A homolog of lariat-debranching enzyme modulates turnover of branched RNA

Garrey, Stephen M.; Katolik, Adam; Prekeris, Mantas; Li, Xueni; York, Kerri; Bernards, Sarah S.; Fields, Stanley; Zhao, Rui; Damha, Masad J.; Hesselberth, Jay R.

doi:10.1261/rna.044602.114

Cited by 34 publications

(44 citation statements)

References 48 publications

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“…S1). This K m value agrees with a previous report of a K d of 0.5 μM for bRNA binding to yDbr1 (31). Electrophoretic analysis of the substrate before and after exposure to Dbr1 indicated that the substrate was completely debranched by Dbr1 (Fig.…”

Section: Resultssupporting

confidence: 92%

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

Clark

Katolik

Roberts

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Intron lariats are circular, branched RNAs (bRNAs) produced during pre-mRNA splicing. Their unusual chemical and topological properties arise from branch-point nucleotides harboring vicinal 2′,5′-and 3′,5′-phosphodiester linkages. The 2′,5′-bonds must be hydrolyzed by the RNA debranching enzyme Dbr1 before spliced introns can be degraded or processed into small nucleolar RNA and microRNA derived from intronic RNA. Here, we measure the activity of Dbr1 from Entamoeba histolytica by using a synthetic, dark-quenched bRNA substrate that fluoresces upon hydrolysis. Purified enzyme contains nearly stoichiometric equivalents of Fe and Zn per polypeptide and demonstrates turnover rates of ∼3 s −1. Similar rates are observed when apo-Dbr1 is reconstituted with Fe(II)+Zn(II) under aerobic conditions. Under anaerobic conditions, a rate of ∼4.0 s −1 is observed when apoenzyme is reconstituted with Fe(II). In contrast, apo-Dbr1 reconstituted with Mn(II) or Fe(II) under aerobic conditions is inactive. Diffraction data from crystals of purified enzyme using X-rays tuned to the Fe absorption edge show Fe partitions primarily to the β-pocket and Zn to the α-pocket. Structures of the catalytic mutant H91A in complex with 7-mer and 16-mer synthetic bRNAs reveal bona fide RNA branchpoints in the Dbr1 active site. A bridging hydroxide is in optimal position for nucleophilic attack of the scissile phosphate. The results clarify uncertainties regarding structure/function relationships in Dbr1 enzymes, and the fluorogenic probe permits high-throughput screening for inhibitors that may hold promise as treatments for retroviral infections and neurodegenerative disease.RNA debranching | intron lariat | enzyme kinetics | X-ray crystallography | Dbr1 T he enzymatic processing of diverse RNA molecules requires selective recognition of their unique physicochemical properties. The sequential trans-esterification reactions catalyzed by the spliceosome yield mature messenger RNA (mRNA) and excised intron lariats (1, 2), the latter of which contain internal branchpoint adenosine nucleotides harboring vicinal 2′,5′-and 3′,5′-phosphodiester linkages (3). Mature mRNA transcripts are exported to the cytosol for protein synthesis, but lariat introns must be linearized before they can be turned over or processed into the subset of small nucleolar RNAs and microRNAs that are derived from intronic RNA (4, 5). The lariat forms when the 2′-hydroxyl group of an adenosine nucleotide near the 3′-end of the intron acts as the nucleophile to attack the 5′-splice site, producing 5′-exon-3′-OH and intron lariat/ 3′-exon intermediates. The 3′-hydroxyl group of the 5′-exon-3′-OH intermediate subsequently acts as the nucleophile to attack the 3′-splice site, resulting in intron excision and exon ligation (6, 7) (Fig. 1A). The resulting vicinal 2′,5′-and 3′,5′-phosphodiester linkages confer unique topological and chemical features to the branchpoint and flanking nucleotides, and these lariats persist in yeast cells lacking active Dbr1 (RNA lariat debranching enzyme) ...

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Section: Resultssupporting

confidence: 92%

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

Clark

Katolik

Roberts

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Notably, a positively charged surface of the N-domain of Cwf19L2 clamps over the lariat junction( Fig. 4c), suggesting a role in modulating the turnover of branched intron 28 ; whereas the bridging helix and the C-domain together bind the BPS/U2 duplex and the 3'-tail of the intron (Fig. 4c); this organization is consistent with the observation that Cwf19L2 may help displace and unwind the BPS/U2 duplex during spliceosome disassembly 49 .…”

Section: The Transition From the P To Ils1 Complexsupporting

confidence: 80%

“…The distinct orientation of the RNaseH-like domain is likely facilitated by its close interactions with Cwf19L2 in human (Cwf19 in S. pombe). Our structural analysis and published studies support the conclusion that Cwf19L2 may have multiple roles during splicing28 . The precise functional annotation of Cwf19L2 requires further biochemical investigation.…”

supporting

confidence: 86%

Structures of the Human Spliceosomes Before and After Release of the Ligated Exon

Zhang

Zhan

Yan

et al. 2018

Preprint

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Pre-mRNA splicing is executed by the spliceosome, which has eight major functional states each with distinct composition. Five of these eight human spliceosomal complexes, all preceding exon ligation, have been structurally characterized. In this study, we report the cryo-electron microscopy structures of the human post-catalytic spliceosome (P complex) and intron lariat spliceosome (ILS) at average resolutions of 3.0 and 2.9 Å, respectively. In the P complex, the ligated exon remains anchored to loop I of U5 small nuclear RNA, and the 3'-splice site is recognized by the junction between the 5'-splice site and the branch point sequence. The ATPase/helicase Prp22, along with the ligated exon and eight other proteins, are dissociated in the P-to-ILS transition. Intriguingly, the ILS complex exists in two distinct conformations, one with the ATPase/helicase Prp43 and one without. Comparison of these three late-stage human spliceosomes reveals mechanistic insights into exon release and spliceosome disassembly.

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“…However, the covalent nature of the branch would present a persistent barrier to the exonuclease activities of the exosome, resulting in a futile cycle of oligo(A) addition and degradation, potentially explaining why the dominant oligo(A) tails at the 3 ′ termini of lariat introns at steady state extended only one nucleotide, as compared with other exosome substrates for which oligo(A) tails are longer and recruitment of the exosome to the oligo(A) tail would generally lead to processive degradation of the RNA (Jia et al 2011;Wlotzka et al 2011). Nevertheless, the exosome embodies not only exonucleolytic activities but also an endonucleolytic activity (Lebreton et al 2008), which could potentially bypass the branched structure and account for the endonucleolytic cleavage of lariats stabilized in a dbr1Δ strain, cleavage that has been observed previously (Ooi et al 1998;Garrey et al 2014) and likely accounts for the population of 3 ′ reads that mapped to the intron body, upstream of the branch (Fig. 3D).…”

Section: Insight Into Lariat Intron Processingmentioning

confidence: 83%

Sequencing of lariat termini in S. cerevisiae reveals 5′ splice sites, branch points, and novel splicing events

Qin

Huang

Wlodaver

et al. 2015

RNA

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Pre-mRNA splicing is a central step in the shaping of the eukaryotic transcriptome and in the regulation of gene expression. Yet, due to a focus on fully processed mRNA, common approaches for defining pre-mRNA splicing genome-wide are suboptimalespecially with respect to defining the branch point sequence, a key cis-element that initiates the chemistry of splicing. Here, we report a complementary intron-centered approach designed to more efficiently, simply, and directly define splicing events genome-wide. Specifically, we developed a method distinguished by deep sequencing of lariat intron termini (LIT-seq). In a test of LIT-seq using the budding yeast Saccharomyces cerevisiae, we not only successfully captured the majority of annotated, expressed splicing events but also uncovered 45 novel splicing events, establishing the sensitivity of LIT-seq. Moreover, our libraries were highly enriched with reads that reported on splice sites; by a simple and direct inspection of sequencing reads, we empirically defined both 5 ′ splice sites and branch sites, as well as their consensus sequences, with nucleotide resolution. Additionally, our study revealed that the 3 ′ termini of lariat introns are subject to nontemplated addition of adenosines, characteristic of signals sensed by 3 ′ to 5 ′ RNA turnover machinery. Collectively, this work defines a novel, genome-wide approach for analyzing splicing with unprecedented depth, specificity, and resolution.

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A homolog of lariat-debranching enzyme modulates turnover of branched RNA

Cited by 34 publications

References 48 publications

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

Structures of the Human Spliceosomes Before and After Release of the Ligated Exon

Sequencing of lariat termini in S. cerevisiae reveals 5′ splice sites, branch points, and novel splicing events

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