ribozymes can efficiently self-cleave the RNA in vitro, they are not able to function in vivo. We have identified a yeast splicing protein that interacts in vivo with our cis-ribozyme by specifically recognizing the ribozyme structure (Lin and Rossi, 1996). This interaction does not occur if different secondary structures are used in place of the ribozyme. The binding of this protein to the ribozyme can account for the inability of ribozymes to efficiently cleave in yeast. Remarkably, when yeast extracts are added to in vitro trans-cleavage reactions, the cleavage ability of the ribozyme is hampered, whereas the addition of mammalian extracts yields an enhancement of the reactions. These results confirm the presence of factor(s) that can block ribozyme function in the yeast intracellular environment.

Base Sequence↗
PubMed1998

Molecular characterization of a novel, widespread nuclear protein that colocalizes with spliceosome components.

We report the identification and molecular characterization of a novel type of constitutive nuclear protein that is present in diverse vertebrate species, from Xenopus laevis to human. The cDNA-deduced amino acid sequence of the Xenopus protein defines a polypeptide of a calculated mass of 146.2 kDa and a isoelectric point of 6.8, with a conspicuous domain enriched in the dipeptide TP (threonine-proline) near its amino terminus. Immunolocalization studies in cultured cells and tissues sections of different origin revealed an exclusive nuclear localization of the protein. The protein is diffusely distributed in the nucleoplasm but concentrated in nuclear speckles, which represent a subnuclear compartment enriched in small nuclear ribonucleoprotein particles and other splicing factors, as confirmed by colocalization with certain splicing factors and Sm proteins. During mitosis, when transcription and splicing are downregulated, the protein is released from the nuclear speckles and transiently dispersed throughout the cytoplasm. Biochemical experiments have shown that the protein is recovered in a approximately 12S complex, and gel filtration studies confirm that the protein is part of a large particle. Immunoprecipitation and Western blot analysis of chromatographic fractions enriched in human U2 small nuclear ribonucleoprotein particles of distinct sizes (12S, 15S, and 17S), reflecting their variable association with splicing factors SF3a and SF3b, strongly suggests that the 146-kDa protein reported here is a constituent of the SF3b complex.

Amino Acid Sequence↗