scholarly journals Structural and biochemical analyses of the DEAD-box ATPase Sub2 in association with THO or Yra1

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yi Ren ◽  
Philip Schmiege ◽  
Günter Blobel
Keyword(s):  
Reaction Mechanisms ◽  
Nuclear Pore ◽  
Closed Conformation ◽  
Dead Box ◽  
Messenger Ribonucleoprotein ◽  
Mrna Binding Protein ◽  
The Dead ◽  
Biochemical Analyses ◽  
Mrna Binding ◽  
Open Configuration

mRNA is cotranscrptionally processed and packaged into messenger ribonucleoprotein particles (mRNPs) in the nucleus. Prior to export through the nuclear pore, mRNPs undergo several obligatory remodeling reactions. In yeast, one of these reactions involves loading of the mRNA-binding protein Yra1 by the DEAD-box ATPase Sub2 as assisted by the hetero-pentameric THO complex. To obtain molecular insights into reaction mechanisms, we determined crystal structures of two relevant complexes: a THO hetero-pentamer bound to Sub2 at 6.0 Å resolution; and Sub2 associated with an ATP analogue, RNA, and a C-terminal fragment of Yra1 (Yra1-C) at 2.6 Å resolution. We found that the 25 nm long THO clamps Sub2 in a half-open configuration; in contrast, when bound to the ATP analogue, RNA and Yra1-C, Sub2 assumes a closed conformation. Both THO and Yra1-C stimulated Sub2’s intrinsic ATPase activity. We propose that THO surveys common landmarks in each nuclear mRNP to localize Sub2 for targeted loading of Yra1.

The DEAD Box Helicase YxiN Maintains a Closed Conformation during ATP Hydrolysis

Biochemistry ◽  
2009 ◽  
Vol 48 (45) ◽  
pp. 10679-10681 ◽  
Author(s):  
Regula Aregger ◽  
Dagmar Klostermeier
Keyword(s):  
Atp Hydrolysis ◽  
Closed Conformation ◽  
Dead Box ◽  
The Dead

scholarly journals Cryo-EM structure of the yeast TREX complex and coordination with the SR-like protein Gbp2

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Yihu Xie ◽  
Bradley P Clarke ◽  
Yong Joon Kim ◽  
Austin L Ivey ◽  
Pate S Hill ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Terminal Domain ◽  
Dead Box ◽  
Open Conformation ◽  
Messenger Ribonucleoprotein ◽  
Evolutionarily Conserved ◽  
Close Proximity ◽  
The Dead

The evolutionarily conserved TREX complex plays central roles during mRNP (messenger ribonucleoprotein) maturation and export from the nucleus to the cytoplasm. In yeast, TREX is composed of the THO sub-complex (Tho2, Hpr1, Tex1, Mft1, and Thp2), the DEAD box ATPase Sub2, and Yra1. Here we present a 3.7 Å cryo-EM structure of the yeast THO•Sub2 complex. The structure reveals the intimate assembly of THO revolving around its largest subunit Tho2. THO stabilizes a semi-open conformation of the Sub2 ATPase via interactions with Tho2. We show that THO interacts with the SR-like protein Gbp2 through both the RS domain and RRM domains of Gbp2. Crosslinking mass spectrometry analysis supports the extensive interactions between THO and Gbp2, further revealing that RRM domains of Gbp2 are in close proximity to the C-terminal domain of Tho2. We propose that THO serves as a landing pad to configure Gbp2 to facilitate its loading onto mRNP.

scholarly journals The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

2009 ◽  
Vol 390 (12) ◽  
Author(s):  
Manuel Hilbert ◽  
Anne R. Karow ◽  
Dagmar Klostermeier
Keyword(s):  
Rna Binding ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Current Data ◽  
Protein Activity ◽  
Closed Conformation ◽  
Dead Box ◽  
Rna Duplexes ◽  
The Dead ◽  
Rna Unwinding

Abstract DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecA-like domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysis- and unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

scholarly journals The nuclear basket mediates perinuclear mRNA scanning in budding yeast

2015 ◽  
Vol 211 (6) ◽  
pp. 1131-1140 ◽  
Author(s):  
Mark-Albert Saroufim ◽  
Pierre Bensidoun ◽  
Pascal Raymond ◽  
Samir Rahman ◽  
Matthew R. Krause ◽  
...  
Keyword(s):  
Nuclear Periphery ◽  
Nuclear Pore ◽  
Messenger Rnas ◽  
Nuclear Pores ◽  
Transport Channel ◽  
Mrna Binding Protein ◽  
Live Cell Microscopy ◽  
Scanning Behavior ◽  
Mrna Binding ◽  
Cell Microscopy

After synthesis and transit through the nucleus, messenger RNAs (mRNAs) are exported to the cytoplasm through the nuclear pore complex (NPC). At the NPC, messenger ribonucleoproteins (mRNPs) first encounter the nuclear basket where mRNP rearrangements are thought to allow access to the transport channel. Here, we use single mRNA resolution live cell microscopy and subdiffraction particle tracking to follow individual mRNAs on their path toward the cytoplasm. We show that when reaching the nuclear periphery, RNAs are not immediately exported but scan along the nuclear periphery, likely to find a nuclear pore allowing export. Deletion or mutation of the nuclear basket proteins MLP1/2 or the mRNA binding protein Nab2 changes the scanning behavior of mRNPs at the nuclear periphery, shortens residency time at nuclear pores, and results in frequent release of mRNAs back into the nucleoplasm. These observations suggest a role for the nuclear basket in providing an interaction platform that keeps RNAs at the periphery, possibly to allow mRNP rearrangements before export.

scholarly journals A Distinct and Parallel Pathway for the Nuclear Import of an mRNA-binding Protein

1997 ◽  
Vol 139 (7) ◽  
pp. 1645-1653 ◽  
Author(s):  
Lucy F. Pemberton ◽  
Jonathan S. Rosenblum ◽  
Günter Blobel
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Binding Protein ◽  
Nuclear Pore ◽  
Polyadenylated Rna ◽  
Mrna Binding Protein ◽  
Parallel Pathway ◽  
Rna Export ◽  
Mrna Binding ◽  
Transport Factors

Three independent pathways of nuclear import have so far been identified in yeast, each mediated by cognate nuclear transport factors, or karyopherins. Here we have characterized a new pathway to the nucleus, mediated by Mtr10p, a protein first identified in a screen for strains defective in polyadenylated RNA export. Mtr10p is shown to be responsible for the nuclear import of the shuttling mRNA-binding protein Npl3p. A complex of Mtr10p and Npl3p was detected in cytosol, and deletion of Mtr10p was shown to lead to the mislocalization of nuclear Npl3p to the cytoplasm, correlating with a block in import. Mtr10p bound peptide repeat-containing nucleoporins and Ran, suggesting that this import pathway involves a docking step at the nuclear pore complex and is Ran dependent. This pathway of Npl3p import is distinct and does not appear to overlap with another known import pathway for an mRNA-binding protein. Thus, at least two parallel pathways function in the import of mRNA-binding proteins, suggesting the need for the coordination of these pathways.

scholarly journals Cryo-EM structure of the yeast TREX complex and coordination with the SR-like protein Gbp2

2021 ◽  
Author(s):  
Yihu Xie ◽  
Bradley P. Clarke ◽  
Yong Joon Kim ◽  
Austin L. Ivey ◽  
Pate S. Hill ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Terminal Domain ◽  
Dead Box ◽  
Open Conformation ◽  
Messenger Ribonucleoprotein ◽  
Evolutionarily Conserved ◽  
Close Proximity ◽  
The Dead

AbstractThe evolutionarily conserved TREX complex plays central roles during mRNP (messenger ribonucleoprotein) maturation and export from the nucleus to the cytoplasm. In yeast, TREX is composed of the THO sub-complex (Tho2, Hpr1, Tex1, Mft1, and Thp2), the DEAD box ATPase Sub2, and Yra1. Here we present a 3.7 Å cryo-EM structure of the yeast THO•Sub2 complex. The structure reveals the intimate assembly of THO revolving around its largest subunit Tho2. THO stabilizes a semi-open conformation of the Sub2 ATPase via interactions with Tho2. We show that THO interacts with the SR-like protein Gbp2 through both the N-terminal domain and RRM domains of Gbp2. Crosslinking mass spectrometry analysis supports the extensive interactions between THO and Gbp2, further revealing that RRM domains of Gbp2 are in close proximity to the C-terminal domain of Tho2. We propose that THO serves as a landing pad to configure Gbp2 to facilitate its loading onto mRNP.

scholarly journals Recruitment of Two Dyneins to an mRNA-Dependent Bicaudal D Transport Complex

2018 ◽  
Author(s):  
Thomas E. Sladewski ◽  
Neil Billington ◽  
M. Yusuf Ali ◽  
Carol S. Bookwalter ◽  
Hailong Lu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Optimal Transport ◽  
Adaptor Proteins ◽  
Full Length ◽  
Messenger Ribonucleoprotein ◽  
Mrna Binding Protein ◽  
Run Lengths ◽  
Transport Complex ◽  
Mrna Binding

AbstractWe investigated the role of binding partners of full-length Drosophila Bicaudal D (BicD) in the activation of dynein-dynactin motility for mRNA transport on microtubules. In single-molecule assays, full-length BicD robustly activated dynein-dynactin only when both the mRNA binding protein Egalitarian (Egl), and K10 mRNA cargo were present. Electron microscopy showed that both Egl and mRNA were needed to disrupt an auto-inhibited, looped BicD conformation that sterically prevents dynein-dynactin binding. In vitro reconstituted messenger ribonucleoprotein (mRNP) complexes with two Egl molecules showed faster speeds and longer run lengths than mRNPs with one Egl, suggesting that cargo binding enhances dynein recruitment. Labeled dynein showed that BicD can recruit two dimeric dyneins to the mRNP, resulting in faster speeds and longer run lengths than with one dynein. The fully reconstituted mRNP provides a model for understanding how adaptor proteins and cargo cooperate to confer optimal transport properties to a dynein-driven transport complex.

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