scholarly journals Pat1 activates late steps in mRNA decay by multiple mechanisms

2019 ◽  
Author(s):  
Joseph H. Lobel ◽  
Ryan W. Tibble ◽  
John D. Gross
Keyword(s):  
Molecular Mechanism ◽  
Mrna Decay ◽  
Substrate Binding ◽  
Mrna Degradation ◽  
Mrna Splicing ◽  
Linear Interaction ◽  
Unknown Mechanism ◽  
Translation Repression ◽  
Mrna Metabolism ◽  
Hydrolysis Of

ABSTRACTPat1 is a hub for mRNA metabolism, acting in pre-mRNA splicing, translation repression and mRNA decay. A critical step in all 5’-3’ mRNA decay pathways is removal of the 5’ cap structure, which precedes and permits digestion of the RNA body by conserved exonucleases. During bulk 5’-3’ decay, the Pat1/Lsm1-7 complex engages mRNA at the 3’ end and promotes hydrolysis of the cap structure by Dcp1/Dcp2 at the 5’ end through an unknown mechanism. We reconstitute Pat1 with 5’ and 3’ decay factors and show how it activates multiple steps in late mRNA decay. First, we find that Pat1 stabilizes binding of the Lsm1-7 complex to RNA using two conserved short-linear interaction motifs. Secondly, Pat1 directly activates decapping by binding elements in the disordered C-terminal extension of Dcp2, alleviating autoinhibition and promoting substrate binding. Our results uncover the molecular mechanism of how separate domains of Pat1 coordinate the assembly and activation of a decapping mRNP that promotes 5’-3’ mRNA degradation.

scholarly journals Pat1 activates late steps in mRNA decay by multiple mechanisms

2019 ◽  
Vol 116 (47) ◽  
pp. 23512-23517 ◽  
Author(s):  
Joseph H. Lobel ◽  
Ryan W. Tibble ◽  
John D. Gross
Keyword(s):  
Molecular Mechanism ◽  
Mrna Decay ◽  
Mrna Degradation ◽  
Mrna Splicing ◽  
Linear Interaction ◽  
Unknown Mechanism ◽  
Translation Repression ◽  
Mrna Metabolism ◽  
Messenger Ribonucleoprotein ◽  
Hydrolysis Of

Pat1 is a hub for mRNA metabolism, acting in pre-mRNA splicing, translation repression, and mRNA decay. A critical step in all 5′-3′ mRNA decay pathways is removal of the 5′ cap structure, which precedes and permits digestion of the RNA body by conserved exonucleases. During bulk 5′-3′ decay, the Pat1/Lsm1-7 complex engages mRNA at the 3′ end and promotes hydrolysis of the cap structure by Dcp1/Dcp2 at the 5′ end through an unknown mechanism. We reconstitute Pat1 with 5′ and 3′ decay factors and show how it activates multiple steps in late mRNA decay. First, we find that Pat1 stabilizes binding of the Lsm1-7 complex to RNA using two conserved short-linear interaction motifs. Second, Pat1 directly activates decapping by binding elements in the disordered C-terminal extension of Dcp2, alleviating autoinhibition and promoting substrate binding. Our results uncover the molecular mechanism of how separate domains of Pat1 coordinate the assembly and activation of a decapping messenger ribonucleoprotein (mRNP) that promotes 5′-3′ mRNA degradation.

scholarly journals dTIS11 Protein-dependent Polysomal Deadenylation Is the Key Step in AU-rich Element-mediated mRNA Decay in Drosophila Cells

2012 ◽  
Vol 287 (42) ◽  
pp. 35527-35538 ◽  
Author(s):  
Caroline Vindry ◽  
Aurélien Lauwers ◽  
David Hutin ◽  
Romuald Soin ◽  
Corinne Wauquier ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Molecular Mechanism ◽  
Mrna Decay ◽  
Degradation Process ◽  
Mrna Degradation ◽  
Decay Process ◽  
Human Protein ◽  
P Bodies ◽  
Drosophila Cells ◽  
Degradation Intermediates

The destabilization of AU-rich element (ARE)-containing mRNAs mediated by proteins of the TIS11 family is conserved among eukaryotes including Drosophila. Previous studies have demonstrated that Tristetraprolin, a human protein of the TIS11 family, induces the degradation of ARE-containing mRNAs through a large variety of mechanisms including deadenylation, decapping, and P-body targeting. We have previously shown that the degradation of the mRNA encoding the antimicrobial peptide Cecropin A1 (CecA1) is controlled by the TIS11 protein (dTIS11) in Drosophila cells. In this study, we used CecA1 mRNA as a model to investigate the molecular mechanism of dTIS11-mediated mRNA decay. We observed that during the biphasic deadenylation and decay process of this mRNA, dTIS11 enhances deadenylation performed by the CCR4-CAF-NOT complex while the mRNA is still associated with ribosomes. Sequencing of mRNA degradation intermediates revealed that the complete deadenylation of the mRNA triggers its decapping and decay in both the 5′-3′ and the 3′-5′ directions. Contrary to the observations made for its mammalian homologs, overexpression of dTIS11 does not promote the localization of ARE-containing mRNAs in P-bodies but rather decreases the accumulation of CecA1 mRNA in these structures by enhancing the degradation process. Therefore, our results suggest that proteins of the TIS11 family may have acquired additional functions in the course of evolution from invertebrates to mammals.

scholarly journals Molecular mechanism for Rabex-5 GEF activation by Rabaptin-5

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Zhe Zhang ◽  
Tianlong Zhang ◽  
Shanshan Wang ◽  
Zhou Gong ◽  
Chun Tang ◽  
...  
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Molecular Mechanism ◽  
Ternary Complex ◽  
Substrate Binding ◽  
Unknown Mechanism ◽  
Substrate Binding Site ◽  
Biochemical Analyses ◽  
Α Helix

Rabex-5 and Rabaptin-5 function together to activate Rab5 and further promote early endosomal fusion in endocytosis. The Rabex-5 GEF activity is autoinhibited by the Rabex-5 CC domain (Rabex-5CC) and activated by the Rabaptin-5 C2-1 domain (Rabaptin-5C21) with yet unknown mechanism. We report here the crystal structures of Rabex-5 in complex with the dimeric Rabaptin-5C21 (Rabaptin-5C212) and in complex with Rabaptin-5C212 and Rab5, along with biophysical and biochemical analyses. We show that Rabex-5CC assumes an amphipathic α-helix which binds weakly to the substrate-binding site of the GEF domain, leading to weak autoinhibition of the GEF activity. Binding of Rabaptin-5C21 to Rabex-5 displaces Rabex-5CC to yield a largely exposed substrate-binding site, leading to release of the GEF activity. In the ternary complex the substrate-binding site of Rabex-5 is completely exposed to bind and activate Rab5. Our results reveal the molecular mechanism for the regulation of the Rabex-5 GEF activity.

scholarly journals Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM

2020 ◽  
Author(s):  
Andrés López-Perrote ◽  
Nele Hug ◽  
Ana González-Corpas ◽  
Carlos F. Rodríguez ◽  
Marina Serna ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Mrna Decay ◽  
Atp Hydrolysis ◽  
Unknown Mechanism ◽  
Macromolecular Complexes ◽  
Wide Range ◽  
Nonsense Mediated Mrna Decay ◽  
Aaa Atpases ◽  
Hydrolysis Of

AbstractNonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1 and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.

scholarly journals Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Andres López-Perrote ◽  
Nele Hug ◽  
Ana González-Corpas ◽  
Carlos F Rodríguez ◽  
Marina Serna ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Mrna Decay ◽  
Atp Hydrolysis ◽  
Unknown Mechanism ◽  
Macromolecular Complexes ◽  
Wide Range ◽  
Nonsense Mediated Mrna Decay ◽  
Aaa Atpases ◽  
Hydrolysis Of

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1, and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.

mRNA degradation by processive 3′-5′ exoribonucleases in Vitro and the implications for prokaryotic mRNA decay in Vivo

1991 ◽  
Vol 221 (1) ◽  
pp. 81-95 ◽  
Author(s):  
Robert S. McLaren ◽  
Sarah F. Newbury ◽  
Geoffrey S.C. Dance ◽  
Helen C. Causton ◽  
Christopher F. Higgins
Keyword(s):  
Mrna Decay ◽  
Mrna Degradation

scholarly journals Influence of Me2SO and incubation time on papain activity studied using fluorogenic substrates.

2001 ◽  
Vol 48 (4) ◽  
pp. 995-1002 ◽  
Author(s):  
M Szabelski ◽  
K Stachowiak ◽  
W Wiczk
Keyword(s):  
Incubation Time ◽  
Active Sites ◽  
Initial Rate ◽  
Substrate Binding ◽  
Rapid Decrease ◽  
Fluorogenic Substrates ◽  
Binding Process ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Papain activity in a buffer containing Me2SO was studied using fluorogenic substrates. It was found that the number of active sites of papain decreases with increasing Me2SO concentration whereas the incubation time, in a buffer containing 3% Me2SO does not affect the number of active sites. However, an increase of papain incubation time in the buffer with 3% Me2SO decreased the initial rate of hydrolysis of Z-Phe-Arg-Amc as well as Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans. Moreover, an increase of Me2SO concentration in working buffer decreased the initial rate of papain-catalysed hydrolysis of both substrates. A rapid decrease of the initial rate (by up to 30%) was observed between 1 and 2% Me2SO. Application of the Michaelis-Menten equation revealed that at the higher Me2SO concentrations the apparent values of k(cat)/Km decreased as a result of Km increase and kcat decrease. However, Me2SO changed the substrate binding process more effectively (Km) than the rate of catalysis k(cat).

scholarly journals Selective inhibition of the p38α MAPK–MK2 axis inhibits inflammatory cues including inflammasome priming signals

2018 ◽  
Vol 215 (5) ◽  
pp. 1315-1325 ◽  
Author(s):  
Chun Wang ◽  
Susan Hockerman ◽  
E. Jon Jacobsen ◽  
Yael Alippe ◽  
Shaun R. Selness ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Clinical Trials ◽  
Mrna Decay ◽  
Selective Inhibition ◽  
Mrna Degradation ◽  
Cytokine Expression ◽  
Clinical Translation ◽  
Tnf Α ◽  
P38α Mapk ◽  
Nlrp3 Expression

p38α activation of multiple effectors may underlie the failure of global p38α inhibitors in clinical trials. A unique inhibitor (CDD-450) was developed that selectively blocked p38α activation of the proinflammatory kinase MK2 while sparing p38α activation of PRAK and ATF2. Next, the hypothesis that the p38α–MK2 complex mediates inflammasome priming cues was tested. CDD-450 had no effect on NLRP3 expression, but it decreased IL-1β expression by promoting IL-1β mRNA degradation. Thus, IL-1β is regulated not only transcriptionally by NF-κB and posttranslationally by the inflammasomes but also posttranscriptionally by p38α–MK2. CDD-450 also accelerated TNF-α and IL-6 mRNA decay, inhibited inflammation in mice with cryopyrinopathy, and was as efficacious as global p38α inhibitors in attenuating arthritis in rats and cytokine expression by cells from patients with cryopyrinopathy and rheumatoid arthritis. These findings have clinical translation implications as CDD-450 offers the potential to avoid tachyphylaxis associated with global p38α inhibitors that may result from their inhibition of non-MK2 substrates involved in antiinflammatory and housekeeping responses.

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