Mutational analysis identifies two separable roles of the Saccharomyces cerevisiae splicing factor Prp18

Bačíková, Dagmar; Horowitz, David S.

doi:10.1017/s1355838202023099

Cited by 21 publications

(8 citation statements)

References 29 publications

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“…Conformational rearrangement, induced by the ATPase Prp16, primes the 5′-exon and 3′SS for the initiation of the second step splicing (35)(36)(37). With the help of the splicing factors Slu7, Cdc40 (Prp17 in S. cerevisiae), Prp18, and the ATPase Prp22, the Step II reaction is completed, forming the post-catalytic spliceosome (P complex) (38)(39)(40)(41)(42)(43)(44)(45). We speculate that the central components of the spliceosomal catalytic center -ISL of U6 snRNA, Helix I of the U2/U6 duplex, and Loop I of U5 snRNA -maintain a generally similar conformation in the B act , B*, C, and P complexes (Fig.…”

Section: Discussionmentioning

confidence: 99%

Structural basis of pre-mRNA splicing

Hang

Wan

Yan

et al. 2015

Science

180

294

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Splicing of precursor messenger RNA is performed by the spliceosome. In the cryogenic electron microscopy structure of the yeast spliceosome, U5 small nuclear ribonucleoprotein acts as a central scaffold onto which U6 and U2 small nuclear RNAs (snRNAs) are intertwined to form a catalytic center next to Loop I of U5 snRNA. Magnesium ions are coordinated by conserved nucleotides in U6 snRNA. The intron lariat is held in place through base-pairing interactions with both U2 and U6 snRNAs, leaving the variable-length middle portion on the solvent-accessible surface of the catalytic center. The protein components of the spliceosome anchor both 5' and 3' ends of the U2 and U6 snRNAs away from the active site, direct the RNA sequences, and allow sufficient flexibility between the ends and the catalytic center. Thus, the spliceosome is in essence a protein-directed ribozyme, with the protein components essential for the delivery of critical RNA molecules into close proximity of one another at the right time for the splicing reaction.

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Section: Discussionmentioning

confidence: 99%

Structural basis of pre-mRNA splicing

Hang

Wan

Yan

et al. 2015

Science

180

294

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“…Specifically, genetic and mutagenesis studies have demonstrated that the carboxy-terminal domain interacts with the protein Slu7 at a weakly conserved face of the domain, and with the protein component of U5 snRNP through the highly conserved loop region linking helices D and E [34, 35]. PRP18 is thought to orient U5 snRNP in a manner that aligns an RNA loop of U5 snRNP with the exon junction; PRP18 thereby guides a specific RNA-RNA interaction through direct protein-protein interactions.…”

Section: Discussionmentioning

confidence: 99%

Structure and Function of Steroid Receptor RNA Activator Protein, the Proposed Partner of SRA Noncoding RNA

McKay

Barthel

et al. 2014

Journal of Molecular Biology

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In a widely accepted model, the steroid receptor RNA activator protein (SRA protein; SRAP) modulates the transcriptional regulatory activity of SRA RNA by binding a specific stem-loop of SRA. We first confirmed that SRAP is present in the nucleus as well as the cytoplasm of MCF-7 breast cancer cells, where it is expressed at the level of about 105 molecules/cell. However, our SRAP-RNA binding experiments, both in vitro with recombinant protein and in cultured cells with plasmid-expressed protein and RNA, did not reveal a specific interaction between SRAP and SRA. We determined the crystal structure of the carboxy-terminal domain of human SRAP and found that it does not have the postulated RRM (RNA recognition motif). The structure is a five-helix bundle that is distinct from known RNA-binding motifs and instead is similar to the carboxy-terminal domain of the yeast spliceosome protein PRP18, which stabilizes specific protein-protein interactions within a multisubunit mRNA splicing complex. SRA binding experiments with this domain gave negative results. Transcriptional regulation by SRA/SRAP was examined with siRNA knockdown. Effects on both specific estrogen-responsive genes and genes identified by RNA-seq as candidates for regulation were examined in MCF-7 cells. Only a small effect (~20% change) on one gene resulting from depletion of SRA/SRAP could be confirmed. We conclude that the current model for SRAP function must be re-evaluated; we suggest SRAP may function in a different context to stabilize specific intermolecular interactions in the nucleus.

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“…PRP18 is an evolutionarily conserved protein, with orthologs found in other plants, yeasts and metazoans ( Figure S1 and Figure S2 ). Prp18 proteins typically have five alpha-helices separated by four loops, with a highly conserved loop between the fourth and fifth helices ( Bacíková and Horowitz 2002 ; Annamalai 2011 ). The A334V mutation we identified in the prp18a-1 mutant is in this highly conserved loop region ( Figure 3 ).…”

Section: Resultsmentioning

confidence: 99%

“…PRP18 is not essential in budding yeast but cells lacking this protein grow slowly and are temperature sensitive ( Bacíková and Horowitz 2002 ). Whether PRP18a is essential in Arabidopsis is unclear.…”

Section: Discussionmentioning

confidence: 99%

A Genetic Screen Identifies PRP18a, a Putative Second Step Splicing Factor Important for Alternative Splicing and a Normal Phenotype inArabidopsis thaliana

Kanno

Lin

Chang

et al. 2018

G3 Genes|Genomes|Genetics

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Splicing of pre-mRNA involves two consecutive trans-esterification steps that take place in the spliceosome, a large dynamic ribonucleoprotein complex situated in the nucleus. In addition to core spliceosomal proteins, each catalytic step requires step-specific factors. Although the Arabidopsis thaliana genome encodes around 430 predicted splicing factors, functional information about these proteins is limited. In a forward genetic screen based on an alternatively-spliced GFP reporter gene in Arabidopsis thaliana, we identified a mutant impaired in putative step II factor PRP18a, which has not yet been investigated for its role in pre-mRNA splicing in plants. Step II entails cleavage at the 3′ splice site accompanied by ligation of the 5′ and 3′ exons and intron removal. In the prp18 mutant, splicing of a U2-type intron with non-canonical AT-AC splice sites in GFP pre-mRNA is reduced while splicing of a canonical GT-AG intron is enhanced, resulting in decreased levels of translatable GFP mRNA and GFP protein. These findings suggest that wild-type PRP18a may in some cases promote splicing at weak, non-canonical splice sites. Analysis of genome-wide changes in alternative splicing in the prp18a mutant identified numerous cases of intron retention and a preponderance of altered 3′ splice sites, suggesting an influence of PRP18a on 3′ splice site selection. The prp18a mutant featured short roots on synthetic medium and small siliques, illustrating that wild-type PRP18a function is needed for a normal phenotype. Our study expands knowledge of plant splicing factors and provides foundational information and resources for further functional studies of PRP18 proteins in plants.

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Mutational analysis identifies two separable roles of the Saccharomyces cerevisiae splicing factor Prp18

Cited by 21 publications

References 29 publications

Structural basis of pre-mRNA splicing

Structural basis of pre-mRNA splicing

Structure and Function of Steroid Receptor RNA Activator Protein, the Proposed Partner of SRA Noncoding RNA

A Genetic Screen Identifies PRP18a, a Putative Second Step Splicing Factor Important for Alternative Splicing and a Normal Phenotype inArabidopsis thaliana

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