Isolation and characterization of the gene encoding yeast debranching enzyme

Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function

“…In addition to reduced splicing efficiency, we note that the level of excised intron product increases in the cwc23TKAN deletion mutant. This apparent inconsistency (less RNA spliced but more intron present) is characteristic of mutations in the splicing apparatus defective in intron release from the spliceosome [e.g., spp382 (Pandit et al 2006)] or subsequent degradation of the released intron [e.g., dbr1 (Chapman and Boeke 1991)]. …”

Prp43 Is an Essential RNA-dependent ATPase Required for Release of Lariat-Intron from the Spliceosome

“…6B). The instructive finding was that addition of T123A resulted in the retention of lariat-intron in a discrete high molecular weight complex sedimenting near the bottom of the gradient (fractions [23][24][25][26][27]. The lariat-intron product cosedimented with the residual lariat-exon 2 splicing intermediate, implying that it remained associated with the spliceosomes.…”

“…Dbr1 specifically cleaves the branched 2Ј-5Ј phosphodiester bond (26 -28). A chromosomal deletion of DBR1 is viable, and excised lariat-introns are present at high levels in the dbr1⌬ cells (26). We prepared whole cell extract from the dbr1⌬ strain and reacted it with 32 Plabeled actin pre-mRNA (Fig.…”

Intronic snoRNA biosynthesis in Saccharomyces cerevisiae depends on the lariat-debranching enzyme: Intron length effects and activity of a precursor snoRNA

“…Schematic structure of box C/D and box H/ACA snoRNAs (adapted from Tollervey & Kiss, 1997)+ A: Box C/D snoRNAs are distinguished by two sequence elements, box C and box D+ Boxes C and D are brought together in the folded pre-snoRNA and form a structure known as the box C/D motif, which is required and sufficient for RNA processing, stability, and nucleolar localization (see text)+ The middle region of some snoRNAs contains a second set of box C and box D sequences, designated boxes C9 and D9, respectively+ In most box C/D snoRNAs, regions complementary to rRNA are located upstream of box D or D9 (indicated by hatched boxes)+ In conjunction with box D or D9, these antisense sequences target rRNA nucleotides for 29-O-methylation+ B: Box H/ACA snoRNAs contain box H, located in the "hinge" region of the snoRNA, and box ACA, a trinucleotide element located three nucleotides upstream of the 39 end (see text)+ Both box elements are required for snoRNA production+ A consensus secondary structure contains stem-loop domains near the 59 and 39 ends+ One or both of these boxes contain short regions complementary to rRNA, which target pseudouridine synthesis in rRNA (⌿)+ Box elements in both classes of snoRNAs are predicted to form protein recognition signals+ iat is completely degraded by exonucleases+ However, if an intron contains a box C/D or box H/ACA snoRNA, the snoRNA segment will be protected from complete degradation, presumably through binding of hypothetical proteins to the box C/D or box H/ACA structural motifs (see also Watkins et al+, 1996;Ganot et al+, 1997b)+ In a recent study, synthesis of intronic snoRNAs in Saccharomyces cerevisiae was shown to depend on the RNA lariat-debranching enzyme Dbr1p (Petfalski et al+, 1998)+ Previous characterization of a dbr1 strain revealed that introns accumulate to very high levels in mutant cells, and that intron lariats are present in the form of "nibbled" lariats, missing the linear sequences on the 39 side of the branch point (Chapman & Boeke, 1991)+ In the snoRNA study, the intronic box C/D snoRNA U24 was found to accumulate almost exclusively as a larger precursor in dbr1 mutants, with electrophoretic mobility characteristic of intron lariats (Chapman & Boeke, 1991;Petfalski et al+, 1998)+ This observation argues that intronic snoRNA production in yeast occurs by a splicing-dependent pathway+ Paradoxically, a different pattern was observed for another intronic box C/D snoRNA examined in this study+ U18 accumulated in both lariat and mature RNA forms at comparable levels, consistent with an alternative, splicing-independent pathway+ With a view to further defining the relationship between intronic snoRNA biogenesis and splicing, we analyzed production of more than 15 different small nucleolar RNAs in the yeast dbr1 mutant, encoded in both natural and nonnatural genomic contexts+ Particular emphasis was made on establishing the nature of events leading to Dbr1p-independent release of intronic snoRNAs from the intron lariat "trap" (see above)+ Another major issue addressed in our study is whether a snoRNA trapped in the lariat retains its biological activity+ This was done by comparing individual rRNA methylation patterns in wild-type and dbr1 mutant cells+…”