Genetic interactions of conserved regions in the DEAD-box protein Prp28p

Latus, Lori J.; Liu, Zheng; Abbott, J. M.

doi:10.1093/nar/25.24.5033

Cited by 31 publications

(23 citation statements)

References 41 publications

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“…Substitution of this Phe in the yeast DEAD box splicing factor Prp28 by glycine or serine elicited ts and cs growth phenotypes (52). We show here that the Prp22 mutant F697A grows at 37°C but is inviable at 30°C.…”

Section: Discussionmentioning

confidence: 70%

Motifs IV and V in the DEAH Box Splicing Factor Prp22 Are Important for RNA Unwinding, and Helicase-defective Prp22 Mutants Are Suppressed by Prp8

Schneider

Campodonico

Schwer

2004

Journal of Biological Chemistry

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The yeast pre-mRNA splicing factor Prp22 is a member of the DEAH box family of nucleic acid-stimulated ATPases and RNA helicases. Here we report a mutational analysis of 16 conserved residues in motifs Ia ( 534 TQPRRVAA 541 ), IV ( 695 LVFLTG 700 ), and V ( 757 TNI-AETSIT 765 ). Mutants T757A, I764A, and T765A were lethal, and F697A cells did not grow at <30°C. The mutant proteins failed to catalyze mRNA release from the spliceosome in vitro, and they were deficient for RNA unwinding. The F697A, I764A, and T765A proteins were active for ATP hydrolysis in the presence of RNA cofactor. The T757A mutant retained basal ATPase activity but was not stimulated by RNA, whereas ATP hydrolysis by T765A was strictly dependent on the RNA cofactor. Thus Thr-757 and Thr-765 in motif V link ATP hydrolysis to the RNA cofactor. To illuminate the mechanism of Prp22-catalyzed mRNA release, we performed a genetic screen to identify extragenic suppressors of the coldsensitive growth defect of a helicase/release-defective Prp22 mutant. We identified one of the suppressors as a missense mutation of PRP8 (R1753K), a protein component of the U5 small nuclear ribonucleoprotein. We show that PRP8-R1753K suppressed multiple helicasedeficient prp22 mutations, including the lethal I764A mutation. Replacing Arg-1753 of Prp8 by either Lys, Ala, Gln, or Glu resulted in suppression of helicase-defective Prp22 mutants. Prp8-Arg1753 mutations by themselves caused temperature-sensitive growth defects in a PRP22 strain. These findings suggest a model whereby Prp22 disrupts an RNA/protein or RNA/RNA interaction in the spliceosome that is normally stabilized by Prp8.Genetic and biochemical analyses implicate at least one member of the DEX(H/D) box protein family in each of the ATP-dependent steps of pre-mRNA splicing (1, 2). Recognition of the 5Ј splice site by U1 and U6 snRNA 1 depends on the DEX(H/D) proteins Prp28 and Brr2, and the association of U2 small nuclear ribonucleoprotein with the branch site is assisted by the DEXD box proteins . Once the spliceosome is assembled, ATP hydrolysis by the DEAH proteins Prp2 and Prp16 drives the first and second transesterification steps, respectively (10, 11). The spliced mRNA and excised lariat-intron products are then released sequentially from the spliceosome by the DEAH proteins Prp22 and Prp43 (12-16). How the DEX(H/D) proteins use ATP to remodel interactions in the spliceosome is not understood, and the exact targets within the spliceosome that are subject to rearrangement by specific DEX(H/D) proteins are not known.DEX(H/D) box proteins are defined by seven colinear peptide motifs (17-19). The conserved residues in motifs I (GKT), II (DEX(H/D)), and VI (QRXGRXGR) are important for catalysis and likely compose the active site for NTP hydrolysis (20, 21). Motif III ((S/T)AT) couples ATP hydrolysis to RNA unwinding in the exemplary DEX(H/D) helicases eIF-4A, NPHII, and Prp22 (22-24). The roles of motifs Ia, IV, and V are less well understood.Many of the DEX(H/D) splicing factors hydrolyze ATP in a manner...

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Section: Discussionmentioning

confidence: 70%

Motifs IV and V in the DEAH Box Splicing Factor Prp22 Are Important for RNA Unwinding, and Helicase-defective Prp22 Mutants Are Suppressed by Prp8

Schneider

Campodonico

Schwer

2004

Journal of Biological Chemistry

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“…In the yeast DEXD/H-box splicing factor Prp28p, mutation of Arg-264 in motif Ia ( 262 PTRELA 267 ) to Glu or Asp results in lethality (49), suggesting that a positive charge may be important at this position. However, a change of the adjacent residue Glu-265 (from Glu to Gln) can be tolerated to a certain extent.…”

Section: Discussionmentioning

confidence: 99%

Insight into the Roles of Helicase Motif Ia by Characterizing Fanconi Anemia Group J Protein (FANCJ) Patient Mutations

Guo

Vidhyasagar

Ding

et al. 2014

Journal of Biological Chemistry

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Background: Two Fanconi anemia patient missense mutations are localized in FANCJ helicase motif Ia. Results: Mutant R251C impairs the DNA binding ability of FANCJ; Q255H uncouples DNA translocation from helicase activity. Conclusion: Helicase motif Ia plays critical roles in FANCJ enzymatic activity and DNA repair. Significance: Helicase motif Ia is involved not only in nucleic acid binding but also ATP binding and coupling ATP hydrolysis to unwinding.

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“…Furthermore, it has been suggested that motif Ia may also be involved in the structural rearrangements that take place during ATP binding and hydrolysis (Schwer and Meszaros 2000;Cordin et al 2006), a concept substantiated by the discovery of interactions between the first residues of motif Ia (PT) and the glutamate of the DEAD motif (Story et al 2001;Zhao et al 2004). In the few studies that have examined motif Ia in bona fide DEAD/ DEAH-box RNA helicases, such as eIF4A (Svitkin et al 2001), Prp28 (Chang et al 1997), Prp43 (Tanaka and Schwer 2006), and the Vaccinia virus NPH-II DExHbox helicase (Gross and Shuman 1998), mutation of the motif often abrogated ATP hydrolysis, RNA binding, and RNA helicase activities in vitro, with concomitant lethal, cold-sensitive/temperaturesensitive, and/or dominant-negative phenotypes in vivo. Similarly, in human eIF4AIII, motif Ia is critical for exon junction complex formation and nonsense-mediated mRNA decay (Shibuya et al 2006).…”

Section: Discussionmentioning

confidence: 99%

The DEAD-box RNA helicase-like Utp25 is an SSU processome component

Charette

Baserga²

2010

RNA

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The SSU processome is a large ribonucleoprotein complex consisting of the U3 snoRNA and at least 43 proteins. A database search, initiated in an effort to discover additional SSU processome components, identified the uncharacterized, conserved and essential yeast nucleolar protein YIL091C/UTP25 as one such candidate. The C-terminal DUF1253 motif, a domain of unknown function, displays limited sequence similarity to DEAD-box RNA helicases. In the absence of the conserved DEAD-box sequence, motif Ia is the only clearly identifiable helicase element. Since the yeast homolog is nucleolar and interacts with components of the SSU processome, we examined its role in pre-rRNA processing. Genetic depletion of Utp25 resulted in slowed growth. Northern analysis of pre-rRNA revealed an 18S rRNA maturation defect at sites A 0 , A 1 , and A 2 . Coimmunoprecipitation confirmed association with U3 snoRNA and with Mpp10, and with components of the t-Utp/UtpA, UtpB, and U3 snoRNP subcomplexes. Mutation of the conserved motif Ia residues resulted in no discernable temperaturesensitive or cold-sensitive growth defects, implying that this motif is dispensable for Utp25 function. A yeast two-hybrid screen of Utp25 against other SSU processome components revealed several interacting proteins, including Mpp10, Utp3, and Utp21, thereby identifying the first interactions among the different subcomplexes of the SSU processome. Furthermore, the DUF1253 domain is required and sufficient for the interaction of Utp25 with Utp3. Thus, Utp25 is a novel SSU processome component that, along with Utp3, forms the first identified interactions among the different SSU processome subcomplexes.

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Genetic interactions of conserved regions in the DEAD-box protein Prp28p

Cited by 31 publications

References 41 publications

Motifs IV and V in the DEAH Box Splicing Factor Prp22 Are Important for RNA Unwinding, and Helicase-defective Prp22 Mutants Are Suppressed by Prp8

Motifs IV and V in the DEAH Box Splicing Factor Prp22 Are Important for RNA Unwinding, and Helicase-defective Prp22 Mutants Are Suppressed by Prp8

Insight into the Roles of Helicase Motif Ia by Characterizing Fanconi Anemia Group J Protein (FANCJ) Patient Mutations

The DEAD-box RNA helicase-like Utp25 is an SSU processome component

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