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At least 1,693 records · Page 94Linked to original sources
PubMed1998

Base pairing with U6atac snRNA is required for 5' splice site activation of U12-dependent introns in vivo.

The minor U12-dependent class of eukaryotic nuclear pre-mRNA introns is spliced by a distinct spliceosomal mechanism that requires the function of U11, U12, U5, U4atac, and U6atac snRNAs. Previous work has shown that U11 snRNA plays a role similar to U1 snRNA in the major class spliceosome by base pairing to the conserved 5' splice site sequence. Here we show that U6atac snRNA also base pairs to the 5' splice site in a manner analogous to that of U6 snRNA in the major class spliceosome. We show that splicing defective mutants of the 5' splice site can be activated for splicing in vivo by the coexpression of compensatory U6atac snRNA mutants. In some cases, maximal restoration of splicing required the coexpression of compensatory U11 snRNA mutants. The allelic specificity of mutant phenotype suppression is consistent with Watson-Crick base pairing between the pre-mRNA and the snRNAs. These results provide support for a model of the RNA-RNA interactions at the core of the U12-dependent spliceosome that is strikingly similar to that of the major class U2-dependent spliceosome.

Base Sequence↗
PubMed1998

A role for U2/U6 helix Ib in 5' splice site selection.

Selection of pre-mRNA splice sites is a highly accurate process involving many trans-acting factors. Recently, we described a role for U6 snRNA position G52 in selection of the first intron nucleotide (+1G). Because some U2 alleles suppress U6-G52 mutations, we investigated whether the corresponding U2 snRNA region also influenced 5' splice site selection. Our results demonstrate that U2 snRNAs mutated at position U23, but not adjacent nucleotides, specifically affect 5' splice site cleavage. Furthermore, all U2 position U23 mutations are synthetic lethal with the thermosensitive U6-G52U allele. Interestingly, the U2-U23C substitution has an unprecedented hyperaccurate splicing phenotype in which cleavage of introns with a +1G substitution is reduced, whereas the strain grows with wild-type kinetics. U2 position U23 forms the first base pair with U6 position A59 in U2/U6 helix Ib. Restoration of the helical structure suppresses 5' splice site cleavage defects, showing an important role for the helix Ib structure in 5' splice site selection. U2/U6 helix Ib and helix II have recently been described as being functionally redundant. This report demonstrates a unique role for helix Ib in 5' splice site selection that is not shared with helix II.

Base Sequence↗
PubMed1999

The identification and characterization of a novel splicing protein, Isy1p, of Saccharomyces cerevisiae.

We have identified a novel splicing factor, Isy1p, through two-hybrid screens for interacting proteins involved in nuclear pre-mRNA splicing. Isy1p was tagged and demonstrated to be part of the splicing machinery, associated with spliceosomes throughout the splicing reactions. At least a portion of the Isy1 protein population is associated with snRNAs; low levels of U5 and U6 snRNAs are coimmunoprecipitated specifically with Isy1p. When the ISY1 gene was knocked out, no defect in vegetative growth was observed. Using a sensitive in vivo splicing assay, however, we observed lower splicing efficiency in the isy1 null mutant compared to wild-type, indicating that Isy1 p is important in the optimization of splicing.

Amino Acid Sequence↗
PubMed1995

Efficient protein-facilitated splicing of the yeast mitochondrial bI5 intron.

The splicing factor CBP2 is required to excise the yeast mitochondrial group I intron bI5 in vivo and at low magnesium ion concentrations in vitro. CBP2 binding is strengthened 20-fold by increasing Mg2+ concentrations from 5 to 40 mM, implying the protein binds, in part, to the same structure as that stabilized by the cation. The same transition is also observed as a cooperative increase in the rate of self-processing between 5 and 40 mM Mg2+, providing strong evidence for an RNA folding transition promoted by either Mg2+ or CBP2. The first step of splicing, guanosine addition at the 5' splice site, is rate limiting for exon ligation. At low (5 mM) magnesium ion, reaction (measured as kcat/Km or kcat) is accelerated 3 orders of magnitude by saturating CBP2. At near-saturating Mg2+ (40 mM), acceleration is 8- and 30-fold, for kcat and kcat/Km, respectively, so high magnesium ion concentrations fail to compensate completely for protein facilitation. Thus, self-splicing proceeds via two additional transitions as compared with reaction of the bI5-CBP2 complex, only the first of which is efficiently promoted by the cation. Guanosine 5'-monophosphate binds (Kd approximately 0.3 mM) with the same affinity to bI5 and the bI5-protein complex, supporting independent binding of the nucleophile and CBP2. Substitution of a phosphorothioate at the 5' splice site and pH profiles provide evidence that kcat is limited by chemistry at low pH and by a conformational step at high pH. Because binding by either Mg2+ or CBP2 increases the rate of chemistry more than the rate of the conformational step, in the physiological pH range (7-7.6) the protein-facilitated reaction is limited by a conformation step while self-splicing reaction is limited by chemistry. We conclude that CBP2 makes manifold contributions to bI5 splicing: binding compensates for at least two structural defects and accelerates the rate of the chemistry.

Base Sequence↗
PubMed1993

Self-splicing of the group I intron from Anabaena pre-tRNA: requirement for base-pairing of the exons in the anticodon stem.

In the cyanobacterium Anabaena, the precursor to tRNA(Leu) has a 249-nucleotide group I intron inserted between the wobble and second bases of the anticodon; the intron self-splices during transcription in vitro [Xu, M. Q., Kathe, S. D., Goodrich-Blair, H., Nierzwicki-Bauer, S. A., & Shub, D. A. (1990) Science 250, 1566-1570]. By studying splicing of isolated pre-tRNA, we confirm that splicing occurs by the two-step transesterification mechanism characteristic of group I introns, resulting in excision of the intron and accurate ligation of the 5' and 3' exons. The first step, guanosine-dependent cleavage of the phosphodiester bond at the 5' splice site, occurs with kcat congruent to 14 min-1 and kcat/Km = 5 x 10(4) M-1 min-1 (32 degrees C, 15 mM MgCl2), unexpectedly efficient for a small group I intron. (kcat/Km is comparable to that of the Tetrahymena pre-rRNA intron, and kcat is an order of magnitude higher than any previously reported for a group I intron). The second step, ligation of the exons, is so slow (k = 0.3 min-1) that it is rate-limiting for splicing in vitro except at very low guanosine concentrations. Disruption of the base pairs that make up the anticodon stem of the tRNA dramatically reduces the rate of the first step of splicing, while compensatory mutations that restore base pairing generally restore activity. We suggest that the very short P1 helix of this pre-tRNA, with only three base pairs preceding the 5' splice site, is unstable without the additional base pairs in the anticodon stem.(ABSTRACT TRUNCATED AT 250 WORDS)

Anabaena↗
PubMed1992

Exon sequence and structure requirements for tRNA splicing in Saccharomyces cerevisiae.

A survey of exon sequence and structure requirements for splicing was undertaken using labeled pre-tRNA substrates prepared by in vitro transcription of bacterial promoter-yeast tRNA(Tyr) gene fusions. Transcription templates were assembled from oligonucleotide cassettes allowing analysis of 22 derivatives affecting each of the potential secondary and certain tertiary interactions in the pre-tRNA. Effects on both excision of the intervening sequence by yeast endonuclease and joining of exons by ligase were examined. Replacements within the D- and T-stems and anticodon stems revealed that while the primary sequences of these segments were not essential for splicing, formation of base-paired structures was required. Replacements which altered the primary sequence while retaining the secondary structure of the aminoacyl stem allowed efficient excision by endonuclease but reduced joining by ligase. Potentially, the effects of changes within these stems may be indirect through effects on adjacent or overall structure. The presence of either structured or unstructured 5' leader and/or 3' trailer sequences had no effect on either splicing step. Alterations in the conserved Levitt tertiary pair (G15/C48), previously implicated in splicing of pre-tRNA(Phe), did not alter splicing of pre-tRNA(Tyr). A precursor in which the small (type I) extra arm in pre-tRNA(Tyr) was replaced with the large (type II) extra arm sequence from tRNA(Ser) was efficiently spliced. These and previous results suggest that only limited features of exon sequence or structure are recognized by the splicing enzymes.

Base Sequence↗
PubMed1998

RNA secondary structure: an important cis-element in rat calcitonin/CGRP pre-messenger RNA splicing.

The calcitonin/CGRP gene pre-mRNA is alternatively spliced in a tissue-specific manner resulting in the formation of calcitonin mRNA in thyroid C-cells and CGRP mRNA in neurons. Computer analysis of the RNA containing the 3' splice acceptor of the calcitonin-specific exon 4 predicts that this region has the potential to form a thermodynamically stable stem-loop. Data from CD spectroscopy and solution phase structure probing with single-strand specific and double-strand specific RNases indicates that RNA in this region is substantially double stranded. In vitro splicing of chimeric human beta-globin/calcitonin transcripts in HeLa nuclear extract was inhibited by base changes predicted to destabilize the stem, while compensatory base changes resulted in splicing at 50% of wild-type levels. Changing the residue opposite the AG dinucleotide adenosine in the stem from G to U, allowing the formation of an A-U basepair, abolished usage of this splice acceptor in vitro. These results indicate that a thermodynamically stable RNA stem-loop forms in vitro at the 3' splice acceptor of exon 4 of the calcitonin/CGRP gene transcript. This RNA secondary structure acts as a novel cis-element involved in proper splice site selection.

Alternative Splicing↗
PubMed1999

RNA and protein catalysis in group II intron splicing and mobility reactions using purified components.

Group II introns encode proteins with reverse transcriptase activity. These proteins also promote RNA splicing (maturase activity) and then, with the excised intron, form a site-specific DNA endonuclease that promotes intron mobility by reverse splicing into DNA followed by target DNA-primed reverse transcription. Here, we used an Escherichia coli expression system for the Lactococcus lactis group II intron Ll.LtrB to show that the intron-encoded protein (LtrA) alone is sufficient for maturase activity, and that RNP particles containing only the LtrA protein and excised intron RNA have site-specific DNA endonuclease and target DNA-primed reverse transcriptase activity. Detailed analysis of the splicing reaction indicates that LtrA is an intron-specific splicing factor that binds to unspliced precursor RNA with a K(d) of </=0.12 pM at 30 degrees C. This binding occurs in a rapid bimolecular reaction, which is followed by a slower step, presumably an RNA conformational change, required for splicing to occur. Our results constitute the first biochemical analysis of protein-dependent splicing of a group II intron and demonstrate that a single intron-encoded protein can interact with the intron RNA to carry out a coordinated series of reactions leading to splicing and mobility.

Bacterial Proteins↗
PubMed1999

Transcript accumulation and utilization of alternate and non-consensus splice sites in rice granule-bound starch synthase are temperature-sensitive and controlled by a single-nucleotide polymorphism.

Granule-bound starch synthase (GBSS), a product of the waxy gene in rice (Oryza sativa L.), is necessary for the synthesis of amylose in the endosperm. In an extended pedigree of 89 rice cultivars, we have previously shown that all cultivars with more than 18% amylose had the sequence AGGTATA at the leader intron 5' splice site, while all cultivars with a lower proportion of amylose had the sequence AGTTATA. This single-nucleotide polymorphism reduces the efficiency of GBSS pre-mRNA processing. It also results in alternate splicing at multiple sites, some of which have non-consensus sequences. Here we demonstrate that this same G-to-T polymorphism is also associated with differential sensitivity to temperature during the period of grain development. Cultivars with the sequence AGTTATA have a substantial increase in accumulation of mature GBSS transcripts at 18 degrees C compared to 25 or 32 degrees C. The selection of leader intron 5' splice sites is also affected by temperature in these cultivars. A 5' splice site -93 upstream from that used in high-amylose varieties predominates at 18 degrees C. At higher temperatures there is increased utilization of a 5' splice site at -I and a non-consensus site at +1. Potential implications of differential 5' splice site selection and associated differences in 3' splice site selection on transcript stability and translational efficiency are discussed.

Alternative Splicing↗
PubMed2003

Inhibition of the group I ribozyme splicing by NADP+.

The coenzyme NADP+ (nicotinamide adenine dinucleotide phosphate) and its analogs were tested for inhibition of the self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td). Of all compounds examined, the 3'-NADP was the most potent inhibitor and the order of inhibitory efficiency for compounds tested was as follows: 3'-NADP+ > NADP+ > NADP(+)-dialdehyde > NADPH > 1,N6-etheno-NADP+. Increasing guanosine concentration up to 40 microM overcame the suppression of self-splicing by NADP+ up to 76% of the level of normal splicing but didn't recover the full splicing activity. Similarly, Mg2+ also served to restore the splicing activity by about 90% at 25 mM concentration above which the splicing started to decline. The kinetic analysis showed that NADP+ acts as a mixed type non-competitive inhibitor for the td intron RNA with a Ki of 4.1 mM. The specificity of the splicing inhibition by NADP+ is predominantly due to increases in Km and decreases in kcat values. The results indicate that the inhibition by NADP+ was guanosine and Mg2+ dependent.

Kinetics↗
PubMed1990

Binding of the Drosophila sex-lethal gene product to the alternative splice site of transformer primary transcript.

Somatic sexual differentiation in Drosophila melanogaster is accomplished by a hierarchy of genes of which one, Sex-lethal (Sxl), is required for the functional female-specific splicing of the transcripts of the immediately downstream regulatory gene, transformer (tra). The first exon of the tra primary transcript is spliced to one of two acceptor sites. Splicing to the upstream site yields a messenger RNA which is neither sex-specific nor functional, but that produced after splicing to the downstream acceptor site yields a functional female-specific mRNA. Here we address the question of how the Sxl gene product determines the alternative splicing of tra primary transcripts. One suggestion is that non-sex-specific splicing to the upstream acceptor is blocked in female flies by sex-specific factors, but neither the identity of the female-specific factors nor the mechanism of the blockage has been specified. We have now performed co-transfection experiments in which Sxl complementary DNA and the tra gene are expressed in Drosophila Kc cells. Moreover, we find that female Sxl-encoded protein binds specifically to the tra transcript at or near the non-sex-specific acceptor site, implying that the female Sxl gene product is the trans-acting factor that regulates the alternative splicing.

Animals↗
PubMed1992

Are vertebrate exons scanned during splice-site selection?

Pairwise recognition of splice sites as a result of a scanning mechanism is an attractive model to explain the coordination of vertebrate splicing. Such a mechanism would predict a polarity-of-site recognition in the scanned unit, but no evidence for a polarity gradient across introns has been found. We have suggested that the exon rather than the intron is the unit of recognition in vertebrates and that polyadenylation and splicing factors interact during recognition of 3'-terminal exons. Interaction is reflected in maximal rates of in vitro polyadenylation. If scanning across the exon is operating during this interaction, then insertion of a 5' splice site should depress polyadenylation. Here we report recognition in vitro and in vivo of a 5' splice site situated within a 3'-terminal exon, and a concomitant depression of polyadenylation and ultraviolet crosslinking of a polyadenylation factor. Decreased crosslinking was only found when the 3' and 5' splice sites were within 300 nucleotides of each other. These results are consistent with an exon scanning mechanism for splice-site selection.

Adenoviridae↗
PubMed1998

Group II intron splicing in vivo by first-step hydrolysis.

Group I, group II and spliceosomal introns splice by two sequential transesterification reactions. For both spliceosomal and group II introns, the first-step reaction occurs by nucleophilic attack on the 5' splice junction by the 2' hydroxyl of an internal adenosine, forming a 2'-5' phosphodiester branch in the intron. The second reaction joins the two exons with a 3'-5' phosphodiester bond and releases intron lariat. In vitro, group II introns can self-splice by an efficient alternative pathway in which the first-step reaction occurs by hydrolysis. The resulting linear splicing intermediate participates in normal second-step reactions, forming spliced exon and linear intron RNAs. Here we show that the group II intron first-step hydrolysis reaction occurs in vivo in place of transesterification in the mitochondria of yeast strains containing branch-site mutations. As expected, the mutations block branching, but surprisingly still allow accurate splicing. This hydrolysis pathway may have been a step in the evolution of splicing mechanisms.

Esterification↗
PubMed1993

Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing.

The U1 small nuclear ribonucleoprotein (snRNP) particle is one of the Sm class of snRNPs essential for splicing of precursor messenger RNA. Mammalian U1 snRNP contains a 165-nucleotide long RNA molecule and at least 11 proteins: the U1-specific 70K proteins A and C, and the common U snRNP proteins (B', B, D1, D2, D3, E, F and G). One of the functions of U1 snRNP is recognition of the 5' splice site, an event that requires both U1 RNA and U1 proteins. The 70K protein is the only heavily phosphorylated U1 protein in the cell. Isolated U1 snRNPs are associated with a kinase activity that selectively phosphorylates the 70K protein in vitro in a reaction requiring ATP. Here we investigate the role of phosphorylation of the 70K protein in the splicing of pre-mRNA. The 70K protein on U1 snRNPs was phosphorylated in vitro with either ATP, or with ATP-gamma S, which gave a thiophosphorylated product that was resistant to dephosphorylation by phosphatases. When HeLa nuclear splicing extracts that had been depleted of endogenous U1 snRNPs were complemented with U1 snRNPs possessing normal phosphorylated 70K protein, mature spliceosomes were generated and the splicing activity of the extracts was fully restored. By contrast, if thiophosphorylated U1 snRNPs were used instead, splicing was completely inhibited, although formation of the mature spliceosome was unaffected. Our data show that the state of phosphorylation of the U1-specific 70K protein is critical for its participation in a pre-catalytic step of the splicing reaction.

Adenosine Triphosphate↗
PubMedDate not supplied

Localization of pre-mRNA splicing in mammalian nuclei.

In mammalian nuclei, precursor messenger RNA splicing factors are distributed non-uniformly. Antibodies directed against structural polypeptides of small nuclear ribonucleoprotein particles (snRNPs) and some non-snRNP splicing factors have shown that these components are concentrated in about 20-50 nuclear 'speckles'. These and other non-homogeneous distributions have been proposed to indicate nuclear 'compartments' that are distinct from the sites of transcription and in which RNA processing occurs. We have tested this idea using a new approach. Previous structural and biochemical data have shown that splicing can occur in association with transcription. Nascent RNA of specific genes can be detected by in situ hybridization as intense spots of nuclear stain which map to the sites of transcription. Here we identify active pre-mRNA splicing sites by localizing the nascent spliced mRNA of specific genes. We find that splicing occurs at the sites of transcription, which are not coincident with intranuclear speckles. We conclude that the nucleus is not compartmentalized with respect to transcription and pre-mRNA splicing.

Actins↗
PubMedDate not supplied

Stimulatory effect of splicing factors on transcriptional elongation.

Transcription and pre-mRNA splicing are tightly coupled gene expression events in eukaryotic cells. An interaction between the carboxy-terminal domain of the largest subunit of RNA polymerase (Pol) II and components of the splicing machinery is postulated to mediate this coupling. Here, we show that splicing factors function directly to promote transcriptional elongation, demonstrating that transcription is more intimately coupled to splicing than previously thought. The spliceosomal U small nuclear ribonucleoproteins (snRNPs) interact with human transcription elongation factor TAT-SF1 (refs 6,7,8,9) and strongly stimulate polymerase elongation when directed to an intron-free human immunodeficiency virus-1 (HIV-1) template. This effect is likely to be mediated through the binding of TAT-SF1 to elongation factor P-TEFb, a proposed component of the transcription elongation complex. Inclusion of splicing signals in the nascent transcript further stimulates transcription, supporting the notion that the recruitment of U snRNPs near the elongating polymerase is important for transcription. Because the TAT-SF1-U snRNP complex also stimulates splicing in vitro, it may serve as a dual-function factor to couple transcription and splicing and to facilitate their reciprocal activation.

Cyclin T↗
PubMed2002

The human 5-HT7 serotonin receptor splice variants: constitutive activity and inverse agonist effects.

1. Using membranes from stably or transiently transfected HEK293 cells cultured in 5-HT-free medium and expressing the recombinant human 5-HT(7) receptor splice variants (h5-HT(7(a)), h5-HT(7(b)) and h5-HT(7(d))), we compared their abilities to constitutively activate adenylyl cyclase (AC). 2. All h5-HT(7) splice variants elevated basal and forskolin-stimulated AC. The basal AC activity was reduced by the 5-HT(7) antagonist methiothepin and this effect was blocked by mesulergine (neutral 5-HT(7) antagonist) indicating that the inhibitory effect of methiothepin is inverse agonism at the 5-HT(7) receptor. 3. Receptor density correlated poorly with constitutive AC activity in stable clonal cell lines and transiently transfected cells. Mean constitutive AC activity as a percentage of forskolin-stimulated AC was significantly higher for the h5-HT(7(b)) splice variant compared to the h5-HT(7(a)) and h5-HT(7(d)) splice variants but only in stable cell lines. 4. All eight 5-HT antagonists tested inhibited constitutive AC activity of all splice variants in a concentration-dependent manner. No differences in inverse agonist potencies (pIC(50)) were observed between the splice variants. The rank order of potencies was in agreement and highly correlated with antagonist potencies (pK(b)) determined by antagonism of 5-HT-stimulated AC activity (methiothepin >metergoline> mesulergine > or = clozapine > or = spiperone > or = ritanserin > methysergide > ketanserin). 5. The efficacy of inverse agonism was not receptor level dependent and varied for several 5-HT antagonists between membrane preparations of transiently and stably transfected cells. 6. It is concluded that the h5-HT(7) splice variants display similar constitutive activity and inverse agonist properties.

Adenylyl Cyclase Inhibitors↗
PubMed2000

Cell type specificity in alternative splicing of the human mismatch repair gene hMSH2.

Human non-polyposis colorectal cancer is caused by germline mutations in the DNA mismatch repair genes hMSH2 and hMHL1. Several alternatively spliced mRNA species of these genes are present in peripheral blood lymphocytes of normal individuals, which can confound RT-PCR based techniques of mutation detection. Using RT-PCR, we compared the pattern of alternative splicing in whole peripheral blood lymphocytes (PBLs), separated T and B cells, lymphoblastoid cell lines (LCLs) from the same individuals, and a variety of tissues. Alternatively spliced forms of hMLH1 lacking exons 9/10, 10/11 and 9/10/11 were found to have similar patterns of expression in T cells, B cells, and LCLs. By contrast, a subset of hMSH2 transcripts, some of which were produced by utilisation of novel splicing motifs, were generally expressed in T but not in B cells. LCLs derived from the same blood samples showed no expression of any hMSH2 splicing variants. The hMSH2 delta ex13 transcript, while absent from LCLs, was expressed in whole PBLs and both T and B cell fractions. This transcript was furthermore largely undetectable in tissues other than mononuclear blood cells. These data provide evidence for tissue specificity in the regulation of alternative splicing in hMSH2. In particular we show that LCLs generally do not express alternatively spliced forms of hMSH2 mRNA and are thus suited for RT-PCR based mutation screening in that gene.

Alternative Splicing↗