Yeast lariat debranching enzyme. Substrate and sequence specificity.

Nam, Karam; Hudson, Robert; Chapman, Karen; Ganeshan, Kanjana; Damha, Masad J.; Boeke, Jef D.

doi:10.1016/s0021-9258(17)32037-9

Cited by 73 publications

(30 citation statements)

References 30 publications

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“…To assess if equivalent mutants in the fission yeast SpPrp16 protein also allowed first step catalysis, we took up primer extension assays. Further, as the inactivation of lariat debranching enzyme dbr1 + could stabilize the lariat intron-3′exon RNA species by impeding its predicted rapid turnover [44], we generated the fission yeast double mutants spprp16F528S dbr1Δ and spprp16G5151A dbr1Δ to carry out growth kinetics and splicing analyses in these strains. The spprp16F528S dbr1Δ double mutant spores were extremely poor growing indicating a synthetic lethal interaction (Figure 4(a), bottom row) while the spprp16G515A dbr1Δ double mutant was viable and mildly temperature sensitive at 37°C (Figure 4(b), bottom row).…”

Section: Spprp16 Facilitates First Step Splicing Catalysismentioning

confidence: 99%

Early splicing functions of fission yeast Prp16 and its unexpected requirement for gene Silencing is governed by intronic features

et al. 2019

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Prp16 is a DEAH box pre-mRNA splicing factor that triggers a key spliceosome conformational switch to facilitate second step splicing in Saccharomyces cerevisiae. However, Prp16 functions are largely unexplored in Schizosaccharomyces pombe, an attractive model with exon-intron architecture more relevant to several other eukaryotes. Here, we generated mis-sense alleles in SpPrp16 whose consequences on genome-wide splicing uncover its nearly global splicing role with only a small subset of unaffected introns. Prp16 dependent and independent intron categories displayed a striking difference in the strength of intronic 5ʹ splice site (5'SS)-U6 snRNA and branch site (BS)-U2 snRNA interactions. Selective weakening of these interactions could convert a Prp16 dependent intron into an independent one. These results point to the role of SpPrp16 in destabilizing 5'SS-U6snRNA and BS-U2snRNA interactions which plausibly trigger structural alterations in the spliceosome to facilitate first step catalysis. Our data suggest that SpPrp16 interactions with early acting factors, its enzymatic activities and association with intronic elements collectively account for efficient and accurate first step catalysis. In addition to splicing derangements in the spprp16F528S mutant, we show that SpPrp16 influences cell cycle progression and centromeric heterochromatinization. We propose that strong 5'SS-U6 snRNA and BS-U2 snRNA complementarity of intron-like elements in noncoding RNAs which lead to complete splicing arrest and impaired Seb1 functions at the pericentromeric loci may cumulatively account for the heterochromatin defects in spprp16F528S cells. These findings suggest that the diverse Prp16 functions within a genome are likely governed by its intronic features that influence splice site-snRNA interaction strength.

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Section: Spprp16 Facilitates First Step Splicing Catalysismentioning

confidence: 99%

Early splicing functions of fission yeast Prp16 and its unexpected requirement for gene Silencing is governed by intronic features

et al. 2019

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“…The branched nature of the molecules may also be confirmed via the yeast debranching enzyme (yDBR), a phosphodiesterase specific to hydrolysis of the 2′,5′-phosphodiester bond of oligonucleotides that contain vicinal 2′,5′-and 3′,5′-phosphodiester linkages (Nam et al, 1994;Ooi et al, 2001). Nucleoside composition analysis of bNA is carried out using snake venom phosphodiesterase (SVPD) according to the method of Eadie et al (1987;UNIT 10.6).…”

Section: Analysis and Purification Of Branched Oligonucleotides Using Anion-exchange Hplcmentioning

confidence: 99%

“…These molecules occur in the cell nucleus, and are formed during the splicing of precursor messenger RNA (pre-mRNA). As such they have many potential applications in nucleic acid biochemistry, particularly as tools for probing the substrate specificity of lariat debranching enzymes, and as tools for studying pre-mRNA splicing (e.g., Nam et al, 1994;Carriero et al, 2001). The assembly of these branched nucleic acids (bNAs) on a solid support can be achieved by following two strategies (Damha and Zabarylo, 1989;Braich and Damha, 1997).…”

mentioning

confidence: 99%

“…These branched molecules all contain a common structural feature, namely vicinal 2′,5′and 3′,5′-phosphodiester bonds at a branch point nucleotide. A novel polypeptide termed the RNA lariat debranching enzyme selectively hydrolyzes the 2′,5′-phosphodiester bond at the branch point of excised intron lariats, thus converting them into linear molecules (Ruskin and Green, 1985;Chapman and Boeke, 1991;Nam et al, 1994).…”

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confidence: 99%

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Solid‐Phase Synthesis of Branched Oligonucleotides

Carriero

Damha

2002

CP Nucleic Acid Chemistry

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Branched nucleic acids (bNAs) have been of particular interest since the discovery of RNA forks and lariats as intermediates of nuclear mRNA splicing, as well as multicopy, single-stranded DNA (msDNA). Such molecules contain the inherent trait of vicinal 2',5'- and 3',5'-phosphodiester linkages. bNAs have many potential applications in nucleic acid biochemistry, particularly as tools for studying the substrate specificity of lariat debranching enzymes, and as biological probes for the investigation of branch recognition during pre-mRNA splicing. The protocols described herein allow for the facile solid-phase synthesis of branched DNA and/or RNA oligonucleotides of varying chain length, containing symmetrical or asymmetrical sequences immediate to an RNA branch point. The synthetic methodology utilizes widely adopted phosphoramidite chemistry. Methods for efficient purification of bNAs via anion-exchange HPLC and PAGE are also illustrated.

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“…Another well-known noncanonical nucleotide is a branched ribonucleotide (branched nucleotide, branchpoint) from which two nucleic acid strands branch out into a 2 ′ -arm and a 3 ′ -arm via vicinal 2 ′ ,5 ′ and 3 ′ ,5 ′ phosphodiester bonds, respectively (Wallace and Edmonds 1983;Nam et al 1994). They are found in spliceosomal and group II intron RNA lariats (Kruger et al 1982;Padgett et al 1984), in 2 ′ ,5 ′ -branched RNA/DNA chimeric molecules (multicopy single-stranded DNAs) (Hsu et al 1989), in mRNA (Nielsen et al 2005), and presumably in the genomic RNA of retroviruses and LTR retrotransposons (Cheng and Menees 2004;Galvis et al 2017).…”

Section: Introductionmentioning

confidence: 99%

Dual coding potential of a 2′,5′-branched ribonucleotide in DNA

Döring

Hurek

2018

RNA

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Branchpoints in RNA templates are highly mutagenic, but it is not known yet whether this also applies to branchpoints in DNA templates. Here, we report how nucleic acid polymerases replicate a 2 ′ ′ ′ ′ ′ ,5 ′ ′ ′ ′ ′ -branched DNA (bDNA) molecule. We constructed long-chained bDNA templates containing a branch guanosine and T7 promoters at both arms by splinted ligation. Quantitative real-time PCR analysis was used to investigate whether a branchpoint blocks DNA synthesis from the two arms in the same manner. We find that the blocking effect of a branchpoint is arm-specific. DNA synthesis from the 2 ′ ′ ′ ′ ′arm is more than 20,000-fold decreased, whereas from the 3 ′ ′ ′ ′ ′ -arm only 15-fold. Our sequence analysis of full-length nucleic acid generated by Taq DNA polymerase, Moloney murine leukemia virus reverse transcriptase, and T7 RNA polymerase from the 2 ′ ′ ′ ′ ′ -arm of bDNA shows that the branched guanine has a dual coding potential and can base-pair with cytosine and guanine. We find that branchpoint templating is influenced by the type of the surrounding nucleic acid and is probably modulated by polymerase and RNase H active sites. We show that the branchpoint bypass by the polymerases from the 3 ′ ′ ′ ′ ′arm of bDNA is predominantly error-free, indicating that bDNA is not as highly mutagenic as 2 ′ ′ ′ ′ ′ ,5 ′ ′ ′ ′ ′ -branched RNA.

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Yeast lariat debranching enzyme. Substrate and sequence specificity.

Cited by 73 publications

References 30 publications

Early splicing functions of fission yeast Prp16 and its unexpected requirement for gene Silencing is governed by intronic features

Early splicing functions of fission yeast Prp16 and its unexpected requirement for gene Silencing is governed by intronic features

Solid‐Phase Synthesis of Branched Oligonucleotides

Dual coding potential of a 2′,5′-branched ribonucleotide in DNA

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