The minimum size for splicing of a vertebrate intron is approximately 70 nucleotides. In Drosophila melanogaster, more than half of the introns are significantly below this minimum yet function well. Such short introns often lack the pyrimidine tract located between the branch point and 3 splice site common to metazoan introns. To investigate if small introns contain special sequences that facilitate their recognition, the sequences and factors required for the splicing of a 59-nucleotide intron from the D. melanogaster mle gene have been examined. This intron contains only a minimal region of interrupted pyrimidines downstream of the branch point. Instead, two longer, uninterrupted C-rich tracts are located between the 5 splice site and branch point. Both of these sequences are required for maximal in vivo and in vitro splicing. The upstream sequences are also required for maximal binding of factors to the 5 splice site, cross-linking of U2AF to precursor RNA, and assembly of the active spliceosome, suggesting that sequences upstream of the branch point influence events at both ends of the small mle intron. Thus, a very short intron lacking a classical pyrimidine tract between the branch point and 3 splice site requires accessory pyrimidine sequences in the short region between the 5 splice site and branch point.Intron/exon architecture varies considerably across the eucaryotic kingdom (8). In vertebrates, introns are larger than exons, with an average size over 1 kb. In lower eucaryotes, introns are often smaller than exons. In fact, lower eucaryotes often have introns smaller than the vertebrate minimum. Drosophila melanogaster contains a mixture of intron sizes, with both very small introns characteristic of lower eucaryotes and larger introns similar to those found in vertebrates. A number of small introns in Drosophila cluster in the 51-to 80-nucleotide (nt) range, with a median of 79 nt (12), a length below the vertebrate limit. It is unclear how such small Drosophila introns function in splicing given the fact that identified splicing factors in Drosophila are similar in size to their vertebrate counterparts.One of the prominent vertebrate splicing consensus sequences is the pyrimidine tract located between the branch point and 3Ј splice site (for a recent review of splicing, see reference 11). This sequence is the binding site for the required splicing factor U2 snRNP auxiliary splicing factor, U2AF (22). In vertebrates, a functional pyrimidine tract has been defined as at least five consecutive uridines or nine consecutive pyrimidines (15), although weaker tracts do exist in known constitutively spliced genes. Large Drosophila introns often possess similar pyrimidine tracts. It has been noted, however, that short Drosophila introns statistically lack such pyrimidine tracts (12), and in this characteristic they resemble introns from Saccharomyces cerevisiae and Schizosaccharomyces pombe (13). Specifically, 51% of large (81-to 5,392-nt) Drosophila introns have a pyrimidine tract downstream of the branch ...
Pietrain swine homozygous for the hal gene (n) associated with porcine stress syndrome (PSS) and a Pietrain-derivative breed, Near Pietrain (NP), with a frequency of .31 for n, were mated to produce reciprocal F1, F2, and purebred NP litters. The halothane challenge test was performed on all 40 parents and 240 progeny to predict their hal genotype and PSS susceptibility. The DNA-based assay for a C to T mutation at base pair 1,843 of the skeletal muscle ryanodine receptor (ryr1) cDNA, which is very highly correlated with PSS, was also determined for these animals. The predicted hal genotypes observed in the progeny differed significantly from the expected Mendelian ratios, and the halothane challenge test consistently overestimated the n/n hal genotype. However, the ryr1 genotypes observed in the progeny did not differ significantly from the expected Mendelian ratios, and this DNA-based assay apparently misidentified only one of the 40 parents. The results of this study indicate that the assay for the ryr1 mutation more accurately predicts both the homozygous and heterozygous forms of the PSS gene than does the halothane challenge test.
One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3 splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3 splice site. Many small introns, especially those in nonvertebrates, lack a classical 3 pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5 splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5 and 3 splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3 pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF-and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.Pre-mRNA splicing is a conserved process occurring in a wide variety of eucaryotes with differing exon/intron architectures (reviewed in references 4, 6, 9, 15, 20, and 26). Vertebrates typically have small exons and large introns. Nonmetazoans frequently have the opposite genetic organization, with introns smaller than the minimum permissible for splicing of a vertebrate intron. Drosophila melanogaster possesses a mixture of these two classes of intron sizes (16,23). In addition, more than half of the small introns in Drosophila are missing a prominent vertebrate splicing signal, the 3Ј polypyrimidine tract (23). For these reasons, Drosophila provides a model system in which to study potential mechanistic variations operating during recognition of splicing signals.In the general model of early vertebrate spliceosome complex assembly, U1 snRNP binds to the 5Ј splice site and U2 snRNP auxiliary factor (U2AF) binds to the 3Ј polypyrimidine tract, thereby facilitating U2 snRNP interaction with the branch point. Various members of the serine/arginine (SR) family of proteins may participate by promoting or stabilizing these interactions (reviewed in references 13, 22, and 31). This family of proteins may also act as exon or intron bridging factors via their SR-mediated interaction with SR domains on the small subunit of U2AF (U2AF 35 ) and the U1 70K protein (32, 33, 38). SF1, originally discovered as an essential splicing factor in reconstitution assays (19), has also been observed to bind to the branch point (7,8). In yeast, BBP (branch point bridging protein), the ortholog to SF1, functions as an intron ...
The rkclutel muscle ryunodinc rcccptor or milligntint hypcrthcnniu-sus~~ptiblc (MHS) pigs contains a mutation ut r&due 615 that is highly corrclutcd with various abnormalities in the rcgulution ol'sarcoplasmic reticulum (SRI Cu?' ch;mncl uctivhy. In isolutcul SR membranes the Argaf3 to Cys"'" rynnodinc rcccptor mutuiion is now shown to bc directly rcsponsiblc Ibr an altcrcd cryptic pcptidc map. due to the elimination of the Arp"" clcuv;~gc site, Furthsrmorc. trypsin tre;nmcnt rclcuscd 86-99 kDu ryunodinc rcccptor fragmsnts encompassing residue 615 from the SR nxmbrunes. WC conclu~Ic hi! the Y6-99 kD;I domain conttiining rcsiduc! 615 is near the cytoplasmie surfax of the ryanodinc receptor and likely near inlporlunl CG' rhitnncl regulatory sites.
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