Visualizing the ai5γ group IIB intron

It has become apparent that much of cellular metabolism is controlled by large well-folded noncoding RNA molecules. In addition to crystallographic approaches, computational methods are needed for visualizing the 3D structure of large RNAs. Here, we modeled the molecular structure of the ai5γ group IIB intron from yeast using the crystal structure of a bacterial group IIC homolog. This was accomplished by adapting strategies for homology and de novo modeling, and creating a new computational tool for RNA refinement. The resulting model was validated experimentally using a combination of structure-guided mutagenesis and RNA structure probing. The model provides major insights into the mechanism and regulation of splicing, such as the position of the branch-site before and after the second step of splicing, and the location of subdomains that control target specificity, underscoring the feasibility of modeling large functional RNA molecules.     

Role of helical constraints of the EBS1–IBS1 duplex of a group II intron on demarcation of the 5′ splice site

Recognition of the 5′ splice site by group II introns involves pairing between an exon binding sequence (EBS) 1 within the ID3 stem–loop of domain 1 and a complementary sequence at the 3′ end of exon 1 (IBS1). To identify the molecular basis for splice site definition of a group IIB ai5γ intron, we probed the solution structure of the ID3 stem–loop alone and upon binding of its IBS1 target by solution NMR. The ID3 stem was structured. The base of the ID3 loop was stacked but displayed a highly flexible EBS1 region. The flexibility of EBS1 appears to be a general feature of the ai5γ and the smaller Oceanobacillus iheyensis (O.i.) intron and may help in effective search of conformational space and prevent errors in splicing as a result of fortuitous base-pairing. Binding of IBS1 results in formation of a structured seven base pair duplex that terminates at the 5′ splice site in spite of the potential for additional A-U and G•U pairs. Comparison of these data with conformational features of EBS1–IBS1 duplexes extracted from published structures suggests that termination of the duplex and definition of the splice site are governed by constraints of the helical geometry within the ID3 loop. This feature and flexibility of the uncomplexed ID3 loop appear to be common for both the ai5γ and O.i. introns and may help to fine-tune elements of recognition in group II introns.     

Dual roles for the Mss116 cofactor during splicing of the ai5γ group II intron

The autocatalytic group II intron ai5γ from Saccharomyces cerevisiae self-splices under high-salt conditions in vitro, but requires the assistance of the DEAD-box protein Mss116 in vivo and under near-physiological conditions in vitro. Here, we show that Mss116 influences the folding mechanism in several ways. By comparing intron precursor RNAs with long (∼300 nt) and short (∼20 nt) exons, we observe that long exon sequences are a major obstacle for self-splicing in vitro. Kinetic analysis indicates that Mss116 not only mitigates the inhibitory effects of long exons, but also assists folding of the intron core. Moreover, a mutation in conserved Motif III that impairs unwinding activity (SAT → AAA) only affects the construct with long exons, suggesting helicase unwinding during exon unfolding, but not in intron folding. Strong parallels between Mss116 and the related protein Cyt-19 from Neurospora crassa suggest that these proteins form a subclass of DEAD-box proteins that possess a versatile repertoire of diverse activities for resolving the folding problems of large RNAs.     

Inhibition of adenosine 5′-triphosphate–creatine phosphotransferase by substrate–anion complexes. Evidence for the transition-state organization of the catalytic site

1. The substrate combination creatine-MgADP does not significantly protect creatine kinase against inhibition by iodoacetamide in the absence of small anions. 2. Small anions can be divided into three groups according to the way in which they affect creatine kinase: I, acetate reversibly increases enzyme activity in the forward reaction but does not affect the rate of inhibition by iodoacetamide in the presence of creatine plus MgADP; II, planar anions and some halides (HCO(3) (-), HCO(2) (-), NO(3) (-), NO(2) (-), Cl(-), Br(-), F(-)) in the presence of creatine plus MgADP protect the enzyme from inhibition by iodoacetamide; III, tetrahedral anions (SO(4) (2-), HPO(4) (2-), ClO(4) (-), BF(4) (-)) and iodide do not affect the rate of inhibition by iodoacetamide in the presence of creatine plus MgADP but may decrease the protection by class II anions under these conditions. Anions of class II and class III also reversibly inhibit enzyme activity. 3. It is concluded that class II anions form a stable and inactive quaternary enzyme-creatine-MgADP-anion complex and this is responsible for the effect attributed by previous workers to the ternary complex lacking anion. Formation of this complex, particularly in the forward reaction, can lead to markedly non-linear enzyme progress curves. Some previous observations are re-appraised in the light of these findings. 4. From the behaviour of chloride and nitrate ions, and the marked lowering of the K(i) values for creatine and MgADP they produce, it is inferred that planar or monoatomic anions act in the quaternary complex by simulating the transferable phosphoryl group in the transition state (or another intermediate state) of the reaction. 5. It is suggested that, in the course of the reaction, the tetrahedral phosphate-binding site for the transferable phosphoryl group of the substrate (that also binds class II and class III anions) changes into a trigonal bipyramid site (also occupied by class II anions). This strains the phosphoryl group to adopt the transitional sp(3)d hybridized state and must contribute significantly to the low activation energy of the reaction. 6. Catalysis is deduced to proceed by an ;in line' transfer reaction and from the effects of class II anions it is possible to estimate the approximate dimensions of the anionic site in the transition-state complex. 7. The specific protecting effect of an equilibrium mixture of substrates against inhibition by iodoacetamide provides further evidence for the conformational change suggested above as a step in the catalytic process.     

The utility of trnK intron 5′ region in phylogenetic analysis of Ulmaceae s. l.

The sequences of chloroplast trn K intron 5' region were determined for seven genera of Ulmaceae s.l. ( 12 species), two genera of Cannabaceae, two of Moraceae and one genus from each of the three families, i.e. , Urticaceae, Eucommiaceae and Malvaceae. The aligned sequences used in PAUP analyses were 805 bp, and the maximum parsimony analysis resulted in a single most parsimonious tree with tree length = 665, CI = 0.7714 and RI = 0.7965. The topology of the tree is relatively strongly supported by the bootstrap test and congruent with those generated from rbc L sequence analysis and cpDNA restriction site analysis. The phylogenetic tree indicated that Ulmaceae s. l. is polyphyletic, and that the monophyletic Ulmaceae s. str. is sister to the other Urticales taxa. Cannabaceae, represented by Humulus and Cannabis, is nested within Celtidaceae, revealing that Celtidaceae is a paraphyletic group. Gironniera and Aphananthe are both clades of Celtidaceae. 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Divalent metal ions tune the self-splicing reaction of the yeast mitochondrial group II intron <Emphasis Type="Italic">Sc</Emphasis>.ai5γ

Group II introns are large ribozymes, consisting of six functionally distinct domains that assemble in the presence of Mg(2+) to the active structure catalyzing a variety of reactions. The first step of intron splicing is well characterized by a Michaelis-Menten-type cleavage reaction using a two-piece group II intron: the substrate RNA, the 5'-exon covalently linked to domains 1, 2, and 3, is cleaved upon addition of domain 5 acting as a catalyst. Here we investigate the effect of Ca(2+), Mn(2+), Ni(2+), Zn(2+), Cd(2+), Pb(2+), and [Co(NH(3))(6)](3+) on the first step of splicing of the Saccharomyces cerevisiae mitochondrial group II intron Sc.ai5gamma. We find that this group II intron is very sensitive to the presence of divalent metal ions other than Mg(2+). For example, the presence of only 5% Ca(2+) relative to Mg(2+) results in a decrease in the maximal turnover rate k (cat) by 50%. Ca(2+) thereby has a twofold effect: this metal ion interferes initially with folding, but then also competes directly with Mg(2+) in the folded state, the latter being indicative of at least one specific Ca(2+) binding pocket interfering directly with catalysis. Similar results are obtained with Mn(2+), Cd(2+), and [Co(NH(3))(6)](3+). Ni(2+) is a much more powerful inhibitor and the presence of either Zn(2+) or Pb(2+) leads to rapid degradation of the RNA. These results show a surprising sensitivity of such a large multidomain RNA on trace amounts of cations other than Mg(2+) and raises the question of biological relevance at least in the case of Ca(2+).     

Evolutionary implications of intron–exon distribution and the properties and sequences of the RPL10A gene in eukaryotes

The RPL10A gene encodes the RPL10 protein, required for joining 40S and 60S subunits into a functional 80S ribosome. This highly conserved gene, ubiquitous across all eukaryotic super-groups, is characterized by a variable number of spliceosomal introns, present in most organisms. These properties facilitate the recognition of orthologs among distant taxa and thus comparative studies of sequences as well as the distribution and properties of introns in taxonomically distant groups of eukaryotes. The present study examined the multiple ways in which RPL10A conservation vs. sequence changes in the gene over the course of evolution, including in exons, introns, and the encoded proteins, can be exploited for evolutionary analysis at different taxonomic levels. At least 25 different positions harboring introns within the RPL10A gene were determined in different taxa, including animals, plants, fungi, and alveolates. Generally, intron positions were found to be well conserved even across different kingdoms. However, certain introns seemed to be restricted to specific groups of organisms. Analyses of several properties of introns, including insertion site, phase, and length, along with exon and intron GC content and exon–intron boundaries, suggested biases within different groups of organisms. The use of a standard primer pair to analyze a portion of the intron-containing RPL10A gene in 12 genera of green algae within Chlorophyta is presented as a case study for evolutionary analyses of introns at intermediate and low taxonomic levels. Our study shows that phylogenetic reconstructions at different depths can be achieved using RPL10A nucleotide sequences from both exons and introns as well as the amino acid sequences of the encoded protein.     

Two different 5′ splice site mutations in the growth hormone gene causing autosomal dominant growth hormone deficiency

Four distinct types of isolated growth hormone deficiency (IGHD) have been described to date. Of these IGHD type II has been defined as having a dominant mode of inheritance. We performed a molecular genetic analysis of two patients clinically characterized as IGHD type II. One of the patients and her father shared a heterozygous G–A transition in the first 5′ donor splice site of intron III. The second father and daughter studied also showed a heterozygous G–A transition in the fifth base from the 5′ donor splice site in the same intron. Both mutations altered the correct splicing of the growth hormone pre-mRNA when the corresponding genes were expressed in COS-7 cells. We propose that both inherited mutations are responsible for IGHD type II in these patients. Received: 7 April 1997 / Accepted: 13 June 1997     

Pyridoxal 5′-phosphate binds to a lysine residue in the adenosine 3′-phosphate 5′-phosphosulfate recognition site of glycolipid sulfotransferase from human renal cancer cells

In the course of characterization of glycolipid sulfotransferase from human renal cancer cells, the manner of inhibition of sulfotransferase activity with pyridoxal 5-phosphate was investigated. Incubation of a partially purified sulfotransferase preparation with pyridoxal 5-phosphate followed by reduction with NaBH₄ resulted in an irreversible inactivation of the enzyme. When adenosine 3-phosphate 5-phosphosulfate was co-incubated with pyridoxal 5-phosphate, the enzyme was protected against this inactivation. Furthermore, pyridoxal 5-phosphate was found to behave as a competitive inhibitor with respect to adenosine 3-phosphate 5-phosphosulfate with aK _i value of 287 µm. These results suggest that pyridoxal 5-phosphate modified a lysine residue in the adenosine 3-phosphate 5-phosphosulfate-recognizing site of the sulfotransferase.     

On the conformational dependence of the proton chemical shifts in nucleosides and nucleotides III. Proton chemical shifts of 5′-nucleotides as a function of different conformational parameters

The variation of the chemical shift of the protons of 5′-UMP and 5′-AMP is calculated as a function of χ_CN, ψ and ? torsion angles. The shift of H8 of 5′-AMP and H6 of 5′-UMP is found to be very sensitive to the value of χ_CN. For the anti conformations the shift of these protons is more sensitive to the value of the rotation about CS′-05′ than about C4′-CS′. For the protons of the ribose the calculations show that for the C2′-endo pucker H3′ and H2′ undergo the largest chemical shift variations when ? and ψ vary. The calculated variations are considered in relation with the role of the conformation of the nucleotides in the chemical shift variation between mono and polynucleotides and between the different helical structures of polynucleotides.     

Regulation of BCL-X splicing reveals a role for the polypyrimidine tract binding protein (PTBP1/hnRNP I) in alternative 5′ splice site selection

Alternative splicing (AS) modulates many physiological and pathological processes. For instance, AS of the BCL-X gene balances cell survival and apoptosis in development and cancer. Herein, we identified the polypyrimidine tract binding protein (PTBP1) as a direct regulator of BCL-X AS. Overexpression of PTBP1 promotes selection of the distal 5′ splice site in BCL-X exon 2, generating the pro-apoptotic BCL-Xs splice variant. Conversely, depletion of PTBP1 enhanced splicing of the anti-apoptotic BCL-XL variant. In vivo cross-linking experiments and site-directed mutagenesis restricted the PTBP1 binding site to a polypyrimidine tract located between the two alternative 5′ splice sites. Binding of PTBP1 to this site was required for its effect on splicing. Notably, a similar function of PTBP1 in the selection of alternative 5′ splice sites was confirmed using the USP5 gene as additional model. Mechanistically, PTBP1 displaces SRSF1 binding from the proximal 5′ splice site, thus repressing its selection. Our study provides a novel mechanism of alternative 5′ splice site selection by PTBP1 and indicates that the presence of a PTBP1 binding site between two alternative 5′ splice sites promotes selection of the distal one, while repressing the proximal site by competing for binding of a positive regulator.     

A novel point mutation in an acceptor splice site of intron 32 (IVS32 A–12→G) but no exon 3 mutations in the glycogen debranching enzyme gene in a homozygous patient with glycogen storage disease type IIIb

Genetic deficiency of the glycogen-debranching enzyme (debrancher) causes glycogen storage disease type III (GSD III), which is divided into two subtypes: IIIa and IIIb. In GSD IIIb, glycogen accumulates only in the liver, whereas both liver and muscles are involved in GSD IIIa. The molecular basis for the differences between the two subtypes has not been fully elucidated. Recently, mutations in exon 3 of the debrancher gene were reported to be specifically associated with GSD IIIb. However, we describe a homozygous GSD IIIb patient without mutations in exon 3. Analysis of the patient’s debrancher cDNA revealed an 11-bp insertion in the normal sequence. An A to G transition at position –12 upstream of the 3′ splice site of intron 32 (IVS 32 A^–12→G) was identified in the patient’s debrancher gene. No mutations were found in exon 3. Mutational analysis of the family showed the patient to be homozygous for this novel mutation as well as three polymorphic markers. Furthermore, the mother was heterozygous and the parents were first cousins. The acceptor splice site mutation created a new 3′ splice site and resulted in insertion of an 11-bp intron sequence between exon 32 and exon 33 in the patient’s debrancher mRNA. The predicted mutant enzyme was truncated by 112 amino acids as a result of premature termination. These findings suggested that a novel IVS 32 A^–12→G mutation caused GSD IIIb in this patient. Received: 1 August 1997 / Accepted: 22 September 1997     

The complete intron/exon structure of Ephydatia mülleri fibrillar collagen gene suggests a mechanism for the evolution of an ancestral gene module

We have completed the analysis of a genomic clone, G238, that contains most of the coding region of the sponge COLF1 fibrillar collagen gene. The main triple helical domain is encoded by 31 exons. Except for the 5 junction exon and the two last 3 exons (126 and 18 base pairs), all these exons are related to a 54-bp unit and begin with an intact glycine codon. A good correlation can be made between this sponge gene and a vertebrate fibrillar collagen gene, revealing the high conservation of the members of this family during evolution. The reconstitution of an ancestral collagen gene can be made by considering all the exon/intron junctions of these genes. We suggest that such an ancestral gene arose from multiple duplications of a 54-bp exon and a (54 + 45)-bp module.Abbreviations used bp base pair(s) - kb kilobase(s) - C-protease the enzyme that cleaves the carboxyl-terminal propeptide     

Secondary structure in polyuridylic acid: Non-classical hydrogen bonding and the function of the ribose 2′-hydroxyl group

The role of non-classical hydrogen bonding in RNA structure has been investigated using polyuridylic acid, which has a labile ordered structure at temperatures near 0 °C, as a model system. By comparing the proton nuclear magnetic resonance spectrum of poly(U) in the transition region with that of uridine and the dimer UpU we find evidence that both the imino N(₃)-H and the ribosyl 2′-OH protons are hydrogen bonded. The characteristics of the former are consistent with participation in N(₃)-HOC bonding primarily between residues in the same strand. As yet we cannot unambiguously assign the acceptor for the 2′-OH in ordered poly(U): because of its apparent stability and the acceptable stereochemistry, we presently favor a bond between ribose 2′-OH and O(1′) connecting adjacent nucleotides of the same strand. This arrangement could contribute to the co-operativity of the poly(U) helix formation. The recently proposed 2′-OHO(1′) interactions in crystalline yeast transfer RNA^Phe suggest similar interactions might play a role in the conformational stability of natural RNAs. A second conformational transition below the major transition in the ultraviolet can be detected in poly(U) by monitoring the H(₆) proton of uracil.