Structural and biochemical studies of the 5′→3′ exoribonuclease Xrn1

Chang, Jeong Ho; Xiang, Song; Xiang, Kehui; Manley, James L.; Tong, Liang

doi:10.1038/nsmb.1984

Cited by 108 publications

(118 citation statements)

References 60 publications

Supporting

Mentioning

117

Contrasting

Order By: Relevance

“…Analyses of the nuclear RNA degradative 5Ј-exoribonuclease Rat1 (nuclear 5Ј-3Ј-exoribonuclease; also known as Xrn2) (41) demonstrate similar secondary structure to FENs (5). In addition, we also note that two recent structures of the corresponding cytosolic enzyme Xrn1 (cytosolic 5Ј-3Ј-exoribonuclease) (39,40) demonstrate that these enzymes conserve the DNA 5Ј-nuclease superfamily active site and that RNA 5Ј-nucleases may in turn may also utilize a similar catalytic mechanism.…”

Section: Discussionmentioning

confidence: 72%

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

Tomlinson

Syson

Sengerová

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

Flap endonucleases (FENs) are divalent metal ion-dependent phosphodiesterases. Metallonucleases are often assigned a "two-metal ion mechanism" where both metals contact the scissile phosphate diester. The spacing of the two metal ions observed in T5FEN structures appears to preclude this mechanism. However, the overall reaction catalyzed by wild type (WT) T5FEN requires three Mg 2؉ ions, implying that a third ion is needed during catalysis, and so a two-metal ion mechanism remains possible. , essential in all life forms, are members of the 5Ј-nuclease superfamily of structure-specific phosphate diesterases that carry out essential divalent metal ion-dependent nucleic acid hydrolysis (1). FENs act upon 5Ј-bifurcated structures known as flaps formed during lagging strand DNA replication and long patch base excision repair. Hydrolysis predominantly occurs one nucleotide into the 5Ј-duplex adjoining the site of bifurcation. An unusual feature of these enzymes is their high density of active site carboxylates that coordinate essential divalent metal ion cofactors. Bacterial and bacteriophage FENs possess eight active site acidic residues, seven of which are conserved in FENs from higher organisms and also in other members of the 5Ј-nuclease superfamily (1-4). In contrast, a typical metallonuclease active site consists of three or four carboxylates (5). However, a subclass of FEN paralogues, typified by Escherichia coli ExoIX, exists in eubacteria and appear to lack three of the metal-binding carboxylates (6).Although the most common mechanism suggested for metal-dependent phosphoryl transferases involves two metal ions in contact with the scissile phosphate diester, this is not universally accepted (5, 7-10). Debate about the validity or otherwise of two-metal ion catalysis has focused on several enzymes including flap endonucleases (11-16). Crystallographic studies of substrate-free FENs have demonstrated two active site-bound metal ions (14,(17)(18)(19). Metal ion 1 (M1) occupies a similar position in all structures. However, there is some variation in the position of the site coordinating a second metal ion (M2), often observed in FEN crystal structures. The distance between the M1 and M2 sites varies, and the separations observed are generally much greater than the 4 Å required for both metals to contact the same phosphate diester. For example, in bacteriophage T5FEN, 5 the M1-M2 separation of two Mn 2ϩ ions is 8 Å (Fig. 1A) (17). However, in a substrate-free structure of hFEN1, two magnesium ions are bound with a separation of 3.4 Å in one of three proteins bound to proliferating cell nuclear antigen (Fig. 1B) , 3-(N-morpholino)propane-sulfonic acid. 5 In early literature, T5FEN was referred to as T5 5Ј-3Ј-exonuclease and T5 5Ј-nuclease. In early literature, T4FEN was referred to as referred to also as T4 RNase H.

show abstract

Section: Discussionmentioning

confidence: 72%

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

Tomlinson

Syson

Sengerová

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…(B) Structure of mammalian XRN1. The catalytic domain (orange boxes) in human XRN1 (NP_061874) spans amino acids 1-595, and a conserved region up to amino acid 1174 is additionally required for enzymatic activity (Chang et al, 2011, reviewed in Nagarajan et al, 2013. A region that binds the decapping machinery is located at the C-terminal 30 amino acids (not depicted) (Braun et al, 2012).…”

Section: Methodsmentioning

confidence: 99%

“…8A). XRN1 is a large molecule and the catalytic domain and additional protein regions required for degradative activity span only two thirds of its length, opening the possibility of additional molecular functions (Chang et al, 2011;Braun et al, 2012;Nagarajan et al, 2013). Remarkably, XRN1 displays several short regions enriched in lysine and/or glutamic acid (Fig.…”

Section: What Is the Nature Of The Sx-bodies?mentioning

confidence: 99%

Synaptic control of mRNA translation by reversible assembly of XRN1 bodies

Luchelli¹,

Thomas²,

Boccaccio³

2015

Journal of Cell Science

View full text Add to dashboard Cite

Repression of mRNA translation is linked to the formation of specific cytosolic foci such as stress granules and processing bodies, which store or degrade mRNAs. In neurons, synaptic activity regulates translation at the post-synapse and this is important for plasticity. Nmethyl-D-aspartate (NMDA) receptor stimulation downregulates translation, and we speculate that this is linked to the formation of unknown mRNA-silencing foci. Here, we show that the 59-39 exoribonuclease XRN1 forms discrete clusters associated with the post-synapse that are different from processing bodies or stress granules, and we named them synaptic XRN1 bodies (SX-bodies). Using primary neurons, we found that the SX-bodies respond to synapse stimulation and that their formation correlates inversely with the local translation rate. SX-bodies increase in size and number upon NMDA stimulation, and metabotropic glutamate receptor activation provokes SX-body dissolution, along with increased translation. The response is specific and the previously described Smaug1 foci and FMRP granules show a different response. Finally, XRN1 knockdown impairs the translational repression triggered by NMDA. Collectively, these observations support a role for the SX-bodies in the reversible masking and silencing of mRNAs at the synapse.

show abstract

“…5A). An analogous internal deletion reduced activity of fungal Xrn1 (Chang et al 2011), indicating a conserved role in catalysis for the linker region.…”

Section: Xrn2 Phosphorylation Enhances Enzymatic Activitymentioning

confidence: 99%

P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates

et al. 2016

Self Cite

View full text Add to dashboard Cite

The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5 ′ -to-3 ′ "torpedo" exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2.

show abstract

Structural and biochemical studies of the 5′→3′ exoribonuclease Xrn1

Cited by 108 publications

References 60 publications

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

Synaptic control of mRNA translation by reversible assembly of XRN1 bodies

P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates

Contact Info

Product

Resources

About