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PubMed2005

Identification and analysis of U5 snRNA variants in Drosophila.

Distinct isoforms of spliceosomal RNAs may be involved in regulating pre-messenger RNA splicing in eukaryotic cells. During a large-scale effort to identify small noncoding RNAs in Drosophila, we isolated a U5 snRNA-like molecule containing a 5' segment identical to that of the canonical (major) U5 snRNA but with a variant Sm binding site and a distinct 3' hairpin sequence. Based on this finding, another six similar U5 snRNA-like sequences were identified within the Drosophila genome by sequence similarity to the invariant loop in the 5' half of U5. Interestingly, although all of these variants are expressed in vivo, each shows a distinct temporal expression profile during Drosophila development, and one is expressed primarily in fly heads. The presence of these U5 snRNA variants within RNP particles suggests their role in splicing and implies a possible connection to regulation of developmental and tissue-specific gene expression.

Amino Acid Sequence↗

PubMed1996

EGF-receptor RNA metabolism in the nucleus of A431 cells.

Epidermal growth factor (EGF) receptor RNA has been shown to be localized around nucleoli in the nucleus of A431 cells (Sibon et al., Histochemistry 101, 223-232 (1994)). Here we have studied the functional implication of this localization. Inhibition of transcription by alpha-amanitin did not influence the localization and amount of EGF-receptor RNA around the nucleolus, indicating that these RNAs represent mainly completed transcripts. Localization of the EGF-receptor genes in A431 cells by in situ hybridization revealed that the majority of the receptor gene clusters are located at the periphery of the nucleus. Next to this virtually all cells studied contain at least one gene cluster in the vicinity of the nucleolus. From these data, it is tempting to suggest that EGF-receptor gene transcription occurs around the nucleolus. In order to obtain information on the site of EGF-receptor RNA splicing, the localization of exon and intron sequences of the EGF-receptor transcripts was studied using a new electron microscopical approach. These labeling studies revealed that both intron and exon sequences were present at the same site around the nucleolus. In addition, exon sequences were also located, around nucleolus separate from intron sequences. All together, these studies suggest that transcription and splicing of the EGF-receptor transcript occurs at the same defined site around the nucleolus in A431 cells.

3T3 Cells↗

PubMed1985

Alternative splicing caused by RNA secondary structure.

mRNA precursors with stable hairpins were constructed by inserting inverted repeats into an adenovirus transcriptional template that encoded the three late leader exons. When the loop of the hairpin contained the second exon and the flanking splice sites, most of the RNA spliced in vitro had the first exon joined directly to the third exon. The remainder was spliced normally. The same types of alternatively spliced RNAs were formed when a similar template was introduced into HeLa cells by transfection. Thus both in extracts and in cells, an exon became optional when sequestered in a hairpin loop. Perhaps a related mechanism creates the alternative splicing patterns of complex transcription units.

Adenoviruses, Human↗

PubMed1987

Morphf mutants of Rous sarcoma virus: nucleotide sequencing analysis suggests that a class of morphf mutants was generated through splicing of a cryptic intron.

The nature of the lesions involved in producing the fusiform phenotype of three mutants (WO101, WO201, and tsST529) of the Schmidt-Ruppin A strain of Rous sarcoma virus (RSV) was determined by molecular cloning and DNA sequencing. WO101 and WO201 contained an in-frame deletion of the v-src region coding for amino acids 116 to 140 of p60v-src. The deleted segment was flanked by consensus splice donor and acceptor sequences and contained an appropriately positioned branchpoint acceptor consensus sequence, suggesting that the deletion occurred through an aberrant RNA splicing event. S1 mapping experiments performed on RNA isolated from chicken cells infected with molecularly cloned wild-type RSV DNA suggested that the splice acceptor involved in the generation of this deletion was utilized at a low frequency (less than 1.0%) in wild-type RSV-infected cells. These results suggested that stable mutations may have arisen in the coding sequence of a eucaryotic viral transforming gene as a result of a probable aberrant RNA splicing event followed by reverse transcription into DNA. ST529 was found to harbor the same deletion present in WO101 and WO201 but also contained a point mutation which resulted in the substitution of lysine for glutamic acid at position 93. This change and the resulting large change in local charge were presumably required for the temperature-sensitive transformation phenotype of ST529. These results, together with other known deletions that produce fusiform mutants, suggested that a region within the amino-terminal one-third coding region of the src gene contributed to a structural domain of p60v-src that was important for controlling some morphological parameters of transformation in cells infected with RSV.

Avian Sarcoma Viruses↗

PubMed1989

Group I introns as mobile genetic elements: facts and mechanistic speculations--a review.

Group I introns form a structural and functional group of introns with widespread but irregular distribution among very diverse organisms and genetic systems. Evidence is now accumulating that several group I introns are mobile genetic elements with properties similar to those originally described for the omega system of Saccharomyces cerevisiae: mobile group I introns encode sequence-specific double-strand (ds) endoDNases, which recognize and cleave intronless genes to insert a copy of the intron by a ds-break repair mechanism. This mechanism results in: the efficient propagation of group I introns into their cognate sites; their maintenance at the site against spontaneous loss; and, perhaps, their transposition to different sites. The spontaneous loss of group I introns occurs with low frequency by an RNA-mediated mechanism. This mechanism eliminates introns defective for mobility and/or for RNA splicing. Mechanisms of intron acquisition and intron loss must create an equilibrium, which explains the irregular distribution of group I introns in various genetic systems. Furthermore, the observed distribution also predicts that horizontal transfer of intron sequences must occur between unrelated species, using vectors yet to be discovered.

Base Sequence↗

PubMed2003

Effects of maturase binding and Mg2+ concentration on group II intron RNA folding investigated by UV cross-linking.

The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase/maturase (LtrA protein) that promotes RNA splicing by stabilizing the catalytically active RNA structure. Here, we mapped 17 UV cross-links induced in both wild-type Ll.LtrB RNA and Ll.LtrB-Delta2486 RNA, which has a branch-point deletion that prevents splicing, and we used these cross-links to follow tertiary structure formation under different conditions in the presence or absence of the LtrA protein. Twelve of the cross-links are long-range, with six near known tertiary interaction sites in the active RNA structure. In a reaction medium containing 0.5 M NH(4)Cl, eight of the 17 cross-links were detected in the absence of Mg(2+) or the presence of EDTA, and all were detected at 5 mM Mg(2+), where efficient splicing requires the LtrA protein. The frequencies of all but four cross-links increased with increasing Mg(2+) concentrations, becoming maximal between 4 and 50 mM Mg(2+), where the intron is self-splicing. These findings suggest that a high Mg(2+) concentration induces self-splicing by globally stabilizing tertiary structure, including key tertiary interactions that are required for catalytic activity. Significantly, the binding of the maturase under protein-dependent splicing conditions (0.5 M NH(4)Cl and 5 mM Mg(2+)) increased the frequency of only nine cross-links, seven of which are long-range, suggesting that, in contrast to a high Mg(2+) concentration, LtrA promotes splicing by stabilizing critical tertiary structure interactions, while leaving other regions of the intron relatively flexible. This difference may contribute to the high rate of protein-dependent splicing, relative to the rate of self-splicing. The propensity of the intron RNA to form tertiary structure even at relatively low Mg(2+) concentrations raises the possibility that the maturase functions at least in part by tertiary structure capture. Finally, an abundant central wheel cross-link, present in >50% of the molecules at 5 mM Mg(2+), suggests models in which group II intron domains I and II are either coaxially stacked or aligned in parallel, bringing the 5'-splice site together with the 3'-splice site and catalytic core elements at JII/III. This and other cross-links provide new constraints for three-dimensional structural modeling of the group II intron catalytic core.

Base Sequence↗

PubMed1988

Analysis of metal-induced mutations altering the expression or structure of a retroviral gene in a mammalian cell line.

Carcinogenic metal compounds, with the exception of chromium(VI), have been found to be poorly mutagenic in both prokaryotic and mammalian cell mutagenesis assays, yet they are clearly clastogenic (Hansen and Stern, 1984). Thus, the role of metals as initiators in carcinogenesis has been difficult to delineate. In an effort to develop a model system capable of assaying DNA damage caused by carcinogenic metals, we have investigated the role of NiCl2, CdCl2, Na2CrO4, and NMU in a murine sarcoma virus-infected mammalian cell line in which expression of the retroviral v-mos gene is growth-temperature regulated. This cell line, designated 6m2, contains a single-copy, stably integrated, mutant Moloney murine sarcoma virus DNA (designated MuSVts110) and is temperature sensitive for morphological transformation due to a conditionally defective viral RNA-splicing event that in turn regulates expression of the viral transforming gene. Mutations affecting the viral DNA in 6m2 cells can be detected if these alterations lead to changes in the structure or expression of the transforming protein encoded by the MuSVts110 v-mos gene. Analysis of the viral proteins from 6m2 'revertant' cell lines (as defined by reversion to the transformed phenotype at all growth temperatures) selected after treatment with the above agents showed that NiCl2, NMU, and Na2CrO4 each induced a different yet specific type of mutation. NiCl2 and NMU each altered the temperature sensitivity of viral RNA splicing, possibly due to base substitution mutations, but did so to distinctly different extents. Na2CrO4 affected the structure of the viral proteins by inducing what appear to be short frameshift mutations that resulted in the temperature-dependent translation of a novel virus-encoded transforming protein, P100gag-mos. CdCl2 also induced frameshift mutations but, in one case, induced a mutation which may result from a deletion of about 300 bases within the MuSVts110 DNA.

Cell Line↗

PubMed1988

The precise structure and coding capacity of mRNAs from early region 2B of human adenovirus serotype 2.

Replication of human adenovirus (Ad) DNA requires three virus-encoded proteins that are coordinately transcribed from a single promoter at early times after infection. The mRNAs for two of these proteins, the precursor to the terminal protein (pTP) and the Ad DNA polymerase (Ad Pol), share several exons, including one encoded near Ad genome coordinate 39. The positions of the splice points of these mRNAs have been mapped by S1 nuclease mapping, by RNA sequencing, and by cDNA cloning. As a result of RNA splicing events, a short open reading frame (ORF) encoded at genome coordinate 39 is connected to the beginning of both the pTP and Ad Pol coding sequences; inclusion of this upstream ORF is essential for expression of functional pTP and Ad Pol proteins.

Adenoviruses, Human↗

PubMed1999

A human papillomavirus E2 transcriptional activator. The interactions with cellular splicing factors and potential function in pre-mRNA processing.

The human papillomavirus (HPV) E2 protein plays an important role in transcriptional regulation of viral genes as well as in viral DNA replication. Unlike most types of HPV, the E2 protein of epidermodysplasia verruciformis (EV)-associated HPVs harbors a relatively long hinged region between the terminal, conserved transactivation and DNA binding/dimerization domains. The sequence of EV-HPV E2 hinge contains multiple arginine/serine (RS) dipeptide repeats which are characteristic of a family of pre-messenger RNA splicing factors, called SR proteins. Here we show that the HPV-5 (an EV-HPV) E2 protein can specifically interact with cellular splicing factors including a set of prototypical SR proteins and two snRNP-associated proteins. Transiently expressed HPV-5 E2 protein colocalizes with a nuclear matrix associated-splicing coactivator in nuclear speckled domains. The RS-rich hinge is essential for E2 transactivator interaction with splicing factors and for its subnuclear localization. Moreover, we present functional evidence for the HPV-5 E2 transactivator, which shows that the RS-rich hinge domain of the E2 protein can facilitate the splicing of precursor messenger RNA made via transactivation by E2 itself. Our results, therefore, suggest that a DNA binding transactivator containing an RS-rich sequence can play a dual role in gene expression.

Gene Expression Regulation↗

PubMed1988

Conserved sequences and structures of group I introns: building an active site for RNA catalysis--a review.

Group I introns fold to form an active site to mediate their own RNA splicing. Sequence elements conserved among the available set of 66 group I introns are compiled. Comparative sequence analysis leads to the prediction of some conserved structural features that have not been widely appreciated. The possible significance of conserved nucleotides within base-paired duplexes is discussed; they might be involved in base triplets or alternate pairing interactions.

Introns↗

PubMed2005

Peptidyl-prolyl cis-trans isomerases (immunophilins) and their roles in parasite biochemistry, host-parasite interaction and antiparasitic drug action.

Immunophilin is the collective name given to the cyclophilin and FK506-binding protein families. As the name suggests, these include the major binding proteins of certain immunosuppressive drugs: cyclophilins for the cyclic peptide cyclosporin A and FK506-binding proteins for the macrolactones FK506 and rapamycin. Both families, although dissimilar in sequence, possess peptidyl-prolyl cis-trans isomerase activity in vitro and can play roles in protein folding and transport, RNA splicing and the regulation of multi-protein complexes in cells. In addition to enzymic activity, many immunophilins act as molecular chaperones. This property may be conferred by the isomerase domain and/or by additional domains. Recent years have seen a great increase in the number of known immunophilin genes in parasitic protozoa and helminths and in many cases their products have been characterised biochemically and their temporal and spatial expression patterns have been examined. Some of these genes represent novel types: one example is a Toxoplasma gondii gene encoding a protein with both cyclophilin and FK506-binding protein domains. Likely roles in protein folding and oligomerisation, RNA splicing and sexual differentiation have been suggested for parasite immunophilins. In addition, unexpected roles in parasite virulence (Mip FK506-binding protein of Trypanosoma cruzi) and host immuno-modulation (e.g. 18-kDa cyclophilin of T. gondii) have been established. Furthermore, in view of the potent antiparasitic activities of cyclosporins, macrolactones and non-immunosuppressive derivatives of these compounds, immunophilins may mediate drug action and/or may themselves represent potential drug targets. Investigation of the mechanisms of action of these agents may lead to the design of potent and selective antimalarial and other antiparasitic drugs. This review discusses the properties of immunophilins in parasites and the 'animal model'Caenorhabditis elegans and relates these to our understanding of the roles of these proteins in cellular biochemistry, host-parasite interaction and the antiparasitic mechanisms of the drugs that bind to them.

Amino Acid Sequence↗

PubMed1988

Synthesis of insulin-like growth factor II (IGF-II) in fetal rat tissues: translation of IGF-II ribonucleic acid and processing of pre-pro-IGF-II.

The single insulin-like growth factor II (IGF-II) gene is transcribed into multiple RNA species in most fetal and neonatal rat tissues. For IGF-II to serve as a local growth factor in fetal tissues, IGF-II RNA must be translated into pre-pro-rat (r) IGF-II, and the biosynthetic precursor processed to smaller biologically active forms. IGF-II RNA extracted from fetal rat liver, muscle, intestine, lung, and stomach, from rat placenta, and from fetal or neonatal mouse liver and lung directed the synthesis of 22,000 mol wt pre-pro-IGF-II in a reticulocyte lysate cell-free translation system. A biosynthetic precursor of this size had been observed previously in translation of RNA from BRL-3A rat liver cells and is predicted by the nucleotide sequence of cDNA clones encoding rIGF-II. Consistent with the developmental pattern of expression of IGF-II RNA observed in hybridization studies, RNA from adult rat liver, muscle, and intestine did not direct the synthesis of pre-pro-rIGF-II. To determine whether the IGF-II biosynthetic precursor was processed to smaller biologically active IGF-II, term fetal rat tissues were extracted with acid-ethanol, the extracts were fractionated by acid gel filtration, and the IGF pools were examined in a RIA specific for IGF-II. Levels of 1-2 micrograms/g were observed in liver, limb, lung, intestine, and brain; lower levels were observed in heart and kidney. In general, the levels of immunoreactive IGF-II corresponded to the levels of IGF-II mRNA. These results suggest that IGF-II mRNA is translated, and pre-pro-IGF-II processed to mature IGF-II in different fetal rat tissues. In contrast to IGF-I, in which alternative RNA splicing generates possible precursor molecules containing different COOH-terminal propeptide segments, we find no evidence for an IGF-II precursor in rat tissues other than 22,000 mol wt pre-pro-rIGF-II.

Animals↗

PubMed1991

A genomic sequence of the Schizosaccharomyces pombe 16 kDa vacuolar H(+)-ATPase.

We have isolated the gene encoding the 16 kDa vacuolar H(+)-ATPase from Schizosaccharomyces pombe. On the basis of RNA splicing signals and amino acid sequence homology with other 16 kDa H(+)-ATPases, the genomic DNA sequence indicated the 16 kDa protein is encoded by five exons. The C-terminal 50 amino acids has more than 90% homology with vacuolar H(+)-ATPases of mammalian cells.

Amino Acid Sequence↗

PubMed2005

The protein phosphatase AtPP2CA negatively regulates abscisic acid signal transduction in Arabidopsis, and effects of abh1 on AtPP2CA mRNA.

To identify new loci in abscisic acid (ABA) signaling, we screened a library of 35ScDNA Arabidopsis (Arabidopsis thaliana)-expressing lines for ABA-insensitive mutants in seed germination assays. One of the identified mutants germinated on 2.5 microm ABA, a concentration that completely inhibits wild-type seed germination. Backcrosses and F2 analyses indicated that the mutant exhibits a dominant phenotype and that the ABA insensitivity was linked to a single T-DNA insertion containing a 35ScDNA fusion. The inserted cDNA corresponds to a full-length cDNA of the AtPP2CA gene, encoding a protein phosphatase type 2C (PP2C). Northern-blot analyses demonstrated that the AtPP2CA transcript is indeed overexpressed in the mutant (named PP2CAox). Two independent homozygous T-DNA insertion lines, pp2ca-1 and pp2ca-2, were recovered from the Arabidopsis Biological Resource Center and shown to lack full-length AtPP2CA expression. A detailed characterization of PP2CAox and the T-DNA disruption mutants demonstrated that, whereas ectopic expression of a 35SAtPP2CA fusion caused ABA insensitivity in seed germination and ABA-induced stomatal closure responses, disruption mutants displayed the opposite phenotype, namely, strong ABA hypersensitivity. Thus our data demonstrate that the PP2CA protein phosphatase is a strong negative regulator of ABA signal transduction. Furthermore, it has been previously shown that the AtPP2CA transcript is down-regulated in the ABA-hypersensitive nuclear mRNA cap-binding protein mutant abh1. We show here that down-regulation of AtPP2CA in abh1 is not due to impaired RNA splicing of AtPP2CA pre-mRNA. Moreover, expression of a 35SAtPP2CA cDNA fusion in abh1 partially suppresses abh1 hypersensitivity, and the data further suggest that additional mechanisms contribute to ABA hypersensitivity of abh1.

Abscisic Acid↗

PubMed2000

The relationship between SMN, the spinal muscular atrophy protein, and nuclear coiled bodies in differentiated tissues and cultured cells.

The spinal muscular atrophy protein, SMN, is a cytoplasmic protein that is also found in distinct nuclear structures called "gems." Gems are closely associated with nuclear coiled bodies and both may have a direct role in snRNP maturation and pre-RNA splicing. There has been some controversy over whether gems and coiled bodies colocalize or form adjacent/independent structures in HeLa and other cultured cells. Using a new panel of antibodies against SMN and antibodies against coilin-p80, a systematic and quantitative study of adult differentiated tissues has shown that gems always colocalize with coiled bodies. In some tissues, a small proportion of coiled bodies (<10%) had no SMN, but independent or adjacent gems were not found. The most striking observation, however, was that many cell types appear to have neither gems nor coiled bodies (e.g., cardiac and smooth muscle, blood vessels, stomach, and spleen) and this expression pattern is conserved across human, rabbit, and pig species. This shows that assembly of distinct nuclear bodies is not essential for RNA splicing and supports the view that they may be storage sites for reserves of essential proteins and snRNPs. Overexpression of SMN in COS-7 cells produced supernumerary nuclear bodies, most of which also contained coilin-p80, confirming the close relationship between gems and coiled bodies. However, when SMN is reduced to very low levels in type I SMA fibroblasts, coiled bodies are still formed. Overall, the data suggest that gem/coiled body formation is not determined by high cytoplasmic SMN concentrations or high metabolic activity alone and that a differentiation-specific factor may control their formation.

Animals↗

PubMed2004

Single-nucleotide polymorphism g.1548G > A (E469K) in human ICAM-1 gene affects mRNA splicing pattern and TPA-induced apoptosis.

The single-nucleotide polymorphism (SNP) g.1548G > A (E469K) in the human intercellular adhesion molecule-1 (ICAM-1) gene has been suggested to have an association with several types of inflammatory diseases. The polymorphism is located at the three-base position upstream of the splice donor site that produces an alternatively spliced short isoform (ICAM-1-S). To clarify its functional relevance, we studied RNA splicing patterns by comparing cells with different genotype (G/G cells and A/A cells). G/G cells expressed a lower amount of ICAM-1-S mRNA than A/A cells. Since ICAM-1-S has no transmembrane or intracellular domain, ICAM-1 signal transduction and cell-cell contact including Fas-FasL interaction may be influenced. In addition, we studied the effect of this change on FLIP-L mRNA and apoptosis. FLIP-L mRNA tended to decrease, while cell death induced by phorbol 12-myristate 13-acetate was increased. These results suggest that the g.1548 polymorphism modifies inflammatory immune responses by changing cell-cell interaction and then regulating apoptosis.

Apoptosis↗

PubMed1991

Nucleotide sequences of avian cardiac and brain SR/ER Ca(2+)-ATPases and functional comparisons with fast twitch Ca(2+)-ATPase. Calcium affinities and inhibitor effects.

Two similar forms of the cardiac/slow Ca(2+)-ATPase (SERCA2a and SERCA2b), differing in sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility, are expressed in chicken heart and brain (Kaprielian, Z., Campbell, A. M., and Fambrough, D. M. (1989) Mol. Brain Res. 6, 55-60). In the current study, cDNAs encoding each form were cloned and sequenced. Chicken SERCA2a is 94% identical to its rabbit homologue, while SERCA2b has an extended carboxyl terminus with 38 of 49 amino acids identical to mammalian homologues. SERCA2b mRNA contains the SERCA2a encoding sequence within its 3'-untranslated region. Chicken genomic DNA sequence reveals that the alternate RNA splicing used to produced SERCA2a and SERCA2b subtypes involves a splice site within an exon. Tissue culture cells expressing the avian SERCA2a, SERCA2b, and SERCA1, each targetting to the endoplasmic reticulum, were used to measure Ca2+ affinities and inhibitor effects; no differences among the three pumps were detected.

Amino Acid Sequence↗

PubMed1997

Requirement of the DEAD-Box protein ded1p for messenger RNA translation.

The DED1 gene, which encodes a putative RNA helicase, has been implicated in nuclear pre-messenger RNA splicing in the yeast Saccharomyces cerevisiae. It is shown here by genetic and biochemical analysis that translation, rather than splicing, is severely impaired in two newly isolated ded1 conditional mutants. Preliminary evidence suggests that the protein Ded1p may be required for the initiation step of translation, as is the distinct DEAD-box protein, eukaryotic initiation factor 4A (eIF4A). The DED1 gene could be functionally replaced by a mouse homolog, PL10, which suggests that the function of Ded1p in translation is evolutionarily conserved.

Animals↗