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Results for “exon dynamics”

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PubMed2003

Alternative splicing of the actin binding domain of human cortactin affects cell migration.

Cortactin is a filamentous actin (F-actin)-binding protein that regulates cytoskeletal dynamics by activating the Arp2/3 complex; it binds to F-actin by means of six N-terminal "cortactin repeats". Gene amplification of 11q13 and consequent overexpression of cortactin in several human cancers is associated with lymph node metastasis. Overexpression as well as tyrosine phosphorylation of cortactin has been reported to enhance cell migration, invasion, and metastasis. Here we report the identification of two alternative splice variants (SV1 and SV2) that affect the cortactin repeats: SV1-cortactin lacks the 6th repeat (exon 11), whereas SV2-cortactin lacks the 5th and 6th repeats (exons 10 and 11). SV-1 cortactin is found co-expressed with wild type (wt)-cortactin in all tissues and cell lines examined, whereas the SV2 isoform is much less abundant. SV1-cortactin binds F-actin and promotes Arp2/3-mediated actin polymerization equally well as wt-cortactin, whereas SV2-cortactin shows reduced F-actin binding and polymerization. Alternative splicing of cortactin does not affect its subcellular localization or growth factor-induced tyrosine phosphorylation. However, cells that overexpress SV1- or SV2-cortactin show significantly reduced cell migration when compared with wt-cortactin-overexpressing cells. Thus, in addition to overexpression and tyrosine phosphorylation, alternative splicing of the F-actin binding domain of cortactin is a new mechanism by which cortactin influences cell migration.

Actins↗

PubMed2001

Pancreatic acinar cell dysfunction in CFTR(-/-) mice is associated with impairments in luminal pH and endocytosis.

BACKGROUND & AIMS: We have previously shown that endocytosis at the apical plasma membrane in pancreatic acinar cells is coupled to ductal bicarbonate secretion into the lumen. We hypothesized that decreased bicarbonate secretion in cystic fibrosis (CF) inhibits apical endocytosis. The aim of this study was to determine in cftr(-/-) mice (1) if the pH of the pancreatic juice is acidic compared with wild-type (WT) controls, (2) if there is a selective block in endocytosis, and (3) if alkalinization of the luminal fluid reverses this defect. METHODS: Fluid secretion and pH of pancreatic juice were measured. Exocytosis, endocytosis, and morphology were compared in pancreatic lobules from cftr(-/-) and WT mice. RESULTS: Pancreatic juice pH was 8.12 +/- 0.06 in WT mice compared with 6.60 +/- 0.04 in cftr(-/-) mice. Although cholecystokinin-stimulated amylase secretion was not significantly different, endocytosis was markedly inhibited in cftr(-/-) compared with WT mice. Cleavage of GP2, a GPI-anchored protein tightly associated with activation of endocytosis, was also decreased. Incubation of lobules from cftr(-/-) mice at pH 8.3 reversed the luminal dilatation. CONCLUSIONS: These data indicate that apical endocytosis is selectively impaired in cftr(-/-) mice, which explains, in part, the luminal dilatation observed at the apical plasma membrane. In vitro alkalinization of luminal fluid led to reversal of defects in membrane dynamics, restored coupled exocytosis and endocytosis, and abolished the luminal dilatation in this animal model of CF. Acidic pH changes in luminal secretions may play a role in the pancreatic membrane dysfunction observed in CF.

Amylases↗

PubMed1995

Dynamic aspects in the expression of the goat insulin-like growth factor-I (IGF-I) gene: diversity in transcription and post-transcription.

Goat IGF-I (gIGF-I) cDNA was cloned using the reverse transcriptase-polymerase chain reaction (RT-PCR). The cDNA, which was homologous to rat class 1 IGF-I cDNA, was 969 bp long. From the open reading frame found in it, we predicted a 154 amino acid protein consisting of a 49 amino acid signal peptide, a 70 amino acid mature IGF-I peptide, and a 35 amino acid E domain (the COOH-terminal peptide). From the gIGF-I gene, we isolated and sequenced some segments containing four exons that encompassed the entire gIGF-I cDNA sequence. Goat liver RNA was analyzed by RT-PCR, and the nucleotides of the RT-PCR products were sequenced and checked with the nucleotide sequences of the segments from the gIGF-I gene. The gene had three leader exons (1W, 1, and 2, from upstream to downstream) and was transcribed into three kinds of mRNAs (classes 1W, 1, and 2). Another RNA species was detected by RT-PCR analysis of exon 1W. We sequenced it and found that in this transcript, the 3'-portion of exon 1 was inserted between exons 1W and 3, resulting in class 1W-1 del. mRNA. That is to say, the gIGF-I gene had three leader exons and four kinds of mature mRNA.

Amino Acid Sequence↗

PubMed2026

Genome-Wide Analysis of the PYL Gene Family and Its Expression Dynamics in Response to Abscisic Acid in Tomato.

The plant hormone abscisic acid (ABA) plays a crucial role throughout the plant life cycle and in adaptive responses to environmental stresses. The pyrabactin resistance 1-like (PYR/PYL/RCAR) proteins act as key regulators in the ABA signal transduction pathway by functioning as direct receptors for ABA. Although PYL genes have been identified in a variety of plant species, their evolutionary and structural characteristics in tomatoes (Solanum lycopersicum) remain elusive. To address this gap, we identified nine SlPYL genes, which were classified into three subfamilies: I (two genes), II (three genes), and III (four genes), and their encoded proteins were predicted to be primarily localized in the cytosol and chloroplast. Structural analysis revealed diverse exon-intron organizations along with five conserved motifs. All identified SlPYLs contained the START domain (PF10604), validating their identity as actual PYL proteins. Prediction of cis-acting regulatory elements in SlPYL's promoter regions was found to be associated with light responsiveness, hormone signaling, stress responses, and plant growth and development. Prediction of post-translational modification sites indicated that SlPYLs are predominantly phosphorylated and acetylated at serine and lysine residues, respectively. Tertiary structure modeling demonstrated conserved three-dimensional architectures among SlPYL proteins, supporting their functional conservation. Expression profiling revealed that specific SlPYL genes exhibit distinct expression patterns across different tissues (root, leaf, and bud) following ABA treatment, indicating functional diversification. Considering the well-established negative correlation between ABA accumulation and bud outgrowth, the ABA-induced differential expression (3~5-fold) of some SlPYL genes (SlPYL3, SlPYL4, SlPYL7, and SlPYL8), particularly in bud tissues after 24 hpt, suggests a potential role in ABA-mediated suppression of bud outgrowth. However, these functional inferences are primarily based on genome-wide computational analyses and expression profiling and therefore require further experimental validation.

Solanum lycopersicum↗

PubMed2000

Homology-based gene structure prediction: simplified matching algorithm using a translated codon (tron) and improved accuracy by allowing for long gaps.

MOTIVATION: Locating protein-coding exons (CDSs) on a eukaryotic genomic DNA sequence is the initial and an essential step in predicting the functions of the genes embedded in that part of the genome. Accurate prediction of CDSs may be achieved by directly matching the DNA sequence with a known protein sequence or profile of a homologous family member(s). RESULTS: A new convention for encoding a DNA sequence into a series of 23 possible letters (translated codon or tron code) was devised to improve this type of analysis. Using this convention, a dynamic programming algorithm was developed to align a DNA sequence and a protein sequence or profile so that the spliced and translated sequence optimally matches the reference the same as the standard protein sequence alignment allowing for long gaps. The objective function also takes account of frameshift errors, coding potentials, and translational initiation, termination and splicing signals. This method was tested on Caenorhabditis elegans genes of known structures. The accuracy of prediction measured in terms of a correlation coefficient (CC) was about 95% at the nucleotide level for the 288 genes tested, and 97. 0% for the 170 genes whose product and closest homologue share more than 30% identical amino acids. We also propose a strategy to improve the accuracy of prediction for a set of paralogous genes by means of iterative gene prediction and reconstruction of the reference profile derived from the predicted sequences. AVAILABILITY: The source codes for the program 'aln' written in ANSI-C and the test data will be available via anonymous FTP at ftp.genome.ad.jp/pub/genomenet/saitama-cc. CONTACT: gotoh@cancer-c.pref.saitama.jp

Algorithms↗

PubMed2002

Quantification of substance p mRNA in human immune cells by real-time reverse transcriptase PCR assay.

We have applied a newly developed real-time reverse transcriptase (RT) PCR (RT-PCR) assay for quantification of substance P (SP) mRNA expression (the SP real-time RT-PCR assay) in human blood monocyte-derived macrophages, peripheral blood lymphocytes, and microglia isolated from fetal brain. The SP real-time RT-PCR assay had a sensitivity of 60 mRNA copies, with a dynamic range of detection between 60 and 600,000 copies of the SP gene transcript per reaction mixture. The coefficient of variation of the threshold cycle number between the SP real-time RT-PCR assays was less than 1.16%. This assay with an SP-specific primer pair efficiently recognizes all four isoforms of preprotachykinin A (the SP precursor) gene transcripts. In order to use this assay to measure the levels of SP mRNA in the human immune cells quantitatively, we designed a specific probe (molecular beacon) derived from exon 3 of the SP gene. We demonstrated that the real-time RT-PCR quantitatively detected SP mRNA in the human immune cells, among which the microglia isolated from fetal brain had the highest levels of SP mRNA. The SP real-time PCR assay yielded reproducible data, as the intra-assay variation was less than 1%. Thus, it is feasible to apply the real-time RT-PCR assay for quantification of SP mRNA levels in human immune cells, as well as in other nonneuronal cells. Since SP is a major modulator of neuroimmunoregulation, this assay has the potential for widespread application for basic and clinical investigations.

Adult↗

PubMed2006

Cooperativity between the hydrophobic and cross-linking domains of elastin.

The principal protein component of the elastic fiber found in elastic tissues is elastin, an amorphous, cross-linked biopolymer that is assembled from a high molecular weight monomer. The hydrophobic and cross-linking domains of elastin have been considered separate and independent, such that changes to one region are not thought to affect the other. However, results from these solid-state 13C NMR experiments demonstrate that cooperativity in protein folding exists between the two domain types. The sequence of the EP20-24-24 polypeptide has three hydrophobic sequences from exons 20 and 24 of the soluble monomer tropoelastin, interspersed with cross-linking domains constructed from exons 21 and 23. In the middle of each cross-linking domain is a "hinge" sequence. When this pentapeptide is replaced with alanines, as in EP20-24-24[23U], its properties are changed. In addition to the expected increase in alpha-helical content and the resulting increase in rigidity of the cross-linking domains, changes to the organization of the hydrophobic regions are also observed. Using one-dimensional CPMAS (cross-polarization with magic angle spinning) techniques, including spectral editing and relaxation measurements, evidence for a change in dynamics to both domain types is observed. Furthermore, it is likely that the methyl groups of the leucines of the hydrophobic domains are also affected by the substitution to the hinge region of the cross-linking sequences. This cooperativity between the two domain types brings new questions to the phenomenon of coacervation in elastin polypeptides and strongly suggests that functional models for the protein must include a role for the cross-linking regions.

Alanine↗

PubMed1995

Evolutionary dynamics of the enhancer region of even-skipped in Drosophila.

We report findings on naturally occurring variation in the regulatory region of even-skipped in Drosophila. This pair-rule gene encodes a homeobox-containing transcription factor, is expressed as a series of seven transverse stripes in developing embryos, and defines parasegmental boundaries. The 5' flanking region of the gene contains a 671-bp enhancer governing stripe 2 expression. The stripe 2 enhancer contains multiple binding sites for four transcription factors that provide positional information in developing blastoderm, the positive regulators bicoid and hunchback and the repressors giant and Kruppel. The study compares polymorphism and divergence in Drosophila melanogaster and Drosophila simulans for the enhancer region, the spacer between the enhancer and the transcription start site, the untranslated leader, the first exon and the intron of eve. Contrary to expectations, we find a relatively high level of variation in the stripe 2 enhancer region, including point substitutions and insertion/deletions in binding sites, and a comparable level of variation in the other noncoding regions. The patterns of variation and divergence within the enhancer region and between regions of the locus fit a model of neutral molecular evolution. We suggest that the multiplicity of binding sites in the enhancer provides a redundancy in function that allows flexibility in the sequence requirements and structural design of the promoter.

Animals↗

PubMed2025

The mutation landscape of Daphnia obtusa reveals evolutionary forces shaping genome stability.

Spontaneous mutations are the primary source of genetic variation and play a central role in shaping evolutionary processes. To investigate mutational dynamics in Daphnia obtusa, we generated a chromosome-level genome assembly spanning 129.4 Mb across 12 chromosomes, encompassing 15,321 predicted protein-coding genes. Leveraging whole-genome sequencing of eight mutation accumulation (MA) lines propagated for an average of 482 generations (spanning over 20 years), we estimated a spontaneous single nucleotide mutation (SNM) rate of 2.23 × 10-9 and an indel mutation rate of 2.75 × 10-10 per site per generation. The SNM spectrum was strongly biased toward C:G > T:A transitions. Comparative analyses with natural population data revealed that exonic mutations observed in the MA lines were significantly less likely to be present in standing variation than intronic or intergenic mutations, suggesting that purifying selection in natural populations acts to remove deleterious alleles. We also identified 48 de novo loss-of-heterozygosity (LOH) events, comprising 8 heterozygous deletions and 40 gene conversion events. The genome-wide gene conversion rate was estimated at 2.62 × 10-5 per heterozygous site per generation. These findings provide a comprehensive view of the mutation spectrum, selective pressures, and mechanisms underlying genome stability in D. obtusa.

Daphnia obtusa↗

PubMed2004

Roles of AUF1 isoforms, HuR and BRF1 in ARE-dependent mRNA turnover studied by RNA interference.

HT1080 cells stably expressing green fluorescent protein (GFP) linked to a 3' terminal AU-rich element (ARE) proved to be a convenient system to study the dynamics of mRNA stability, as changes in mRNA levels are reflected in increased or decreased fluorescence intensity. This study examined whether mRNA stability can be regulated by small interfering RNAs (siRNAs) targeted to AU-binding proteins (AUBPs), which in turn should reveal their intrinsic role as stabilizers or destabilizers of ARE-mRNAs. Indeed, siRNAs targeting HuR or BRF1 decreased or increased fluorescence, respectively. This effect was abolished if cells were treated with both siRNAs, thus indicating antagonistic control of ARE-mRNA stability. Unexpectedly, downregulation of all four AUF1 isoforms by targeting common exons did not affect fluorescence whereas selective downregulation of p40AUF1/p45AUF1 strongly increased fluorescence by stabilizing the GFP-ARE reporter mRNA. This observation was fully confirmed by the finding that only selective reduction of p40AUF1/p45AUF1 induced the production of GM-CSF, an endogenous target of AUF1. These data suggest that the relative levels of individual isoforms, rather than the absolute amount of AUF1, determine the net mRNA stability of ARE-containing transcripts, consistent with the differing ARE-binding capacities of the isoforms.

Antigens, Surface↗

PubMed2001

L- and M-cone-driven electroretinograms in Stargardt's macular dystrophy-fundus flavimaculatus.

PURPOSE: To study the dynamics of the long (L)- and middle (M)-wavelength-sensitive cone-driven pathways and their interactions in patients with Stargardt's macular dystrophy-fundus flavimaculatus (SMD-FF) and to correlate them with other clinical parameters and individual genotypes. METHODS: Forty-seven patients with SMD-FF participated in the study. In addition to standard 30-Hz flicker electroretinograms (30-Hz fERG), ERG responses were measured to stimuli that modulated exclusively the L or the M cones (L/M cones) or the two simultaneously. Blood samples were screened for mutations in the 50 exons of the ABCA4 gene. RESULTS: Patients with SMD-FF did not show a decrease in the mean L/M-cone-driven ERG sensitivity, but there was a significant increase in the interindividual variability. The mean L-/M-cone weighting ratio was normal. However, the L-cone-driven ERG was significantly phase delayed, whereas the M-cone-driven ERG was significantly phase advanced. These phase changes were significantly correlated with disease duration. The amplitude and implicit time of the standard 30-Hz fERG both correlated significantly with the L/M-cone-driven ERG sensitivity and with the phase difference between the L/M-cone-driven ERGs, indicating the complex origin of the standard 30-Hz fERG. Probable disease-associated mutations in the ABCA4 gene were found in 40 of 45 patients, suggesting that they form a genetically fairly uniform SMD-FF study group. There was no correlation between the genotype and the L/M-cone-driven ERGS: CONCLUSIONS: The changes in L/M-cone-driven ERG sensitivity and phase possibly represent two independent disease processes. The phase changes are similar to those found in patients with retinitis pigmentosa and possibly are a general feature of retinal dystrophies.

ATP-Binding Cassette Transporters↗

PubMed2004

Doublecortin kinase-2, a novel doublecortin-related protein kinase associated with terminal segments of axons and dendrites.

The microtubule (MT)-associated DCX protein plays an essential role in the development of the mammalian cerebral cortex. We report on the identification of a protein kinase, doublecortin kinase-2 (DCK2), with a domain (DC) highly homologous to DCX. DCK2 has MT binding activity associated with its DC domain and protein kinase activity mediated by a kinase domain, organized in a structure in which the two domains are functionally independent. Overexpression of DCK2 stabilizes the MT cytoskeleton against cold-induced depolymerization. Autophosphorylation of DCK2 strongly reduces its affinity for MTs. DCK2 and DCX mRNAs are nervous system-specific and are expressed during the period of cerebrocortical lamination. DCX is down-regulated postnatally, whereas DCK2 persists in abundance into adulthood, suggesting that the DC sequence has previously unrecognized functions in the mature nervous system. In sympathetic neurons, DCK2 is localized to the cell body and to the terminal segments of axons and dendrites. DCK2 may represent a phosphorylation-dependent switch for the reversible control of MT dynamics in the vicinity of neuronal growth cones.

Amino Acid Sequence↗

PubMed2001

Developmental changes of tau protein and mRNA in cultured rat brain oligodendrocytes.

Oligodendrocytes elaborate an extensive network of multibranched processes and flat membranous sheets. Microtubules (MT) participate in the elaboration and stabilization of myelin-forming processes and are essential for cellular sorting processes. Microtubule-associated proteins (MAPs) are involved in the regulation and stabilization of the dynamic MT network. It has been shown previously that oligodendrocytes express the MAP tau, a phosphoprotein most abundant in neurons of the CNS. In this article, we demonstrate for the first time that oligodendrocytes contain all six tau isoforms, and that tau mRNA and protein expression is developmentally regulated. Immunoblot analysis reveals that tau protein is more abundant, and mature isoforms are more prominent at later stages of development. During the first week of culture maturation, a marked decrease in phosphorylation is observable. Using an RT-PCR approach, we can show that oligodendrocytes express small amounts of exon 3 containing isoforms and that during culture maturation, tau mRNA splice products with 3 MT-binding domains (3R) decrease and mRNA with 4 MT-binding domains (4R) increase. In situ hybridization study demonstrates that tau mRNA is present in precursor cells and in mature oligodendrocytes. Tau mRNA is actively transported into the cellular processes, is specifically present in the primary and some of the secondary processes, enriched at the turning and branching points and the growing tips, and often appears as small patches. Hence, localized tau translation at specific sites in the cellular extensions might contribute to the regulation of MT stability during process formation, early axonal contact establishment, and myelination.

Aging↗

PubMed1994

Optimally parsing a sequence into different classes based on multiple types of evidence.

We consider the problem of parsing a sequence into different classes of subsequences. Two common examples are finding the exons and introns in genomic sequences and identifying the secondary structure domains of protein sequences. In each case there are various types of evidence that are relevant to the classification, but none are completely reliable, so we expect some weighted average of all the evidence to provide improved classifications. For example, in the problem of identifying coding regions in genomic DNA, the combined use of evidence such as codon bias and splice junction patterns can give more reliable predictions than either type of evidence alone. We show three main results: 1. For a given weighting of the evidence a dynamic programming algorithm returns the optimal parse and any number of sub-optimal parses. 2. For a given weighting of the evidence a dynamic programming algorithm determines the probability of the optimal parse and any number of sub-optimal parses under a natural Boltzmann-Gibbs distribution over the set of possible parses. 3. Given a set of sequences with known correct parses, a dynamic programming algorithm allows one to apply gradient descent to obtain the weights that maximize the probability of the correct parses of these sequences.

Algorithms↗

PubMed2001

Transcriptional and translational mechanisms of cytochrome b5 reductase isoenzyme generation in humans.

Cytochrome b5 reductase (b5R) is an essential enzyme that exists in soluble and membrane-bound isoforms, each with specific functions. In the rat, the two forms are generated from alternative transcripts differing in the first exons. In contrast, the biogenesis of b5R isoforms in the human is not yet well understood. In the present study we have detected three novel alternative exons, designated 1S, S' and 1B, located between the first alternative exon 1M and the common second exon in the human b5R gene. Accordingly, multiple M-type, S-type and SS'-type and B-type transcripts are generated. All types of human b5R transcript are expressed ubiquitously. An analysis of in vitro translation products demonstrated an alternative use of different AUG initiators resulting in the production of various human b5R protein isoforms. Our results indicate that the organization of the 5' region of the b5R gene is not conserved between rodents and humans. Insertion of Alu elements into the human b5R gene, in particular just upstream of the S/S' region, could be responsible for dynamic events of gene rearrangement during evolution.

Alternative Splicing↗

PubMed2001

Generation of a database containing discordant intron positions in eukaryotic genes (MIDB).

MOTIVATION: Intron sliding is the relocation of intron-exon boundaries over short distances and is often also referred to as intron slippage or intron migration or intron drift. We have generated a database containing discordant intron positions in homologous genes (MIDB--Mismatched Intron DataBase). Discordant intron positions are those that are either closely located in homologous genes (within a window of 10 nucleotides) or an intron position that is present in one gene but not in any of its homologs. The MIDB database aims at systematically collecting information about mismatched introns in the genes from GenBank and organizing it into a form useful for understanding the genomics and dynamics of introns thereby helping understand the evolution of genes. RESULTS: Intron displacement or sliding is critically important for explaining the present distribution of introns among orthologous and paralogous genes. MIDB allows examining of intron movements and allows mapping of intron positions from homologous proteins onto a single sequence. The database is of potential use for molecular biologists in general and for researchers who are interested in gene evolution and eukaryotic gene structure. Partial analysis of this database allowed us to identify a few putative cases of intron sliding. AVAILABILITY: http://intron.bic.nus.edu.sg/midb/midb.html

Amino Acid Sequence↗

PubMed1995

Three distinct axonal transport rates for tau, tubulin, and other microtubule-associated proteins: evidence for dynamic interactions of tau with microtubules in vivo.

Microtubule-associated proteins (MAPs), such as tau, modulate neuronal shape and process outgrowth by influencing the stability and organization of microtubules. The dynamic nature of MAP-microtubule interactions in vivo, however, is poorly understood. Here, we have assessed the stability of these interactions by investigating the synthesis and axoplasmic transport of tau in relation to that of tubulin and other MAPs within retinal ganglion cells of normal adult mice in vivo. Using immunoprecipitation and Western blot analysis with anti-tau monoclonal and polyclonal antibodies, we unequivocally identified in optic axons a family of 50-60 kDa tau isoforms and a second 90-95 KDa tau family, the members of which were shown to contain the domain of tau encoded by exon 4A. To measure the rates of translocation of tau proteins in vivo, we injected mice with 35S-methionine intravitreously and, after 6-30 d, quantitated the radiolabeled tau isoforms immunoprecipitated from eight consecutive 1.1 mm segments of the nerve and optic tract and separated by electrophoresis. Linear regression analysis of protein transport along optic axons showed that the tau isoforms advanced at a rate of 0.2-0.4 mm/d, and other radiolabeled MAPs, identified by their association with taxol-stabilized microtubules, moved three- to fivefold more rapidly. By contrast, tubulins advanced at 0.1-0.2 mm/d, significantly more slowly than tau or other MAPs. These studies establish that tau is not cotransported with tubulin or microtubules, indicating that associations of tau with microtubules within axons are not as stable as previously believed. Our findings also reveal differences among various MAPs in their interactions with microtubules and provide evidence that assembly and reorganization of the microtubule network is an active process even after axons establish connections and fully mature.

Animals↗

PubMed1997

Multiple tertiary interactions involving domain II of group II self-splicing introns.

The ribozyme core of group II introns is organized into six domains of secondary structure. Of these, domain II was long thought to be relatively unimportant for group II self-splicing. However, we now demonstrate the existence, in both major subdivisions of the group II family, of essential tertiary interactions involving domain II. theta-theta' is a novel tertiary interaction between the terminal loop of the IC1 stem of domain I and the basal stem of domain II. The theta-theta' interaction appears to stabilize the group II ribozyme core: it is essential for efficient self-splicing at elevated temperatures but, as shown by the use of a bimolecular reaction system, molecules with a defective theta-theta' contact are not affected in catalysis. An interaction, eta-eta', between domains II and VI of subgroup IIB introns was recently reported to mediate a conformational rearrangement between the two steps of the self-splicing reaction. We now show that domains II and VI of subgroup IIA introns also contact each other, although in a somewhat different way. Reinforcement of the eta-eta' interaction of a subgroup IIA intron prevents the use of a specific 2'-hydroxyl group in domain VI to initiate splicing by transesterification at the 5' splice site; the 5' intron-exon junction is hydrolyzed instead. Since disruption of eta-eta' has exactly opposite effects, and promotes reversal of the first transesterification step, it is concluded that formation of eta-eta' mediates a conformational change in subgroup IIA introns as well. Just like the eta-eta' interaction of subgroup IIB introns, the eta-eta' interaction of subgroup IIA introns (and the theta-theta' interaction) involves terminal loops of the GNRA family and their RNA receptors. Therefore, these motifs are used by nature not only to stabilize three-dimensional RNA architectures, but also in situations that require dynamic interactions.

Base Sequence↗