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PubMed1997

The first intron of the myelin proteolipid protein gene confers cell type-specific expression by a transcriptional repression mechanism in non-expressing cell types.

Chimeric genes containing portions of the mouse myelin proteolipid protein (PLP) gene fused to the lacZ reporter gene were used to detect the effect of PLP intron 1 sequences on cell type-specific expression. A transfected fusion gene containing PLP intron 1 sequences was expressed in an oligodendrocyte cell line but not in a liver cell line, consistent with endogenous PLP gene expression. However, an analogous fusion gene missing the first intron was expressed in either oligodendrocyte or liver transfected cells. These studies suggest that transcriptional repressor element(s) located in PLP intron 1 are important in extinguishing expression in non-glial cell types and that the promoter alone functions in an indiscriminate manner. This moderately large intron (>8 kb) was sequenced to aid in future fine mapping of these cell-specific regulatory element(s).

Animals↗
PubMed1999

Intron splice sites of Papilio glaucus PglRh3 corroborate insect opsin phylogeny.

Full-length cDNA clones encoding the PglRh3 opsin from the tiger swallowtail butterfly Papilio glaucus were isolated from cDNA synthesized from adult head tissue total RNA. This cDNA consists of 1679 nucleotides and contains a single open reading frame predicted to be 379 amino acids in length. PCR amplification of genomic DNA with primers spanning the coding region yielded a single 2760bp fragment which was sequenced. The PglRh3 gene has nine exons and eight introns, four of which are in unique locations relative to the positions of introns in other known insect opsin sequences. Phylogenetic analyses of amino acid and nucleotide sequence data places PglRh3 within a clade of insect visual pigments thought to be sensitive to long wavelengths of light. The genomic structure of PglRh3 is the first characterized from a member of this opsin clade. Three PglRh3 intron positions are shared with Drosophila Rh1, and one of these is also shared with Drosophila Rh2. By contrast, none of the known intron locations in a clade of anciently diverged ultraviolet- and blue-sensitive visual pigments are shared by P. glaucus PglRh3, Drosophila Rh1 or Rh2. The placement of introns within opsin genes therefore independently supports the clustering of a putatively long-wavelength-sensitive clade with a clade of blue-green-sensitive visual pigments.

Amino Acid Sequence↗
PubMed2000

Two promoters regulate transcription of the mouse folylpolyglutamate synthetase gene three tightly clustered Sp1 sites within the first intron markedly enhance activity of promoter B.

The process of polyglutamylation mediated by folylpolyglutamate synthetase (FPGS) in mammalian cells has nutritional and pharmacological importance. In murine cells, FPGS expression is controlled by two promoters that, as we show here, vary substantially in their efficiency, at least in the context of a reporter gene assay. Characteristics of the most efficient promoter (promoter B) were examined in the present studies. Insertion in pGL3 of a 1635 bp segment of upstream sequence including the most upstream exon (B1c), intron B1c and only 26 bp of the more downstream exon Bla resulted in a 15-20-fold increase in transcription in NIH3T3 and Hep1-6 cells compared with the promoterless vector. Deletion analysis of DNA sequence upstream of exon B1c showed that transcription was regulated by putative cis active elements only within two distally located upstream segments which when deleted cumulatively increased transcription three- to four-fold. However, deletion of the 56 bp intron B1c immediately downstream of the most upstream exon (Blc) resulted in 1/10 the rate of transcription. Primer extension analysis with NIH3T3 cells revealed start sites for transcription appreciably upstream of and within exon B1c as well as downstream in exon B1a. This result is consistent with the frequent occurrence in murine cells of an FPGS variant (variant III) incorporating exon B1c [Roy et al., J. Biol. Chem. 271 (1996) 23820; 272 (1997) 5587]. Site-directed mutagenesis and DNAse I footprinting revealed that three canonical GC boxes, either overlapping or tightly clustered within intron B1c, bound Sp1 and markedly enhanced transcription, accounting for the maximal promoter B activity. Moreover, in a cellular background devoid of Sp1 activity, we demonstrate that Spl can induce high levels of promoter B activity in pGL3 transfectants, but only when intron B1c is included within the reporter gene construct used. These results suggest that the unusually tight cluster of active Sp1 sites within intron B1c are essential and sufficient for maximal activity of this promoter. These tightly clustered sites appear to act as an enhancer element in promoting transcription and efficiently stabilize transcription initiation complexes at both distal and proximal start sites.

3T3 Cells↗
PubMed2000

Exon-intron organization of the human gp130 gene.

The exon-intron organization and sequences of the exon-intron boundaries of the human gp130 transmembrane receptor gene have been determined using genomic DNAs as samples. The gp130 gene comprises 17 exons and 16 introns. The positions of the exon-intron boundaries show good correlation to the functional/homology regions of gp130. Exons 3-17 code for the gp130 protein, and each subdomain of the receptor is encoded by a set of exons. The coding potential of exons and the intron phasing of the human gp130 gene conform to the patterns observed previously for other cytokine receptor genes. This supports the notions that the gp130 gene evolved from the same ancestral gene that gave rise to other members of the cytokine receptor family.

Antigens, CD↗
PubMed1999

Tissue-specific alternative mRNA splicing of phenylethanolamine N-methyltransferase (PNMT) during development by intron retention.

The expression of phenylethanolamine N-methyl transferase (EC 2. 1.1.2.8, PNMT), the final enzyme in the cascade of catecholamine synthesis, is differentially regulated in adrenergic neurons in the brain and in adrenal chromaffin cells. Using reverse transcription-polymerase chain reaction-based techniques, we detected in the prenatal developing rat brainstem, two species of PNMT mRNA which were produced by a rare alternative splicing mechanism known as intron retention. The spliced, intronless message was downregulated postnatally, while the intron-retained mRNA species continued to be constitutively expressed through adulthood. By contrast in the adrenals, at all stages of development examined, only the intronless message was expressed. In line with previous reports on the failure of glucocorticoids to induce PNMT expression in the brain, the pattern of PNMT splicing in brainstem explants was not affected by the presence of the synthetic glucocorticoid dexamethasone. Undifferentiated sympathoadrenal PC12 pheochromocytoma cells expressed very low basal levels of both mRNA variants, accompanied by a very low basal PNMT enzymatic activity. Exposure of PC12 cells to dexamethasone resulted in the upregulation of only the spliced mRNA variant concomitant with a 3-fold increase in PNMT enzymatic activity. In contrast, treatment of PC 12 cells with nerve growth factor (NGF) enhanced the expression of both the intron-retained and the intronless mRNA species without changes in the basal enzyme activity. This latter result suggests that the translation of the intronless mRNA species may be regulated by the intron-retained mRNA species, which by itself may yield a truncated, yet enzymatically functional translational product. Our data suggest that the tissue-specific regulation of PNMT expression is based on a rare alternative splicing mechanism termed intron retention, and that in the adrenal, but not in the brain, this mechanism is sensitive to regulation by glucocorticoids. Thus, this system is uniquely suited for studying the hormonal control of tissue-specific splicing in the nervous system.

Adrenal Medulla↗
PubMed2004

Evolutionary conservation of a 2-kb intronic sequence flanking a tissue-specific alternative exon in the PTBP2 gene.

nPTB is a member of the polypyrimidine tract-binding (PTB) protein family, which participates in alternative pre-mRNA processing. Tissue-specific splicing of exon 10 in nPTB (HGMW-approved symbol PTBP2) may play an important role in regulating the functional activity of nPTB in neuronal versus nonneuronal cells. In this study, we found that 297 consecutive intronic nucleotides flanking this alternatively spliced exon 10 were identical between human, green monkey, mouse, rat, and pig, while 207 consecutive intronic nucleotides were identical between human and bird DNA. In addition, a 2-kb sequence spanning this intron region showed 85 and 70% conservation in mammal and bird DNA, respectively. Unexpected intergenic sequence conservation between human and mouse genomes has recently been identified. We have now identified intragenic (intronic) sequence conservation from mammals to birds. The striking conservation of this large segment of flanking intronic sequence suggests an important role in tissue-specific splice site selection and may function in regulating the production of functional nPTB.

Alternative Splicing↗
PubMed2002

The evolution of spliceosomal introns.

Although the widespread proliferation of introns in eukaryotic protein-coding genes remains one of the most poorly understood aspects of genomic architecture, major advances have emerged recently from large-scale genome sequencing projects and functional analyses of mRNA-processing events. Evidence supports the idea that spliceosomal introns were not only present in the stem eukaryote but diverged into at least two distinct classes very early in eukaryotic evolution. Some rough estimates of intron turnover rates are provided, and a testable hypothesis for the origin of new introns is proposed. In light of recent findings on the molecular natural history of splicing, various aspects of the phylogenetic and physical distributions of introns can now be interpreted in a theoretical framework that jointly considers the population-genetic roles of mutation, random genetic drift, and natural selection.

Animals↗
PubMed1991

The recent origins of introns.

Accumulating evidence that introns are highly restricted in their phylogenetic distribution strongly supports the view that introns were inserted late in eukaryotic evolution into preformed genes and, hence, that exon-shuffling played no role in the assembly of primordial genes. Potential mechanisms of intron insertion and the possible evolution of nuclear introns and their splicing machinery from self-splicing group II introns are also discussed.

Animal Population Groups↗
PubMed2003

Phylogenetic utility of the second intron of LEAFY in Neillia and Stephanandra (Rosaceae) and implications for the origin of Stephanandra.

A homeotic gene, LEAFY, has been suggested to be a single-copy gene in diploid angiosperms. Nucleotide sequences of the second intron of this gene, along with those of several regions of the chloroplast genome (trnL-trnF, trnD-trnY-trnE-trnT, and matK-trnK) and nuclear ribosomal ITS, were obtained from the species of Neillia and Stephanandra to examine the phylogenetic utility of the intron and to elucidate the phylogenetic relationships among species of the two genera. PCR amplification of the second intron of LEAFY using universal degenerate primers produced PCR products in sufficient quantity for successful direct sequencing. The length of the intron ranged from 591 to 622 base pairs (bp) in Neillia and Stephanandra, except in N. thibetica (ca. 1370 bp), and sequence analysis of this region from multiple accessions revealed low levels of infraspecific variation. Comparison of the LEAFY data with ITS and cpDNA data demonstrated that the LEAFY intron was the most variable and useful for phylogenetic analysis at the species level, providing many more phylogenetically informative characters per 100 bp (7.4) than either ITS (3.2) or cpDNA (0.7). Phylogenetic analyses of LEAFY data using both maximum parsimony and likelihood methods generated well supported and highly resolved gene trees with few homoplasies (CI=0.97). Stephanandra is monophyletic and is nested within Neillia in both LEAFY and cpDNA trees, while the relationship is poorly resolved by ITS data. LEAFY and cpDNA data, however, strongly conflicted with each other with respect to the position of Stephanandra: LEAFY trees placed Stephanandra as sister to the ((N. affinis, N. gracilis), N. thyrsiflora) clade whereas cpDNA data suggested Stephanandra is sister to N. uekii. Both gene trees, however, are nearly identical to each other when Stephanandra is excluded. A hybrid origin of Stephanandra is suggested as a plausible hypothesis to explain the incongruence between LEAFY and cpDNA data sets, though gene duplication/loss and lineage sorting events cannot be ruled out as possibilities.

Amino Acid Sequence↗
PubMed2001

CRS1 is a novel group II intron splicing factor that was derived from a domain of ancient origin.

Protein-dependent group II intron splicing provides a forum for exploring the roles of proteins in facilitating RNA-catalyzed reactions. The maize nuclear gene crs1 is required for the splicing of the group II intron in the chloroplast atpF gene. Here we report the molecular cloning of the crs1 gene and an initial biochemical characterization of its gene product. Several observations support the notion that CRS1 is a bona fide group II intron splicing factor. First, CRS1 is found in a ribonucleoprotein complex in the chloroplast, and cofractionation data provide evidence that this complex includes atpF intron RNA. Second, CRS1 is highly basic and includes a repeated domain with features suggestive of a novel RNA-binding domain. This domain is related to a conserved free-standing open reading frame of unknown function found in both the eubacteria and archaea. crs1 is the founding member of a gene family in plants that was derived by duplication and divergence of this primitive gene. In addition to its previously established role in atpF intron splicing, new genetic data implicate crs1 in chloroplast translation. The chloroplast splicing and translation functions of crs1 may be mediated by the distinct protein products of two crs1 mRNA forms that result from alternative splicing of the crs1 pre-mRNA.

Alternative Splicing↗
PubMed2002

Solution structure of an RNA fragment with the P7/P9.0 region and the 3'-terminal guanosine of the tetrahymena group I intron.

In the second step of the two consecutive transesterifications of the self-splicing reaction of the group I intron, the conserved guanosine at the 3' terminus of the intron (omegaG) binds to the guanosine-binding site (GBS) in the intron. In the present study, we designed a 22-nt model RNA (GBS/omegaG) including the GBS and omegaG from the Tetrahymena group I intron, and determined the solution structure by NMR methods. In this structure, omegaG is recognized by the formation of a base triple with the G264 x C311 base pair, and this recognition is stabilized by the stacking interaction between omegaG and C262. The bulged structure at A263 causes a large helical twist angle (40 +/- 80) between the G264 x C311 and C262 x G312 base pairs. We named this type of binding pocket with a bulge and a large twist, formed on the major groove, a "Bulge-and-Twist" (BT) pocket. With another twist angle between the C262 x G312 and G413 x C313 base pairs (45 +/- 100), the axis of GBS/omegaG is kinked at the GBS region. This kinked axis superimposes well on that of the corresponding region in the structure model built on a 5.0 A resolution electron density map (Golden et al., Science, 1998, 282:345-358). This compact structure of the GBS is also consistent with previous biochemical studies on group I introns. The BT pockets are also found in the arginine-binding site of the HIV-TAR RNA, and within the 16S rRNA and the 23S rRNA.

Animals↗
PubMed1998

Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo.

The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA-protein interactions in HIV both in vivo and in vitro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td) intron in vivo. All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro. Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA. In the absence of translation, two interactions between sequences in the upstream exon and the 5' and 3' splice sites trap the pre-mRNA in splicing-incompetent conformations. Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B. Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA. Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation. This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E. coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing is caused by an alternative structural element containing a cryptic 5' splice site, which serves as a substrate for the ribozyme. Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.

Animals↗
PubMed1995

Conversion of a group II intron into a new multiple-turnover ribozyme that selectively cleaves oligonucleotides: elucidation of reaction mechanism and structure/function relationships.

The self-splicing ai5g group II intron was transformed into a three-part ribozyme that site-specifically cleaves small oligonucleotide substrates with multiple turnover. The ribozyme is composed of intron domain 1 (D1, 425 nucleotides), with catalytically essential domain 5 (D5, 58 nucleotides) provided separately as a reaction cofactor. Together, the D1/D5 complex cleaves small substrates analogous in sequence to the 5'-splice site of the intron. Activity of the ribozyme was studied using a combination of single- and multiple-turnover experiments in which the concentrations of the RNA components were varied in order to probe their individual role in the overall mechanism. Values for kcat, Km, and kcat/Km were the same within experimental error for the two enzymological approaches. These kinetic analyses reveal that the ribozyme utilizes a classic Michaelis-Menten reaction mechanism in which the chemical step of catalysis (kcat = kchem = approximately 0.03 min-1 at full saturation) is rate limiting for the overall reaction. The D1/D5 complex binds tightly to the substrate (Km = 6.3 nM) and specifically recognizes sequences both 5' and 3' to the ribozyme cleavage site. These studies represent the first quantitative analysis of group II recognition and affinity for the 5'-splice site. As observed in previous studies on the role of D5 RNA, D5 binds tightly to the ternary complex (Km = 870 nM). The second-order rate constant for RNA cleavage (kcat/Km = 3.3 x 10(6)) is an order of magnitude slower than that observed for other ribozymes in this mechanistic class, all of which are rate-limited by steps other than chemistry. That kcat equals kchem in this ribozyme is supported by the overall kinetic analysis and by the observation that an Rp phosphorothioate is cleaved approximately 3-fold more slowly than a phosphate at the cleavage site. These studies represent a preliminary examination of stereochemical preference by a group II intron active site in the transition state. The substrate specificity, reaction conditions, and mutational sensitivity of this ribozyme are consistent with a reaction analogous to the first step of group II intron self-splicing, although its stereochemical preference is analogous to a second-step reversal.

Base Sequence↗
PubMed2000

Tri-partite assay for studying exon ligation by the ai5gamma group II intron.

Group II introns self-splice via a two-step mechanism: cleavage at the 5' splice site followed by exon ligation at the 3' splice site. The second step has been difficult to study in vitro because it is generally faster than the first. Herein we describe development and partial kinetic characterization of a novel assay for studying the second step in isolation. In this system, a truncated linear intron (nucleotides 1-881) mediates exon ligation between two oligonucleotide substrates: a 19 nt 5' exon and a 3' substrate consisting of the last 6 nucleotides of the intron plus a 6 nucleotide 3' exon. We found that neither the exact structure of domain 6 nor the identity of nucleotides flanking the 3' splice site is critical for accurate 3' splice site choice by the ai5gamma group II intron. The multiple turnover k(cat) (0.14 min(-)(1)) is slower than the single turnover k(obs) (0.6-0.7 min(-)(1)), consistent with rate-limiting product release under steady-state conditions. Decreased single turnover rates at lower pHs were more consistent with loss of catalytic activity than with rate-limiting chemistry. Binding of the 3' substrate (K(m) = 2.6 microM) could be improved by changing a long-range A:U base pair involving the last intronic nucleotide (the gamma-gamma' interaction) to G:C (K(m(3)(')(substrate)) = 1 microM).

Base Sequence↗
PubMed2001

Recognition elements for 5' exon substrate binding to the Candida albicans group I intron.

A group I intron precursor and ribozyme were cloned from the large subunit rRNA of the human pathogen Candida albicans. Both the precursor and ribozyme are functional as determined from in vitro assays. Comparisons of dissociation constants for oligonucleotide binding to the ribozyme and to a hexanucleotide mimic of its internal guide sequence lead to a model for recognition of the 5' exon substrate by this intron. In particular, tertiary contacts with the P1 helix that help align the splice site include three 2'-hydroxyl groups, a G.U pair that occurs at the intron's splice junction, and a G.A pair. The free energy contribution that each interaction contributes to tertiary binding is determined. When the G.A pair is replaced with a G-C pair, tertiary interactions to 5' exon mimic 2'-hydroxyl groups are significantly weakened. When the G.A pair is replaced with a G.U pair, tertiary interactions are retained and binding is 10-fold tighter. These results expand our knowledge of substrate recognition by group I introns, and also provide a basis for rational design of oligonucleotide-based therapeutics for targeting group I introns by binding enhancement by tertiary interactions and suicide inhibition strategies.

Base Sequence↗
PubMed2000

A tertiary interaction that links active-site domains to the 5' splice site of a group II intron.

Group II introns are self-splicing RNAs that are commonly found in the genes of plants, fungi, yeast and bacteria. Little is known about the tertiary structure of group II introns, which are among the largest natural ribozymes. The most conserved region of the intron is domain 5 (D5), which, together with domain 1 (D1), is required for all reactions catalysed by the intron. Despite the importance of D5, its spatial relationship and tertiary contacts to other active-site constituents have remained obscure. Furthermore, D5 has never been placed directly at a site of catalysis by the intron. Here we show that a set of tertiary interactions (lambda-lambda') links catalytically essential regions of D5 and D1, creating the framework for an active-site and anchoring it at the 5' splice site. Highly conserved elements similar to components of the lambda-lambda' interaction are found in the eukaryotic spliceosome.

Base Pairing↗
PubMed1997

Trans-activation of group II intron splicing by nuclear U5 snRNA.

Similarities between RNA splicing during autocatalytic excision of group II introns and pre-mRNA processing led to the hypothesis that group II introns might be the evolutionary predecessors of spliceosomal small nuclear RNAs. The ID3 subdomain stem-loop structure of group II introns, the proposed analogue of the spliceosomal U5 snRNA, is thought to be essential for 5' splice site recognition and anchoring of the free 5' exon. Using the group II intron bI1 we have analysed the role of ID3 in splicing. In the absence of ID3 the 5' splice site was recognized accurately and efficiently, but exon anchoring was greatly reduced. This step was restored in the presence of RNA fragments consisting of either the terminal stem-loop structure of ID3 or spliceosomal U5 snRNA. This suggests that the predominant role of both RNAs is to anchor the 5' exon during exon ligation. Furthermore, as U5 complements for the loss of ID3, a similar network of structural RNAs may form the catalytic core of both group II introns and spliceosomes.

Binding Sites↗
PubMed2000

Minimal catalytic domain of a group I self-splicing intron RNA.

The self-splicing intron ribozymes have been regarded as primitive forms of the splicing machinery for eukaryotic pre-mRNAs. The splicing activity of group I self-splicing introns is dependent on an absolutely conserved and exceptionally densely packed core region composed of two helical domains, P3-P7 and P4-P6, that are connected rigidly via base triples. Here we show that a mutant group I intron ribozyme lacking both the P4-P6 domain and the base triples can perform the phosphoester transfer reactions required for splicing at both the 5' and 3' splice sites, demonstrating that the elements required for splicing are concentrated in the stacked helical P3-P7 domain. This finding establishes that the conserved core of the intron consists of two physically and functionally separable components, and we present a model showing the architecture of a prototype of this class of intron and the course of its molecular evolution.

Base Sequence↗