Unusual branch point selection in processing of human growth hormone pre-mRNA.

Intron A of the human growth hormone gene does not contain an A residue within 56 nucleotides preceding the 3' splice site. The analysis of the excised intron lariat revealed a C residue 28 nucleotides upstream from the 3' splice site as the major branch acceptor nucleotide. Two additional minor branched nucleotides were identified as U residues at positions -22 and -36. An adenosine substitution at position -22 results in lariat formation solely to this nucleotide. Therefore, C and U residues can function efficiently as natural branch acceptors, but an A residue is preferred if available in the proper region. In addition, the data strongly reinforce the importance of the distance constraint for lariat formation. To explain selection of the branch acceptor nucleotide, potential base-pairing interactions of branch point sequences with the U2 RNA are discussed.     

Splicing and spliceosome formation of the yeast MATa1 transcript require a minimum distance from the 5'' splice site to the internal branch acceptor site.

Small deletions of 6, 7, and 12 nucleotides introduced between the 5' splice site and the internal branch acceptor site of the first intron of the yeast MATa1 gene completely abolish accurate splicing in vitro in these constructs. Splicing only occurs at an alternative 5' splice site which was found in the first exon of the MATa1 gene and which is used both in vivo and in vitro. The splicing defect cannot be cured by expanding the distance from the branch point to the 3' splice site. If the alternative 5' splice site is deleted as well in these constructs, neither spliced products nor spliceosomes are formed. Our findings especially lead to the conclusion that a minimum distance between the 5' splice site and the internal branch acceptor site of the intron is required for the formation of splicing complexes and for accurate splicing.     

Yeast pre-mRNA splicing requires a minimum distance between the 5'' splice site and the internal branch acceptor site.

We have generated several deletions within the intron of a yeast actin gene construct which have lead to different splicing efficiencies as measured by Northern blot (RNA blot) and primer extension analyses. Our data especially demonstrate that a minimum distance from the 5' splice site to the internal branch acceptor site is required for accurate and efficient splicing. In a construct in which splicing was completely abolished, splicing could be restored by expanding the distance from the 5' splice site to the internal branch acceptor site with heterologous sequences. Alternative splicing, i.e., exon skipping and the use of a cryptic 5' splice site, was observed when the mRNA precursor was derived from a tandem repeat of a truncated intron with flanking exon sequences.     

分支位点在真核基因受体位点识别中的作用

真核基因受体位点识别是剪接位点识别的一部分,也是基因识别中的重要环节,一直受到研究人员的关注。已有的研究结果显示受体位点的识别与分支位点有关,然而关于分支位点和受体位点识别的关系问题,目前还无人将其作为专门的问题予以深入研究。从受体位点识别出发,选取不同的受体位点序列长度,以神经网络为识别工具,对分支位点在受体位点识别中的作用做了深入研究和分析。实验结果表明,受体位点序列的特征信息集中在分支位点一例,因此分支位点在受体位点识别中具有重要作用。研究结果为受体位点识别问题中序列特征提取提供了依据。     

The role of tunneling in enzyme catalysis of C-H activation

Recent data from studies of enzyme catalyzed hydrogen transfer reactions implicate a new theoretical context in which to understand C-H activation. This is much closer to the Marcus theory of electron transfer, in that environmental factors influence the probability of effective wave function overlap from donor to acceptor atoms. The larger size of hydrogen and the availability of three isotopes (H, D and T) introduce a dimension to the kinetic analysis that is not available for electron transfer. This concerns the role of gating between donor and acceptor atoms, in particular whether the system in question is able to tune distance between reactants to achieve maximal tunneling efficiency. Analysis of enzyme systems is providing increasing evidence of a role for active site residues in optimizing the inter-nuclear distance for nuclear tunneling. The ease with which this optimization can be perturbed, through site-specific mutagenesis or an alteration in reaction conditions, is also readily apparent from an analysis of the changes in the temperature dependence of hydrogen isotope effects.     

The role of tunneling in enzyme catalysis of C-H activation

Recent data from studies of enzyme catalyzed hydrogen transfer reactions implicate a new theoretical context in which to understand C-H activation. This is much closer to the Marcus theory of electron transfer, in that environmental factors influence the probability of effective wave function overlap from donor to acceptor atoms. The larger size of hydrogen and the availability of three isotopes (H, D and T) introduce a dimension to the kinetic analysis that is not available for electron transfer. This concerns the role of gating between donor and acceptor atoms, in particular whether the system in question is able to tune distance between reactants to achieve maximal tunneling efficiency. Analysis of enzyme systems is providing increasing evidence of a role for active site residues in optimizing the inter-nuclear distance for nuclear tunneling. The ease with which this optimization can be perturbed, through site-specific mutagenesis or an alteration in reaction conditions, is also readily apparent from an analysis of the changes in the temperature dependence of hydrogen isotope effects.     

Interterminal distance and flexibility of a triantennary glycopeptide as measured by resonance energy transfer

Three geometric isomers of a single triantennary glycopeptide, each containing two fluorophores attached to terminal positions in the molecule, were used to probe distance and flexibility of the oligosaccharide in solution. A dansyl group (energy acceptor) was attached to the C6 of Gal at either position 6', 6, or 8, and a naphthyl-2-acetyl group (energy donor) was coupled to the N terminus of the Ala-Asn peptide. (formula; see text) Resonance energy-transfer measurements revealed an average distance of approximately 22, 18, and 17 A between the donor and the acceptor attached to either the 6, 8, or 6' Gal residue, respectively. The lifetime of the donor's emission was nearly a single-exponential decay of 27 ns (96%), whereas the decay of the donor with proximally attached acceptor was fit by nonlinear least-squares analysis to a multiexponential for each glycopeptide probe. Fitting with a Lorentzian function revealed spatially distinct donor/acceptor distances presumably arising from glycopeptide branch flexibility. The results suggest that the acceptor located at Gal 8 is the most rigid relative to the donor with a single population of distances centered at 18.4 A. In contrast, the acceptor attached to either Gal 6' or 6 displayed two populations of different distances from the donor. The Gal 6 isomer contained a major population with average donor/acceptor separation distance of 21.7 A and a minor population with average separation distance of 9.7 A. Similarly, the Gal 6' isomer showed a major population with donor/acceptor separation distance of 18.3 A and a minor population with separation distance of 11.7 A. These data support the earlier conclusions that the Man alpha(1----6)Man linkage found in the core pentasaccharide of all branched N-linked oligosaccharides is flexible. In addition, the data suggest that the branch containing Gal 6 is also flexible in the triantennary glycopeptide.     

Mutational analysis of 3' splice site selection during trans-splicing

trans-Splicing is essential for mRNA maturation in trypanosomatids. A conserved AG dinucleotide serves as the 3' splice acceptor site, and analysis of native processing sites suggests that selection of this site is determined according to a 5'-3' scanning model. A series of stable gene replacement lines were generated that carried point mutations at or near the 3' splice site within the intergenic region separating CUB2.65, the calmodulin-ubiquitin associated gene, and FUS1, the ubiquitin fusion gene of Trypanosoma cruzi. In one stable line, the elimination of the native 3' splice acceptor site led to the accumulation of Y-branched splicing intermediates, which served as templates for mapping the first trans-splicing branch points in T. cruzi. In other lines, point mutations shifted the position of the first consensus AG dinucleotide either upstream or downstream of the wild-type 3' splice acceptor site in this intergenic region. Consistent with the scanning model, the first AG dinucleotide downstream of the branch points was used as the predominant 3' splice acceptor site. In all of the stable lines, the point mutations affected splicing efficiency in this region.     

A branch point consensus from Arabidopsis found by non-circular analysis allows for better prediction of acceptor sites.

Little knowledge exists about branch points in plants; it has even been claimed that plant introns lack conserved branch point sequences similar to those found in vertebrate introns. A putative branch point consensus sequence for Arabidopsis thaliana resembling the well known metazoan consensus sequence has been proposed, but this is based on search of sequences similar to those in yeast and metazoa. Here we present a novel consensus sequence found by a non-circular approach. A hidden Markov model with a fixed A nucleotide was trained on sequences upstream of the acceptor site. The consensus found by the Markov model shares features with the metazoan consensus, but differs in its details from the consensus proposed earlier. Despite the fact that branch point consensus sequences in plants are weak, we show that a prediction scheme incorporating them leads to a substantial improvement in the recognition of true acceptor sites; the false positive rate being reduced by a factor of 2. We take this as an indication that the consensus found here is the genuine one and that the branch point does play a role in the proper recognition of the acceptor site in plants.     

Mutually exclusive splicing of alpha-tropomyosin exons enforced by an unusual lariat branch point location: implications for constitutive splicing

Alternative splicing of alpha-tropomyosin pre-mRNA involves mutually exclusive utilization of exons 2 and 3, exon 3 being preferentially selected in most cells. This mutually exclusive behavior is enforced by absolute incompatibility between the adjacent splice sites of the two exons, due to close proximity of the exon 3 branch point to exon 2. The branch point, with an associated polypyrimidine tract, is in an unusual location, 177 nt upstream of the acceptor, only 42 nt from the exon 2 splice donor site. Splicing of exon 2 to 3 is consequently blocked prior to formation of an active spliceosome complex. This block to splicing can be relieved by insertion of spacer elements that increase the donor site-branch point separation to 51-59 nt. The unconventional relative location of the constitutive cis splicing elements therefore provides a simple mechanistic basis for strict mutually exclusive splicing. These results not only demonstrate that the branch point is not specified by proximity to the splice acceptor site, but rather suggest that it is the acceptor site which is specified relative to the branch point.     

Identification,characterization and molecular phylogeny of U12-dependent introns in the Arabidopsis thaliana genome

U12-dependent introns are spliced by the minor U12-type spliceosome and occur in a variety of eukaryotic organisms, including Arabidopsis. In this study, a set of putative U12-dependent introns was compiled from a large collection of cDNA/EST- confirmed introns in the Arabidopsis thaliana genome by means of high-throughput bioinformatic analysis combined with manual scrutiny. A total of 165 U12-type introns were identified based upon stringent criteria. This number of sequences well exceeds the total number of U12-type introns previously reported for plants and allows a more thorough statistical analysis of U12-type signals. Of particular note is the discovery that the distance between the branch site adenosine and the acceptor site ranges from 10 to 39 nt, significantly longer than the previously postulated limit of 21 bp. Further analysis indicates that, in addition to the spacing constraint, the sequence context of the potential acceptor site may have an important role in 3′ splice site selection. Several alternative splicing events involving U12-type introns were also captured in this study, providing evidence that U12-dependent acceptor sites can also be recognized by the U2-type spliceosome. Furthermore, phylogenetic analysis suggests that both U12-type AT-AC and U12-type GT-AG introns occurred in Na⁺/H⁺ antiporters in a progenitor of animals and plants.     

Characterization of an alternative splicing by a NAGNAG splice acceptor site in the porcine KIT gene

The KIT gene has been shown to have multiple functions in hematopoiesis, melanogenesis, and gametogenesis. In addition, mutations of this gene cause pigmentation disorders in humans and mice and are responsible for coat color differences in pigs. While characterizing polymorphisms in the porcine KIT gene, we detected alternative splicing (AS) of the NAGNAG splice acceptor site at the boundary of intron 4 and exon 5. This AS event generated the E and I isoforms, characterized by insertion or deletion, respectively, of CAG at the borders of coding sequence. AS patterns measured in tissue samples from two randomly selected animals did not identified any tissue-specific outcomes. Analysis of AS patterns using three breeds demonstrated that Landrace and Large White pigs expressed both the E and I isoforms. In contrast, a subset of specimens from Korean Native Pigs (KNP) yielded a single I isoform. Alignment of the sequence from several species revealed that the region between the branch point sequence (BPS) and 3′ acceptor site is conserved. However, it is appeared that the selection of either the proximal or distal splice site varied between species. To test the breed specificity the NAGNAG splice acceptor site, we constructed two lineages of minigenes from KNP and Landrace pigs harboring breed-specific mutations. The minigene splicing assay demonstrated that both types of minigenes expressed both the E and I isoforms in two host cell lines, and no differences were detected in the AS pattern between the two breeds. We conclude that the AS at the NAGNAG splice acceptor site on intron 4/exon 5 in the porcine KIT gene is the result of noise selection at the splice site by the splicing machinery. Therefore, this AS event in the porcine KIT gene is unlikely to have any relationship with the coat color variations of Landrace and KNP breeds.