Pyrimidine Stretch Research Articles

Coordinated tissue-specific regulation of adjacent alternative 3' splice sites in C. elegans.

Adjacent alternative 3′ splice sites, those separated by ≤18 nucleotides, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron's 3′ end depends upon sequence elements that define the branchpoint, polypyrimidine tract, and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched Caenorhabditis elegans samples, we identify hundreds of introns with adjacent alternative 3′ splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is monodirectional, with somatic cells preferring to splice at the distal 3′ splice site (furthest from the 5′ end of the intron) and germline cells showing a distinct shift toward usage of the adjacent proximal 3′ splice site (closer to the 5′ end of the intron). Splicing patterns in somatic cells follow C. elegans consensus rules of 3′ splice site definition; a short stretch of pyrimidines preceding an AG dinucleotide. Splicing in germline cells occurs at proximal 3′ splice sites that lack a preceding polypyrimidine tract, and in three instances the germline-specific site lacks the AG dinucleotide. We provide evidence that use of germline-specific proximal 3′ splice sites is conserved across Caenorhabditis species. We propose that there are differences between germline and somatic cells in the way that the basal splicing machinery functions to determine the intron terminus.

Synthesis, Improved Antisense Activity and Structural Rationale for the Divergent RNA Affinities of 3′-Fluoro Hexitol Nucleic Acid (FHNA and Ara-FHNA) Modified Oligonucleotides

The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F···H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py)···H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.

The NADP-dependent Glutamate Dehydrogenase Gene from the Astaxanthin Producer Xanthophyllomyces dendrorhous: Use of Its Promoter for Controlled Gene Expression

The gdhA gene encoding the NADP-dependent glutamate dehydrogenase (GDH) activity from Xanthophyllomyces dendrorhous has been cloned and characterized, and its promoter used for controlled gene expression in this red-pigmented heterobasidiomycetous yeast. We determined the nucleotide sequence of a 4701 bp DNA genomic fragment, showing an open reading frame of 1871 bp interrupted by five introns with fungal consensus splice-site junctions. The predicted protein (455 amino acids; 49 kDa) revealed high identity to GDHs, especially to those from the fungi Cryptococcus neoformans (70%), Sclerotinia sclerotiorum (66%), and several species of Aspergillus (66-67%). Gene phylogenies support the grouping of X. dendrorhous GDH close to those from the majority of the filamentous fungi. The promoter region of the gdhA gene (PgdhA) contains a TATA-like box and two large pyrimidine stretches. The use of PgdhA for gene expression was validated by electrotransformation of X. dendrorhous using an in-frame fusion with the hygromycin resistance gene (hygR) as a reporter. X. dendrorhous transformants were able to grow in YEME complex medium and in Czapek minimal medium supplemented with 50 microg/ml hygromycin, but gene expression in Czapek medium was repressed when using ammonium acetate as a nitrogen source. PgdhA is a valuable tool for controlled gene expression in Basidiomycetes.

Recognition of Oxidized Thymine Base on the Single-Stranded DNA by Replication Protein A

Replication protein A (RAP) is a eukaryotic single-stranded DNA binding protein involved in DNA replication, repair, and recombination. Recent studies indicate that RPA preferentially binds the damaged sites rather than the undamaged sites. Therefore, RPA is thought to be a member of repair factories or a sensor of lesion on DNA. To obtain further information of behavior of RPA against the oxidized lesion, we studied the binding affinity of RPA for the single-stranded DNA containing 5-formyluracil, a major lesion of thymine base yielded by the oxidation, using several synthetic oligonucleotides. The affinity of RPA for oligonucleotides was determined by gel shift assay. Results suggest that the surrounding sequence of 5-formyluracil may affect the affinity for RPA, and that the 5-formyluracil on the purine stretch but not the pyrimidine stretch increases the affinity for RPA. Results of affinity labeling experiment of RPA with the oligonucleotides containing 5-formyluracil indicate that RPA1 subunit may directly recognize and bind to the 5-formyluracil on the single-stranded DNA.

The γ-actin encoding gene from the β-carotene producer Blakeslea trispora

We determined the nucleotide sequence of a 4599-bp DNA genomic fragment including the γ-actin encoding gene from Blakeslea trispora, showing an open reading frame of 1561 bp interrupted by four introns with fungal consensus splice-site junctions. The untranslated regions of the actA gene contain a consensus TATA box, a CCAAT motif, a large pyrimidine stretch, and the polyadenylation sequence AATAAA. The predicted protein (375 amino acids) revealed high identity to γ-actins from fungi (>90%), and gene phylogenies support the grouping of B. trispora actin close to those from the majority of the filamentous fungi. actA transcript (1.4 kb) level in β-carotene producing conditions was faintly higher than carRA (1.9 kb) and slightly lower than carB (1.8 kb) β-carotene biosynthetic genes. The use of the actA promoter (P actA) for heterologous gene expression was ascertained by the transformation of gene fusions with the bleomycin resistance gene ( ble R) from Streptoalloteichus hindustanus and the geneticin resistance marker ( aphI) from Tn903, into Escherichia coli and Acremonium chrysogenum.

Short pyrimidine stretches containing mixed base PNAs are versatile tools to induce translation elongation arrest and truncated protein synthesis.

Recently, we showed that antisense peptide nucleic acids (PNA) containing a short pyrimidine stretch (C(4)TC(3)) invade Ha-ras mRNA hairpin structures to form highly stable duplex and triplex complexes that contribute to the arrest of translation elongation. The antisense PNA targeted to codon 74 of Ha-ras was designed to bind in antiparallel configuration (the N-terminal of the PNA faces the 3'-end of target mRNA), as PNA/RNA duplexes are most stable in this configuration. In order to show that different sequences in the coding region could be targeted successfully with antisense PNAs, we extended our study to three other purine-rich targets. We show that the tridecamer PNA (targeted to codon 149) containing a CTC(3)T pyrimidine stretch forms with the complementary oligoribonucleotide (ORN) a stable (PNA)(2)/ORN triplex at neutral pH (T(m) = 50 degrees C) and arrests Ha-ras mRNA translation elongation. Interestingly, the thermal stability of triplexes formed with PNAs designed to bind to the complementary ORN in a parallel orientation (the N-terminal of the PNA faces the 5'-end of target) was higher than that formed with antiparallel oriented PNAs (T(m) = 58 degrees C). Because parallel and antiparallel PNAs form stable triplexes with target sequence, they act as translation elongation blockers. These duplex-forming and partly triplex-forming PNAs targeted to Ha-ras mRNA also arrested translation elongation at specific polypurine sites contained in the mRNA coding for HIV-integrase protein. Furthermore, the tridecamer PNA containing the C(3)TC(4) motif was more active than a bis-PNA in which the Hoogsteen recognizing strand was linked to the Watson-Crick recognizing strand by a flexible linker. Pyrimidine-rich, short PNAs that form very stable duplexes with target Ha-ras mRNA inhibit translation by a mechanism that does not involve ribosome elongation arrest, whereas PNAs forming duplex and triplex structures arrest ribosome elongation. The remarkable efficacy of the tridecamer PNAs in arresting translation elongation of HIV-1 integrase mRNA is explained by their ability to form stable triplexes at neutral pH with short purine sequences.

Bias of purine stretches in sequenced chromosomes

We examined more than 700 DNA sequences (full length chromosomes and plasmids) for stretches of purines (R) or pyrimidines (Y) and alternating YR stretches; such regions will likely adopt structures which are different from the canonical B-form. Since one turn of the DNA helix is roughly 10 bp, we measured the fraction of each genome which contains purine (or pyrimidine) tracts of lengths of 10 bp or longer (hereafter referred to as ‘purine tracts’), as well as stretches of alternating pyrimidines/purine (‘pyr/pur tracts’) of the same length. Using this criteria, a random sequence would be expected to contain 1.0% of purine tracts and also 1.0% of the alternating pyr/pur tracts. In the vast majority of cases, there are more purine tracts than would be expected from a random sequence, with an average of 3.5%, significantly larger than the expectation value. The fraction of the chromosomes containing pyr/pur tracts was slightly less than expected, with an average of 0.8%. One of the most surprising findings is a clear difference in the length distributions of the regions studied between prokaryotes and eukaryotes. Whereas short-range correlations can explain the length distributions in prokaryotes, in eukaryotes there is an abundance of long stretches of purines or alternating purine/pyrimidine tracts, which cannot be explained in this way; these sequences are likely to play an important role in eukaryotic chromosome organisation.

Structural and phylogenetic analysis of the gamma-actin encoding gene from the penicillin-producing fungus Penicillium chrysogenum.

The nucleotide sequence of a 2994-bp genomic fragment, including the gamma-actin encoding gene from Penicillium chrysogenum, has been determined, showing an open reading frame (ORF) of 1756 bp interrupted by five introns with fungal consensus splice-site junctions. The 5' untranslated region contains a consensus TATA box, five CAAT motifs, and two large pyrimidine stretches. The predicted protein (375 amino acids) revealed high identity to gamma-actins from fungi (>90%), and gene phylogenies support the grouping of P. chrysogenum actin close to those from the majority of the filamentous fungi. The actA gene is present as a single copy in the genome of P. chrysogenum, and its expression is constitutive during penicillin fermentation, showing a single 1.4-kb transcript.

The gene encoding gamma-actin from the cephalosporin producer Acremonium chrysogenum.

The nucleotide sequence of a 3240-bp genomic fragment including the gamma-actin-encoding gene from Acremonium chrysogenum has been determined, showing an open reading frame of 1691 bp, interrupted by five introns with fungal consensus splice-site junctions. The untranslated regions of the actA gene contain a consensus TATA box, a CCAAT motif, pyrimidine stretches and the polyadenylation sequence AATAA. The predicted protein (375 amino acids) revealed high identity to gamma-actins from fungi (> 90%). Gene phylogenies constructed using DNA and protein sequences support the grouping of A. chrysogenum actin close to those from the majority of the filamentous fungi. The actA gene is present as a single copy in the genome of A. chrysogenum; and its expression level, opposite to pcbC and cefEF cephalosporin biosynthetic genes, was steady during cephalosporin fermentation, showing a single 1.4-kb transcript.

An ikaros-containing chromatin-remodeling complex in adult-type erythroid cells.

We have previously described a SWI/SNF-related protein complex (PYR complex) that is restricted to definitive (adult-type) hematopoietic cells and that specifically binds DNA sequences containing long stretches of pyrimidines. Deletion of an intergenic DNA-binding site for this complex from a human beta-globin locus construct results in delayed human gamma- to beta-globin switching in transgenic mice, suggesting that the PYR complex acts to facilitate the switch. We now show that PYR complex DNA-binding activity also copurifies with subunits of a second type of chromatin-remodeling complex, nucleosome-remodeling deacetylase (NuRD), that has been shown to have both nucleosome-remodeling and histone deacetylase activities. Gel supershift assays using antibodies to the ATPase-helicase subunit of the NuRD complex, Mi-2 (CHD4), confirm that Mi-2 is a component of the PYR complex. In addition, we show that the hematopoietic cell-restricted zinc finger protein Ikaros copurifies with PYR complex DNA-binding activity and that antibodies to Ikaros also supershift the complex. We also show that NuRD and SWI/SNF components coimmunopurify with each other as well as with Ikaros. Competition gel shift experiments using partially purified PYR complex and recombinant Ikaros protein indicate that Ikaros functions as a DNA-binding subunit of the PYR complex. Our results suggest that Ikaros targets two types of chromatin-remodeling factors-activators (SWI/SNF) and repressors (NuRD)-in a single complex (PYR complex) to the beta-globin locus in adult erythroid cells. At the time of the switch from fetal to adult globin production, the PYR complex is assembled and may function to repress gamma-globin gene expression and facilitate gamma- to beta-globin switching.

La protein has a positive effect on the translation of TOP mRNAs in vivo.

In vertebrates, the mRNAs encoding ribosomal proteins, as well as other proteins implicated in translation, are characterized by a 5'-untranslated region (5'-UTR), including a stretch of pyrimidines at the 5'-end. The 5'-terminal oligopyrimidine (5'-TOP) sequence, which is involved in the growth-dependent translational regulation characteristic of this class of genes (so-called TOP genes), has been shown to specifically bind the La protein in vitro, suggesting that La might be implicated in translational regulation in vivo. In order to substantiate this hypothesis, we have examined the effect of La on TOP mRNA translational control in both stable and transient transfection experiments. In particular we have constructed and analyzed three stably transfected Xenopus cell lines inducible for overexpression of wild-type La or of putative dominant negative mutated forms. Moreover, La-expressing plasmids have been transiently co-transfected together with a plasmid expressing a reporter TOP mRNA in a human cell line. Our results suggest that in vivo La protein plays a positive role in the translation of TOP mRNA. They also suggest that the function of La is to counteract translational repression exerted by a negative factor, possibly cellular nucleic acid binding protein (CNBP), which has been previously shown to bind the 5'-UTR downstream from the 5'-TOP sequence.

Analysis of the pseudoautosomal X-linked geneSYBL1in bipolar affective disorder: Description of a new candidate allele for psychiatric disorders

The absence of father-to-son transmission has been observed in a subset of families with bipolar disorder (BPD), suggestive of a susceptibility gene on the sex-linked portion of the X chromosome. This is supported by some genetic linkage studies that have provided evidence for a susceptibility locus near Xq28. We have analyzed one candidate gene on Xq28, SYBL1, which maps to the Xq pseudoautosomal region (PAR). SYBL1 encodes a member of the synaptobrevin family of proteins that is involved in synaptic vesicle docking and membrane transport. Genes in the PAR generally escape X-chromosome inactivation and have an active homolog on the Y chromosome, which would result in an increase in same-sex concordance in paternal transmitted traits. However, SYBL1 is neither expressed on the Y chromosome nor the inactive X chromosome and would therefore be expected to show typical sex-linked transmission. We have screened SYBL1 for mutations that could be tested as candidate alleles in the development of BPD. Following single-strand conformation polymorphism (SSCP) analysis and DNA sequencing, four single nucleotide polymorphisms were detected: a silent mutation at codon 108, two intron mutations without any obvious biological significance, and a G-->C transversion in the polypyrimidine tract at the 3' splice acceptor site preceding exon 8. This polymorphism, which creates a perfect 16/16 stretch of pyrimidines, was analyzed in 110 patients with BPD not selected for sex-linked transmission and 119 control subjects. The results show a statistical trend toward an increase in the frequency of the C allele in males with BPD but not females. Males: chi(2) = 3.46, 1 df, p =.06; Females: chi(2) =.20, 1 df, p =.66.

The Expression of Poly(A)-binding Protein Gene Is Translationally Regulated in a Growth-dependent Fashion through a 5′-Terminal Oligopyrimidine Tract Motif

Poly(A)-binding protein (PABP) is an important regulator of gene expression that has been implicated in control of translation initiation. Here we report the isolation and the initial structural and functional characterization of the human PABP gene. Delineation of the promoter region revealed that it directs the initiation of transcription at consecutive C residues within a stretch of pyrimidines. A study of the translational behavior of the corresponding mRNA demonstrates that it is translationally repressed upon growth arrest of cultured mouse fibroblasts and translationally activated in regenerating rat liver. Furthermore, transfection experiments show that the first 32 nucleotides of PABP mRNA are sufficient to confer growth-dependent translational control on a heterologous mRNA. Substitution of the C residue at the cap site by purines abolishes the translational control of the chimeric mRNA. These features have established PABP mRNA as a new member of the terminal oligopyrimidine tract mRNA family. Members of this family are known to encode for components of the translational apparatus and to contain an oligopyrimidine tract at the 5' terminus (5'TOP). This motif mediates their translational control in a growth-dependent manner.

A novel isoform ratio switch of the polypyrimidine tract binding protein

In this report we present evidence for a novel switch in the ratio of the two major isoforms of the polypyrimidine tract binding protein (PTB) in two related prostate cancer cell lines. The existence of different isoforms of PTB is thought to be the result of alternative splicing. We used UV cross-linking to identify a PTB doublet in the DT3 cell line, which is a rat prostate epithelial cancer line that is androgen-dependent and nonmetastatic. The AT3 cell line, a metastatic, androgen-independent cell line derived from the same tumor as the DT3 cells, was noted here to have a different isoform ratio of PTB. The two most prevalent isoforms of PTB were found to bind to an RNA probe containing a pyrimidine stretch. Western blot analysis demonstrated that these isoforms are indeed expressed differently in the two cell lines and that the observed binding is the result of this differential expression. These two cell lines are derived from the original Dunning prostate tumor, which is a model for studying tumor progression in the prostate. This ratio switch may be an important event in tumor progression in this model system of prostate cancer.

Yeast DNA helicase A: cloning, expression, purification, and enzymatic characterization.

We have cloned and expressed the yeast DNA helicase A in Escherichia coli at a high level (approximately 30 mg/L of culture) in soluble form. We describe here a simple two-step purification protocol that produces reasonable quantities of homogeneous enzyme. In denaturing gel electrophoresis the enzyme behaved as a approximately 90 kDa protein. The native structure, determined by gel-filtration studies, appeared to be hexameric and its quaternary structure was salt (NaCl) dependent. In low-salt buffers (containing 50 mM NaCl), the enzyme eluted in a single activity peak at an elution volume that appeared to correlate with a possible hexameric structure. In higher salt buffer (containing greater than 150 mM NaCl), the enzyme eluted as smaller assemblies (monomer/dimer). The recombinant helicase A was able to hydrolyze ATP or dATP with equal efficiency. The ATPase activity of the enzyme was absolutely DNA-dependent. The nucleotidase activities were comparable to those of the native enzyme. Kinetic analysis of the ATPase activity demonstrated that the Km of the enzyme was approximately 90 microM and the rate of ATP hydrolysis was approximately 20 ATP s-1 molecule-1. DNA sequences containing pyrimidine stretches were more effective activators than those containing purine stretches. However, poly(dC) appeared to be the most effective activator of the ATPase activity. The ATPase activity was inhibited by salt (NaCl) above 50 mM with a half-maximal inhibition at approximately 110 mM. It is likely that the active state of helicase A is hexameric. The helicase activity of the recombinant enzyme was stimulated significantly by the yeast replication protein A (RPA) and to a lower extent by the single-stranded DNA binding protein of E. coli (SSB). The DNA helicase migrated on a DNA template in a 5' --> 3' direction. Helicase A appeared to share a number of enzymatic characteristics including directionality, stimulation by RPA/SSB, and quaternary structure (monomer-hexamer) dynamics that are common to known replicative helicases such as DnaB helicase and the SV40 T-antigen.

Triplex-forming oligonucleotides trigger conformation changes of a target hairpin sequence.

We used a DNA duplex formed between the 5' end of a 69mer (69T) and an 11mer (OL7) as a substrate for BamHI. The former oligonucleotide folds into a hairpin structure, the stem of which contains a stretch of pyrimidines in one strand and consequently a stretch of purines in the other strand. The oligomer 69T was used as a target for complementary oligodeoxypyrimidines made of 10 nt (OL1), 16 nt (OL5) or 26 nt (OL2) which can engage the same 10 pyrimidine-purine-pyrimidine triplets with the 69T hairpin stem. Although the binding site of OL7 did not overlap that of OL1, OL2 or OL5, the BamHI activity on 69T-OL7 complexes was drastically modified in the presence of these triplex-forming oligomers: OL1 abolished the cleavage by BamHI whereas OL5 and OL2 strongly increased it. Using footprinting assays and point-mutated oligonucleotides we demonstrated that these variations were due to different conformations of the 69T-OL7 complex induced by the binding of oligomers OL1, OL2 or OL5. Therefore, oligonucleotides can act as structural switchers, offering one additional mode for modulating gene expression.

Solution structure of an antiparallel purine motif triplex containing a T·CG pyrimidine base triple

Background: Triplex formation is an approach of potential use in regulating and mapping of gene sequences. However, such applications have been limited to homogeneous sequences consisting of stretches of purines or pyrimidines. Understanding how heterogeneous duplexes are recognized by a third strand oligonucleotide at the atomic resolution level is an essential step toward broadening the application of triplex formation into biochemical and biomedical areas. Results The solution structure of an antiparallel triplex (RRY6) containing a site of inversion (i.e. a T within a homopurine stretch, forming a T·CG base triple) has been determined using NMR-restrained computations in the presence of explicit water. The results reveal that within the RRY6 triplex the conformation of the duplex is mostly B-like and that of the third strand exhibits significant variations in interbase separations and backbone torsion angles. A major displacement of the inversion site T sugar in a 5′-direction, accompanied by the tilt of the T base in T·CG, was observed. The T·CG base triple contains a single hydrogen bond between T O4 and the exposed C amino proton and is stabilized by a number of interstrand and sequential van der Waal contacts. The structural comparisons of RRY6 with two related triplexes indicate localized perturbation at the non-classical base triple site. Various triplexes contain sugars in the C2′-endo family and the global features of their duplexes are similar. Conclusion This study provides valuable information concerning the molecular basis of the specific recognition of a Watson–Crick base paired C residue at the inversion sites in the antiparallel triplex and should lead to general rules for designing triplexes containing heterogeneous sequences.

Conformational influence of the ribose 2'-hydroxyl group: Crystal structures of DNA-RNA chimeric duplexes

We have crystallized three double-helical DNA-RNA chimeric duplexes and determined their structures by X-ray crystallography at resolutions between 2 and 2.25 A. The two self-complementary duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC).r(GCG)d(TATACCC), were found to adopt A-type conformations. The crystal structures are non-isomorphous, and the crystallographic environments for the three chimeras are different. A number of intramolecular interactions of the ribose 2'-hydroxyl groups contribute to the stabilization of the A-conformation. Hydrogen bonds between 2'-hydroxyls and 5'-oxygens or phosphate oxygens, in addition to the previously observed hydrogen bonds to 1'-oxygens of adjacent riboses and deoxyriboses, are observed in the DNA-RNA chimeric duplexes. The crystalline chimeric duplexes do not show a transition between the DNA A- and B-conformations. CD spectra suggest that the Okazaki fragment assumes an A-conformation in solution as well. In this molecule the three RNA residues may therefore lock the complete decamer in the A-conformation. Crystals of an all-DNA strand with the same sequence as the self-complementary chimeras show a morphology which is different from those of the chimera crystals. Moreover, the oligonucleotide does not match any of the sequence characteristics of DNAs usually adopting the A-conformation in the crystalline state (e.g., octamers with short alternating stretches of purines and pyrimidines). In DNA-RNA chimeric duplexes, it is therefore possible that a single RNA residue can drive the conformational equilibrium toward the A-conformation.

Species-specific signals for the splicing of a short Drosophila intron in vitro.

The effects of branchpoint sequence, the pyrimidine stretch, and intron size on the splicing efficiency of the Drosophila white gene second intron were examined in nuclear extracts from Drosophila and human cells. This 74-nucleotide intron is typical of many Drosophila introns in that it lacks a significant pyrimidine stretch and is below the minimum size required for splicing in human nuclear extracts. Alteration of sequences of adjacent to the 3' splice site to create a pyrimidine stretch was necessary for splicing in human, but not Drosophila, extracts. Increasing the size of this intron with insertions between the 5' splice site and the branchpoint greatly reduced the efficiency of splicing of introns longer than 79 nucleotides in Drosophila extracts but had an opposite effect in human extracts, in which introns longer than 78 nucleotides were spliced with much greater efficiency. The white-apricot copia insertion is immediately adjacent to the branchpoint normally used in the splicing of this intron, and a copia long terminal repeat insertion prevents splicing in Drosophila, but not human, extracts. However, a consensus branchpoint does not restore the splicing of introns containing the copia long terminal repeat, and alteration of the wild-type branchpoint sequence alone does not eliminate splicing. These results demonstrate species specificity of splicing signals, particularly pyrimidine stretch and size requirements, and raise the possibility that variant mechanisms not found in mammals may operate in the splicing of small introns in Drosophila and possibly other species.

Species-specific signals for the splicing of a short Drosophila intron in vitro.

The effects of branchpoint sequence, the pyrimidine stretch, and intron size on the splicing efficiency of the Drosophila white gene second intron were examined in nuclear extracts from Drosophila and human cells. This 74-nucleotide intron is typical of many Drosophila introns in that it lacks a significant pyrimidine stretch and is below the minimum size required for splicing in human nuclear extracts. Alteration of sequences of adjacent to the 3' splice site to create a pyrimidine stretch was necessary for splicing in human, but not Drosophila, extracts. Increasing the size of this intron with insertions between the 5' splice site and the branchpoint greatly reduced the efficiency of splicing of introns longer than 79 nucleotides in Drosophila extracts but had an opposite effect in human extracts, in which introns longer than 78 nucleotides were spliced with much greater efficiency. The white-apricot copia insertion is immediately adjacent to the branchpoint normally used in the splicing of this intron, and a copia long terminal repeat insertion prevents splicing in Drosophila, but not human, extracts. However, a consensus branchpoint does not restore the splicing of introns containing the copia long terminal repeat, and alteration of the wild-type branchpoint sequence alone does not eliminate splicing. These results demonstrate species specificity of splicing signals, particularly pyrimidine stretch and size requirements, and raise the possibility that variant mechanisms not found in mammals may operate in the splicing of small introns in Drosophila and possibly other species.