Chapter 2 A Kinetic Assay to Monitor RNA Decapping Under Single- Turnover Conditions

Jones, Brittnee N.; Quang-Dang, Duc-Uy; Oku, Yuko; Gross, John D.

doi:10.1016/s0076-6879(08)02602-5

Cited by 23 publications

(38 citation statements)

References 47 publications

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“…Indeed, deadenylation is often the rate-limiting step of mRNA decay (Shyu et al 1991;Muhlrad and Parker 1992;Decker and Parker 1993). Interestingly, the in vivo decapping rates are slower than those measured in vitro with purified enzyme and substrate (Jones et al 2008). This discrepancy likely reflects competitive binding of other proteins to the cap in vivo, such as eIF4E, or perhaps limiting concentration of decapping enzyme.…”

Section: Discussionmentioning

confidence: 99%

A quantitative assay for measuring mRNA decapping by splinted ligation reverse transcription polymerase chain reaction: qSL-RT-PCR

Blewett¹,

Coller²,

Goldstrohm³

2011

RNA

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The degradation of messenger RNA is a critical node of gene regulation. A major pathway of mRNA decay is initiated by shortening of the poly(A) tail, followed by removal of the 59 cap structure (decapping) and subsequent degradation. Decapping is an important determinate in the destruction of many transcripts. Detailed kinetic analysis of in vivo decapping rates is necessary to understand how this step is regulated. Importantly, the product of decapping is recalcitrant for investigation, in part due to its transient nature. As such, little in vivo kinetic information is available. Here we report the development of an assay that measures decapping of mRNAs by combining splinted ligation and quantitative RT-PCR (qSL-RT-PCR). We apply this method to determine the decapping rate constant for a natural mRNA in vivo for the first time. The qSL-RT-PCR assay may be adapted for use on any mRNA, providing a new tool to study regulation of mRNA decay.

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Section: Discussionmentioning

confidence: 99%

A quantitative assay for measuring mRNA decapping by splinted ligation reverse transcription polymerase chain reaction: qSL-RT-PCR

Blewett¹,

Coller²,

Goldstrohm³

2011

RNA

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“…biochem.ucl.ac.uk/hansen/fuda) and CPMG dispersion curves were fit using cpmg_fitd8 (a gift from D. Korzhnev and L. Kay, University of Toronto, Toronto, ON). Enzyme kinetics were measured under single-turnover conditions as described (9,17,57). Structure figures were made using PyMOL (http://pymol.org).…”

Section: Methodsmentioning

confidence: 99%

Interdomain dynamics and coactivation of the mRNA decapping enzyme Dcp2 are mediated by a gatekeeper tryptophan

Floor

Borja²,

Gross³

2012

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

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Conformational dynamics in bilobed enzymes can be used to regulate their activity. One such enzyme, the eukaryotic decapping enzyme Dcp2, controls the half-life of mRNA by cleaving the 5′ cap structure, which exposes a monophosphate that is efficiently degraded by exonucleases. Decapping by Dcp2 is thought to be controlled by an open-to-closed transition involving formation of a composite active site with two domains sandwiching substrate, but many details of this process are not understood. Here, using NMR spectroscopy and enzyme kinetics, we show that Trp43 of Schizosaccharomyces pombe Dcp2 is a conserved gatekeeper of this open-to-closed transition. We find that Dcp2 samples multiple conformations in solution on the millisecond-microsecond timescale. Mutation of the gatekeeper tryptophan abolishes the dynamic behavior of Dcp2 and attenuates coactivation by a yeast enhancer of decapping (Edc1). Our results determine the dynamics of the open-to-closed transition in Dcp2, suggest a structural pathway for coactivation, predict that Dcp1 directly contacts the catalytic domain of Dcp2, and show that coactivation of decapping by Dcp2 is linked to formation of the composite active site.enzyme dynamics | methyl groups | mRNA decay | protein NMR C onformational dynamics in enzymes often comprise the ratelimiting step in the catalytic cycle and thus are prime targets for regulatory cofactors (1-5). Bilobed proteins frequently use an open-to-closed transition to coordinate catalysis on their substrates following cellular cues such as posttranslational modifications or macromolecular interactions (6-8). A recent model proposes that the eukaryotic mRNA decapping enzyme Dcp2 is regulated by such a transition, where a composite active site is formed using conserved surfaces on each of the two N-terminal domains (9). According to this model, stimulating or inhibiting this conformational transition could regulate decapping. However, the structural details of this composite active site and the timescale of interconversion between closed and open states of Dcp2 are currently unknown. Moreover, whether coactivators use the composite active site to effect decapping is unclear.Degradation of eukaryotic mRNA is critical to many biological processes including development (10), stress response (11), clearance of the products of pervasive transcription (12), and quality control of gene expression (13). For example, it has been suggested that microRNAs (miRNAs) act primarily by destabilizing messages and it is known that Dcp2 is a vital component of miRNA-induced mRNA decay (14, 15). Further, an entire class of unstable transcripts was recently discovered that is sensitive to the exonuclease Xrn1, whose members are therefore likely products of decapping (12, 16). Each of the variety of pathways that utilize decapping relies on coactivator proteins that are believed to recruit messages to the decapping machinery and activate it.A model of decapping coactivation is emerging following recent work on the Saccharomyces cerevisiae coactivator...

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“…Decapping complexes in the presence or absence of Edc3 were incubated at 4°C for 30 min prior to initiation of the reactions. Time courses were monitored and analyzed as described previously (22). Dcp1-Dcp2(1-245) and Dcp1-Dcp2(1-315) were coexpressed in E. coli and purified as described previously (10).…”

Section: Microscopymentioning

confidence: 99%

Identification and Analysis of the Interaction between Edc3 and Dcp2 in Saccharomyces cerevisiae

Harigaya

Jones²,

Muhlrad

et al. 2010

Molecular and Cellular Biology

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Cap hydrolysis is a critical control point in the life of eukaryotic mRNAs and is catalyzed by the evolutionarily conserved Dcp1-Dcp2 complex. In Saccharomyces cerevisiae, decapping is modulated by several factors, including the Lsm family protein Edc3, which directly binds to Dcp2. We show that Edc3 binding to Dcp2 is mediated by a short peptide sequence located C terminal to the catalytic domain of Dcp2. This sequence is required for Edc3 to stimulate decapping activity of Dcp2 in vitro, for Dcp2 to efficiently accumulate in P-bodies, and for efficient degradation of the RPS28B mRNA, whose decay is enhanced by Edc3. In contrast, degradation of YRA1 pre-mRNA, another Edc3-regulated transcript, occurs independently from this region, suggesting that the effect of Edc3 on YRA1 is independent of its interaction with Dcp2. Deletion of the sequence also results in a subtle but significant defect in turnover of the MFA2pG reporter transcript, which is not affected by deletion of EDC3, suggesting that the region affects some other aspect of Dcp2 function in addition to binding Edc3. These results raise a model for Dcp2 recruitment to specific mRNAs where regions outside the catalytic core promote the formation of different complexes involved in mRNA decapping.

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Chapter 2 A Kinetic Assay to Monitor RNA Decapping Under Single- Turnover Conditions

Cited by 23 publications

References 47 publications

A quantitative assay for measuring mRNA decapping by splinted ligation reverse transcription polymerase chain reaction: qSL-RT-PCR

A quantitative assay for measuring mRNA decapping by splinted ligation reverse transcription polymerase chain reaction: qSL-RT-PCR

Interdomain dynamics and coactivation of the mRNA decapping enzyme Dcp2 are mediated by a gatekeeper tryptophan

Identification and Analysis of the Interaction between Edc3 and Dcp2 in Saccharomyces cerevisiae

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