Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

Özalp, Veli Cengiz; Pedersen, Tina Ravn; Nielsen, Lise Junker; Olsen, Lars Folke

doi:10.1074/jbc.m110.155119

Cited by 106 publications

(130 citation statements)

References 43 publications

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“…Thus, Brr2 as modeled in the cryo-EM structure could not adopt the autoinhibited state described here, as its loading on U4 snRNA would be in conflict with the autoinhibitory position of the plug. The complex used for cryo-EM analysis disintegrated in an uncharacterized fashion upon addition of 1 mM ATP (Nguyen et al 2015), showing that additional mechanisms must exist to prevent wasteful tri-snRNP disruption in vivo, where ATP levels range between 1 and 2.8 mM (Ozalp et al 2010). Consistent with this proposal, we observed that yeast tri-snRNPs in extracts remain stable in the presence of ATP.…”

Section: Discussionsupporting

confidence: 81%

The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

et al. 2015

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The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 disnRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.[Keywords: pre-mRNA splicing; RNA helicase structure and function; remodeling of RNA-protein complexes; spliceosome catalytic activation; X-ray crystallography] Supplemental material is available for this article.Received September 14, 2015; revised version accepted November 13, 2015.Splicing entails the removal of noncoding sequences (introns) from primary transcripts and the concomitant ligation of neighboring coding regions (exons). It is mediated by a highly dynamic, multimegadalton RNA protein (RNP) molecular machine, the spliceosome, which consists of five small nuclear RNPs (snRNPs; U1, U2, U4, U5, and U6 in the case of the major spliceosome) and numerous non-snRNPs (Wahl et al. 2009). For each round of splicing, a spliceosome is assembled de novo on a substrate by the stepwise recruitment of snRNPs and nonsnRNPs. After assembly of a precatalytic complex, the spliceosome is catalytically activated and carries out the two consecutive steps of a splicing reaction before it is disassembled and its subunits are recycled. Each assembly, activation, catalysis, and disassembly step involves profound rearrangements of the spliceosomal RNP interaction networks, mediated predominantly by eight conserved superfamily 2 (SF2) NTPases/RNA helicases (Staley and Guthrie 1998). The most extensive rearrangements occur during spliceosome activation. In the precatalytic spliceosome, U4 and U6 snRNPs form a disnRNP by base-pairing of their snRNAs and are associated with U5 snRNP via protein-protein interactions. During spliceosome activation, the U5 snRNP-specific Brr2 helicase unwinds the U4/U6 ...

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Section: Discussionsupporting

confidence: 81%

The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

et al. 2015

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“…S6). Because cellular ATP concentrations are only about twofold higher than ADP concentrations (62), these data suggest that Rok1 is bound to ADP in vivo, rendering this annealing activity relevant.…”

Section: A2 A2mentioning

confidence: 85%

Cofactor-dependent specificity of a DEAD-box protein

Young

Khoshnevis

Karbstein

2013

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

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DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.RNA helicases | annealing D EAD-box proteins are RNA-binding ATPases that are involved in all aspects of RNA metabolism: translation initiation, pre-mRNA splicing, mRNA export and decay, and ribosome biogenesis. In these processes, their functions include RNA duplex unwinding, RNA-protein complex remodeling, RNA duplex annealing, and ATP-dependent RNA binding (1-5).In vivo, these enzymes have specific functions that often involve the recognition of specific RNAs and the discrimination against a myriad of nonsubstrates. Nevertheless, only DbpA, a DEAD-box protein involved in bacterial ribosome assembly (6-8), has sequence-specific ATPase activity (6, 9), which arises from unique sequences in its C terminus (10). Such sequence specificity has not been demonstrated for any other DEAD-box protein and is consistent with their highly conserved structures and the almost universal conservation of residues that contact the RNA. Furthermore, analysis of the structures of RNA-bound DEAD-box proteins reveals that RNA contacts are made with the sugarphosphate backbone (11-17), largely precluding sequence-specific interactions between DEAD-box proteins and RNA.Many DEAD-box and related DEAH-box proteins function in conjunction with cofactors (ref. 18 and references therein). These cofactors can regulate the activity of DEAD-box or DEAH-box proteins by stimulating or inhibiting their ATPase, helicase, RNAbinding, or nucleotide-binding activities. Interestingly, cofactors are often RNA-binding proteins (19)(20)(21)(22)(23)(24)(25). We and others therefore postulated that these cofactors could als...

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“…It is not known when during the cycle Dbp5 becomes bound to ATP. As the intracellular concentration of ATP is high (>1 mM) (Ozalp et al 2010), Dbp5 might bind ATP soon after dissociation of ADP. However, because Gle1-IP 6 promotes ATP binding in vitro (Noble et al 2011), Dbp5 might remain in the apo form until it moves from Nup159 to Gle1.…”

Section: Discussionmentioning

confidence: 99%

The Dbp5 cycle at the nuclear pore complex during mRNA export I: dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1

Hodge¹,

Tran²,

Noble³

et al. 2011

Genes Dev.

101

143

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Nuclear export of messenger RNA (mRNA) occurs by translocation of mRNA/protein complexes (mRNPs) through nuclear pore complexes (NPCs). The DEAD-box protein Dbp5 mediates export by triggering removal of mRNP proteins in a spatially controlled manner. This requires Dbp5 interaction with Nup159 in NPC cytoplasmic filaments and activation of Dbp5's ATPase activity by Gle1 bound to inositol hexakisphosphate (IP 6 ). However, the precise sequence of events within this mechanism has not been fully defined. Here we analyze dbp5 mutants that alter ATP binding, ATP hydrolysis, or RNA binding. We found that ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs. Interestingly, mutants defective for RNA binding are dominant-negative (DN) for mRNA export in yeast and human cells. We show that the DN phenotype stems from competition with wild-type Dbp5 for Gle1 at NPCs. The Dbp5-Gle1 interaction is limiting for export and, importantly, can be independent of Nup159. Fluorescence recovery after photobleaching experiments in yeast show a very dynamic association between Dbp5 and NPCs, averaging <1 sec, similar to reported NPC translocation rates for mRNPs. This work reveals critical steps in the Gle1-IP 6 /Dbp5/Nup159 cycle, and suggests that the number of remodeling events mediated by a single Dbp5 is limited.[Keywords: nucleocytoplasmic transport; DEAD-box proteins; nuclear pore complex; FRAP; dominant-negative mutants] Supplemental material is available for this article. Messenger RNAs (mRNAs) are produced in the nucleus through a series of highly coordinated steps that include transcription, processing, and assembly with proteins to form a messenger ribonucleoprotein complex (mRNP) (for reviews, see Pandit et al. 2008;Zhong et al. 2009; Licatalosi and Darnell 2010). Some of the factors that participate in these steps associate with RNA polymerase II during transcription (Cho et al. 1997;Moore and Proudfoot 2009;Perales and Bentley 2009), facilitating coordination by positioning these factors to interact with nascent RNAs upon recognition of key sequence elements or structures. This results in the formation of mRNPs that are exported to the cytoplasm for translation after premRNA processing has been completed. mRNPs are exported through nuclear pore complexes (NPCs), large macromolecular structures (>60 MDa) embedded in the nuclear envelope (NE) (for reviews, see D'Angelo and Hetzer 2008;Hetzer and Wente 2009;Wente and Rout 2010). NPCs have eightfold radial symmetry perpendicular to the NE plane, and the NPC core has twofold symmetry in the NE plane. Attached to the core

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Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

Cited by 106 publications

References 43 publications

The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

Cofactor-dependent specificity of a DEAD-box protein

The Dbp5 cycle at the nuclear pore complex during mRNA export I: dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1

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