Structural analysis of the spliceosomal RNA helicase Prp28 from the thermophilic eukaryoteChaetomium thermophilum

Prp28 (pre-mRNA-splicing ATP-dependent RNA helicase 28) is a spliceosomal DEAD-box helicase which is involved in two steps of spliceosome assembly. It is required for the formation of commitment complex 2 in an ATP-independent manner as well as for the formation of the pre-catalytic spliceosome, which in contrast is ATP-dependent. During the latter step, Prp28 is crucial for the integration of the U4/U6·U5 tri-snRNP since it displaces the U1 snRNP and allows the U6 snRNP to base-pair with the 5′-splice site. Here, the crystal structure of Prp28 from the thermophilic fungusChaetomium thermophilumis reported at 3.2 Å resolution and is compared with the available structures of homologues.

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p68 RNA Helicase Is an Essential Human Splicing Factor That Acts at the U1 snRNA-5′ Splice Site Duplex

Molecular and Cellular Biology ◽

10.1128/mcb.22.15.5443-5450.2002 ◽

2002 ◽

Vol 22 (15) ◽

pp. 5443-5450 ◽

Cited By ~ 87

Author(s):

Zhi-Ren Liu

Keyword(s):

Splice Site ◽

Early Stage ◽

Rna Helicase ◽

Mrna Splicing ◽

Cross Linking ◽

Spliceosome Assembly ◽

U1 Snrna ◽

U1 Snrnp ◽

P68 Rna Helicase

ABSTRACT Modulation of the interaction between U1 snRNP and the 5′ splice site (5′ss) is a key event that governs 5′ss recognition and spliceosome assembly. Using the methylene blue-mediated cross-linking method (Z. R. Liu, A. M. Wilkie, M. J. Clemens, and C. W. Smith, RNA 2:611-621, 1996), a 65-kDa protein (p65) was shown to interact with the U1-5′ss duplex during spliceosome assembly (Z. R. Liu, B. Sargueil, and C. W. Smith, Mol. Cell. Biol. 18:6910-6920, 1998). In this report, p65 was identified as p68 RNA helicase and shown to be essential for in vitro pre-mRNA splicing. Depletion of endogenous p68 RNA helicase does not affect the loading of the U1 snRNP to the 5′ss during early stage of splicing. However, dissociation of the U1 from the 5′ss is largely inhibited. The data suggest that p68 RNA helicase functions in destabilizing the U1-5′ss interactions. Furthermore, depletion of p68 RNA helicase arrested spliceosome assembly at the prespliceosome stage, suggesting that p68 may play a role in the transition from prespliceosome to spliceosome.

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Structural and functional analysis of the RNA helicase Prp43 from the thermophilic eukaryoteChaetomium thermophilum

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15024498 ◽

2016 ◽

Vol 72 (2) ◽

pp. 112-120 ◽

Cited By ~ 18

Author(s):

Marcel J. Tauchert ◽

Jean-Baptiste Fourmann ◽

Henning Christian ◽

Reinhard Lührmann ◽

Ralf Ficner

Keyword(s):

Crystal Structure ◽

Saccharomyces Cerevisiae ◽

Functional Analysis ◽

Rna Helicase ◽

Mrna Splicing ◽

Rna Helicases ◽

Cellular Processes ◽

Chaetomium Thermophilum ◽

Rrna Maturation

RNA helicases are indispensable for all organisms in each domain of life and have implications in numerous cellular processes. The DEAH-box RNA helicase Prp43 is involved in pre-mRNA splicing as well as rRNA maturation. Here, the crystal structure ofChaetomium thermophilumPrp43 at 2.9 Å resolution is revealed. Furthermore, it is demonstrated that Prp43 fromC. thermophilumis capable of functionally replacing its orthologue fromSaccharomyces cerevisiaein spliceosomal disassembly assays.

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Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.97.24.13080 ◽

2000 ◽

Vol 97 (24) ◽

pp. 13080-13085 ◽

Cited By ~ 187

Author(s):

J. M. Caruthers ◽

E. R. Johnson ◽

D. B. McKay

Keyword(s):

Crystal Structure ◽

Rna Helicase ◽

Initiation Factor ◽

Dead Box

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Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714006439 ◽

2014 ◽

Vol 70 (6) ◽

pp. 1622-1630 ◽

Cited By ~ 14

Author(s):

Sina Möhlmann ◽

Rebecca Mathew ◽

Piotr Neumann ◽

Andreas Schmitt ◽

Reinhard Lührmann ◽

...

Keyword(s):

Binding Activity ◽

Mrna Splicing ◽

Structural Basis ◽

Atp Binding ◽

Helicase Domain ◽

Crucial Step ◽

Closed Conformation ◽

Dead Box ◽

Open Conformation ◽

U1 Snrnp

The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

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Crystal structure of the N-terminal RecA-like domain of a DEAD-box RNA helicase, the -like gene B protein

Journal of Structural Biology ◽

10.1016/j.jsb.2005.01.006 ◽

2005 ◽

Vol 150 (1) ◽

pp. 58-68 ◽

Cited By ~ 12

Author(s):

K KURIMOTO ◽

Y MUTO ◽

N OBAYASHI ◽

T TERADA ◽

M SHIROUZU ◽

...

Keyword(s):

Crystal Structure ◽

Rna Helicase ◽

Dead Box

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Structural modelling studies and immunoprophylactic potential ofBrugia malayiDEAD Box RNA helicase

Parasitology ◽

10.1017/s0031182013000322 ◽

2013 ◽

Vol 140 (8) ◽

pp. 1016-1025 ◽

Cited By ~ 1

Author(s):

MEGHNA SINGH ◽

NIDHI SHRIVASTAVA ◽

UZMA SAQIB ◽

MOHAMMAD IMRAN SIDDIQI ◽

SHAILJA MISRA-BHATTACHARYA

Keyword(s):

Adverse Outcome ◽

3D Structure ◽

Rna Helicase ◽

Mrna Splicing ◽

Oxygen Species ◽

Rna Helicases ◽

Infective Larvae ◽

Dead Box ◽

Modelling Studies ◽

Mastomys Coucha

SUMMARYDEAD Box RNA helicases are essential enzymes that are involved in RNA metabolic processes such as transcription, pre-mRNA splicing, translation initiation and RNA decay. We have previously over-expressed and biochemically characterized an immunodominant cDNA clone encoding DEAD box RNA helicase (BmL3-Helicase) isolated by immunoscreening of the larval stage cDNA library ofBrugia malayi.In the current study, the 3D structure was determined and the immunoprophylactic efficacy of BmL3-Helicase was investigated by immunizingMastomys couchawith the recombinant protein and subsequently challenging withB. malayiinfective larvae. The immunization had an adverse outcome on the establishment of challenged larvae resulting in a 67·4% reduction in adult parasite recovery, a 86·7% decrease in the microfilarial density and profound sterility of the recovered female worms. The immune response thus generated was investigated by measuring the levels of specific antibodies including IgG subclasses, reactive oxygen species and cytokines.

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Mechanism of 5ʹ splice site transfer for human spliceosome activation

Science ◽

10.1126/science.aax3289 ◽

2019 ◽

Vol 364 (6438) ◽

pp. 362-367 ◽

Cited By ~ 30

Author(s):

Clément Charenton ◽

Max E. Wilkinson ◽

Kiyoshi Nagai

Keyword(s):

Splice Site ◽

Messenger Rna ◽

Catalytic Center ◽

U6 Snrna ◽

Dead Box ◽

Cryo Electron Microscopy ◽

Branch Point Sequence ◽

U1 Snrnp ◽

Spliceosome Activation ◽

Small Nuclear Ribonucleoproteins

The prespliceosome, comprising U1 and U2 small nuclear ribonucleoproteins (snRNPs) bound to the precursor messenger RNA 5ʹ splice site (5ʹSS) and branch point sequence, associates with the U4/U6.U5 tri-snRNP to form the fully assembled precatalytic pre–B spliceosome. Here, we report cryo–electron microscopy structures of the human pre–B complex captured before U1 snRNP dissociation at 3.3-angstrom core resolution and the human tri-snRNP at 2.9-angstrom resolution. U1 snRNP inserts the 5ʹSS–U1 snRNA helix between the two RecA domains of the Prp28 DEAD-box helicase. Adenosine 5ʹ-triphosphate–dependent closure of the Prp28 RecA domains releases the 5ʹSS to pair with the nearby U6 ACAGAGA-box sequence presented as a mobile loop. The structures suggest that formation of the 5ʹSS-ACAGAGA helix triggers remodeling of an intricate protein-RNA network to induce Brr2 helicase relocation to its loading sequence in U4 snRNA, enabling Brr2 to unwind the U4/U6 snRNA duplex to allow U6 snRNA to form the catalytic center of the spliceosome.

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