Crystal Structure of Prp5p Reveals Interdomain Interactions that Impact Spliceosome Assembly

Zhang, Zhimin; Yang, Fei; Zhang, Jinru; Tang, Qing; Li, Jie; Gu, Jing; Zhou, Jiahai; Xu, Yufan

doi:10.1016/j.celrep.2013.10.047

Cited by 26 publications

(36 citation statements)

References 57 publications

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“…The spliceosomes are then directed to a discard pathway mediated by Prp5 using the energy from ATP hydrolysis. Mutations that reduce the ATPase activity of Prp5 slow down Prp5 action, allowing more time for duplex formation between U2 and the branch site (Xu and Query 2007;Zhang et al 2013). Supporting this hypothesis, mutations that increase the ATPase activity of Prp5 were shown to deteriorate the growth defect of branch site mutants .…”

Section: Discussionsupporting

confidence: 54%

A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence

Liang

Cheng

2015

Genes Dev.

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The DEAD-box RNA helicase Prp5 is required for the formation of the prespliceosome through an ATP-dependent function to remodel U2 small nuclear ribonucleoprotein particles (snRNPs) and an ATP-independent function of unknown mechanism. Prp5 has also been implicated in proofreading the branch site sequence, but the molecular mechanism has not been well characterized. Using actin precursor mRNA (pre-mRNA) carrying branch site mutations, we identified a Prp5-containing prespliceosome with Prp5 directly bound to U2 small nuclear RNA (snRNA). Prp5 is in contact with U2 in regions on and near the branchpoint-interacting stem-loop (BSL), suggesting that Prp5 may function in stabilizing the BSL. Regardless of its ATPase activity, Prp5 mutants that suppress branch site mutations associate with the spliceosome less tightly and allow more tri-snRNP binding for the reaction to proceed. Our results suggest a novel mechanism for how Prp5 functions in prespliceosome formation and proofreading of the branch site sequence. Prp5 binds to the spliceosome in association with U2 by interacting with the BSL and is released upon the base-pairing of U2 with the branch site to allow the recruitment of the tri-snRNP. Mutations impairing U2-branch site base-pairing retard Prp5 release and impede tri-snRNP association. Prp5 mutations that destabilize the Prp5-U2 interaction suppress branch site mutations by allowing progression of the pathway.[Keywords: Prp5; splicing; branch site; U2; proofreading] Supplemental material is available for this article.Received October 4, 2014; revised version accepted November 10, 2014.Introns of precursor mRNAs (pre-mRNAs) are removed via two steps of transesterification reaction, catalyzed by the spliceosome, which consists of five small nuclear RNAs (snRNAs; U1, U2, U4/U6, U5) and many protein factors. The snRNAs, each complexed with a number of proteins to form a small nuclear ribonucleoprotein particle (snRNP), bind to the pre-mRNA sequentially in an order of U1 and U2 and then U4/U6.U5 as a preformed trisnRNP (Brow 2002;Wahl et al. 2009;Will and L€ uhrmann 2011). Spliceosome assembly initiates with the binding of U1 to the 59 splice, forming the commitment complex (CC), followed by binding of U2 to the branch site to form the prespliceosome. Respective recognition of the 59 splice site and the branch site by U1 and U2 is mediated through RNA base-pairing interactions between the snRNAs and the intronic sequence. Prior to U2 addition, a Msl5-Mud2 heterodimer binds to the branch site and interacts with U1 snRNP, bridging the interaction between the 59 splice site and the branch site (Berglund et al. 1997(Berglund et al. , 1998Wang et al. 2008). Msl5-Mud2 is displaced from the branch site before U2 snRNA can base-pair with the branch site sequence in the formation of the prespliceosome. Following binding of the tri-snRNP, the spliceosome undergoes a major structural rearrangement to release U1 and U4 and forms new base pairs between U6 and the 59 splice site and between U2 and U6. A protein complex assoc...

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Section: Discussionsupporting

confidence: 54%

A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence

Liang

Cheng

2015

Genes Dev.

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“…(Bottom) Prp5 has been proposed to undergo a conformational change to promote splicing. The open structure (left) represents the structure determined by X-ray crystallography (pdb 4LJY) while the closed structure (right) is believed to be necessary for ATP hydrolysis and was modeled based on structures of other DEAD-box proteins (coordinates for the closed structure were obtained from Yong-Zhen Xu and Charles Query) (53). Positions of the Prp5 mutations used in this study are noted.…”

Section: Resultsmentioning

confidence: 99%

SF3b1 mutations associated with myelodysplastic syndromes alter the fidelity of branchsite selection in yeast

et al. 2017

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RNA and protein components of the spliceosome work together to identify the 5΄ splice site, the 3΄ splice site, and the branchsite (BS) of nascent pre-mRNA. SF3b1 plays a key role in recruiting the U2 snRNP to the BS. Mutations in human SF3b1 have been linked to many diseases such as myelodysplasia (MDS) and cancer. We have used SF3b1 mutations associated with MDS to interrogate the role of the yeast ortholog, Hsh155, in BS selection and splicing. These alleles change how the spliceosome recognizes the BS and alter splicing when nonconsensus nucleotides are present at the −2, −1 and +1 positions relative to the branchpoint adenosine. This indicates that changes in BS usage observed in humans with SF3b1 mutations may result from perturbation of a conserved mechanism of BS recognition. Notably, different HSH155 alleles elicit disparate effects on splicing: some increase the fidelity of BS selection while others decrease fidelity. Our data support a model wherein conformational changes in SF3b1 promote U2 association with the BS independently of the action of the DEAD-box ATPase Prp5. We propose that SF3b1 functions to stabilize weak U2/BS duplexes to drive spliceosome assembly and splicing.

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“…Therefore, DDX3 contains a cryptic second binding site for the HELICc domain on the DEAD-domain, which is present in solution and is inhibitory for function. S. cerevisiae Prp5p was also crystallized in an inhibitory conformation, and destabilization of this "twisted" state accelerated catalysis (54). It is possible that specific proteins may bind and stabilize inhibitory conformations of DEAD-box proteins to negatively regulate catalysis, as opposed to the many MIF4G domains that bind and activate catalysis (55)(56)(57)(58)(59).…”

Section: Discussionmentioning

confidence: 99%

“…However, this region forms part of a ␤-sheet in the ADP-bound structure of DDX19 (PDB 3EWS) (60). In the crystal structure of S. cerevisiae Prp5p, an NTE helix stabilizes the twisted conformation by interacting with the DEAD and HELICc domains (54). Therefore, structural plasticity at the N-terminal boundary of the DEAD domain may be a feature common to many DEAD-box proteins.…”

Section: Discussionmentioning

confidence: 99%

Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3

Floor

Condon

Sharma

et al. 2016

Journal of Biological Chemistry

112

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DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEADbox proteins.DEAD-box proteins are ATP-dependent RNA-binding proteins that remodel RNA structures and RNA-protein complexes, stably clamp RNA, and promote fluidity within RNA granules (1-3). The human DEAD-box protein DDX3 (encoded by DDX3X) and its yeast ortholog Ded1p have been implicated in numerous functions including translation initiation (4 -12). Messenger RNA molecules containing especially long or structured 5Ј leader sequences are particularly sensitive to DDX3 activity (6,7,12,13). DDX3X is frequently mutated in numerous cancer types (5), such as chronic lymphocytic leukemia (14 -16), natural killer/T-cell lymphoma (17), head and neck squamous cell carcinoma (18,19), and lung cancer (20). DDX3X is also one of the most frequently mutated genes in the highly malignant brain tumor medulloblastoma (21-24). In medulloblastoma, many mutations are predicted to inactivate DDX3, and some have been demonstrated to diminish activities in vitro (17,25).DEAD-box proteins are defined by 12 different motifs that function in ATP binding or hydrolysis and RNA binding, or couple ATP and RNA binding (1). Outside of these conserved motifs, each DEAD-box protein subfamily has unique tails that lie N-or C-terminal to the helicase core and contain elements that define the unique properties of that subfamily. For example, DDX21 has a GUCT domain in its C-terminal extension (26, 27), DDX5 has tandem P68HR domains in its C-terminal extension, and DDX43 has a KH1 domain in its N-terminal extension. However, as the tails of each DEAD-box protein subfamily are idiosyncratic, whereas the cores are very similar (28,29), it is essential to study individual subfamilies of DEAD-box proteins in detail to understand the role of subfamily-specific tails.DDX3 is a member of the Ded1/DDX...

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Crystal Structure of Prp5p Reveals Interdomain Interactions that Impact Spliceosome Assembly

Cited by 26 publications

References 57 publications

A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence

A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence

SF3b1 mutations associated with myelodysplastic syndromes alter the fidelity of branchsite selection in yeast

Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3

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