Recombinant Saccharomyces cerevisiae Pre-mRNA-splicing factor 18 (PRP18)

What is PRP18 and what is its primary function in Saccharomyces cerevisiae?

PRP18 is a U5 snRNP-associated protein involved in pre-mRNA splicing in Saccharomyces cerevisiae. Its primary function is to stabilize the interaction between exons and the spliceosome during the second step of splicing, which involves exon ligation after intron removal. PRP18 specifically helps maintain the proper alignment of exons with loop 1 of the U5 snRNA, facilitating the joining of exons during the catalytic reaction . This stabilization is particularly important when the exonic sequences at splice junctions are suboptimal, demonstrating PRP18's critical role in ensuring splicing fidelity across diverse transcript sequences .

How conserved is PRP18 across different yeast species and humans?

PRP18 shows significant conservation across fungal and mammalian species, particularly in its C-terminal region. Comparative analysis reveals that S. pombe SpPrp18 shares 35% identity and 58% similarity with S. cerevisiae ScPrp18, with a similar degree of relatedness to human hPrp18 . The most conserved element is the C-terminal five-helical bundle structure with a highly conserved region (CR) loop between helices 4 and 5 . Notably, both S. pombe SpPrp18 and human hPrp18 contain a splicing factor motif in their N-terminal regions that is absent in S. cerevisiae ScPrp18, suggesting evolutionary divergence in certain functional domains .

What is the structural organization of PRP18 protein?

PRP18 is characterized by a distinctive structural organization:

• The C-terminal region forms a five-helix bundle, which is the most conserved part of the protein across species

• Between helices 4 and 5 lies a highly conserved region (CR) loop that is crucial for function

• The N-terminal region is less conserved, with S. cerevisiae PRP18 lacking the splicing factor motif found in S. pombe and human orthologs

• Studies with truncated versions have shown that an N-terminally truncated ScPrp18 (ScPrp18Δ79) lacking 79 residues remains functional for in vitro splicing

The protein's crystal structure (at least for the truncated version) reveals a compact five-helix bundle fold that serves as the functional core for interactions with other splicing components .

Is PRP18 essential for viability in S. cerevisiae?

Unlike many splicing factors, PRP18 is not essential for viability in S. cerevisiae. Deletion mutants (prp18Δ) are viable but exhibit temperature-sensitive growth, with cells showing arrested growth at elevated temperatures . This temperature sensitivity correlates with a splicing defect, specifically in the second step of pre-mRNA splicing both in vivo and in vitro . The non-essential nature of PRP18 in budding yeast contrasts with its potentially more critical role in other organisms, making S. cerevisiae an excellent model system for studying PRP18 function without the complications of lethality.

What phenotypic consequences result from PRP18 mutations?

Mutation or deletion of PRP18 leads to several distinct phenotypes:

• Temperature sensitivity: prp18Δ cells in S. cerevisiae show growth defects at elevated temperatures

• Splicing defects: Accumulation of splicing intermediates occurs, specifically those involved in the second step of splicing

• Exon sequence sensitivity: In yeast expressing PRP18 lacking its conserved region (prp18ΔCR), splicing becomes highly dependent on the exonic bases at splice junctions

• Cell cycle effects: In S. pombe, PRP18 depletion causes cell cycle arrest before S phase, indicating a role in regulating cell cycle progression through its effect on splicing specific transcripts involved in G1-S transition

How does the conserved region of PRP18 contribute to splicing efficiency?

The conserved region (CR) of PRP18 plays a critical role in stabilizing the interaction between exons and the U5 snRNA during the second step of splicing. Experimental evidence demonstrates that when the CR is deleted (prp18ΔCR), splicing becomes highly dependent on the sequence of exonic bases near splice junctions .

Mechanistically, the CR helps stabilize the interaction of exon ends with loop 1 of U5 snRNA. In prp18ΔCR yeast, only pre-mRNAs with optimal base-pairing potential with U5 snRNA can be efficiently spliced. Specifically:

• At the 3' end of exon 1, position -1 shows a strong preference for adenosine (A) in prp18ΔCR mutants, with 100% of efficiently spliced transcripts containing A at this position

• At the 5' end of exon 2, position +1 also strongly favors adenosine

• These preferences correlate with potential base-pairing to U residues in loop 1 of U5 snRNA

This suggests a model where PRP18's conserved region functions to compensate for suboptimal exon sequences, allowing the spliceosome to process a wider variety of transcripts efficiently .

What experimental approaches can be used to study PRP18 function in splicing?

Several complementary experimental approaches have proven effective for studying PRP18 function:

• Genetic manipulation systems:

  • Creation of deletion mutants (prp18Δ)

  • Generation of point mutations in conserved regions (e.g., prp18-5 with V194R mutation)

  • Construction of conditional mutants using regulatable promoters like nmt81 in S. pombe

• In vitro splicing assays:

  • Cell-free splicing extracts from wild-type and mutant strains

  • Use of model pre-mRNA substrates to analyze step-specific defects

  • Immunodepletion of PRP18 from splicing extracts to assess function

• Exon sequence libraries:

  • Random mutagenesis of exonic sequences at splice junctions

  • Screening using reporter systems like ACT1-CUP1 where copper resistance indicates splicing efficiency

  • Analysis of sequence preferences in wild-type versus mutant backgrounds

• Structural biology approaches:

  • Crystal structure determination of truncated proteins

  • Homology modeling for comparative structural analysis

• Global splicing analysis:

  • RNA-seq to identify intron-specific splicing defects

  • Primer extension assays to detect accumulation of specific splicing intermediates

How does recombinant PRP18 interact with other splicing factors?

Recombinant PRP18 participates in a network of protein-protein interactions that are essential for its function in the spliceosome:

• Interaction with Slu7: The N-terminal region of S. cerevisiae PRP18, specifically helices 1 and 2, mediates critical interactions with the splicing factor Slu7 . This interaction is required for proper spliceosomal association of PRP18.

• Association with U5 snRNP: PRP18 associates with the U5 snRNP complex. The globular domain of PRP18 is involved in stabilizing U5 snRNA interactions with exonic sequences after the first catalytic reaction of splicing .

• Species-specific interactions: The interaction patterns appear to differ between species. For example, human PRP18 (hPrp18) cannot rescue the temperature-sensitive phenotype of S. cerevisiae prp18Δ cells, suggesting differences in spliceosomal associations between these orthologs .

• Functional coordination with other factors: Genetic interaction studies show connections between PRP18 and early-acting splicing factors. For instance, the S. pombe prp18-5 mutant shows genetic interaction with the spppr2-1 mutant, which affects the early-acting U2AF59 protein, suggesting functional coordination between early and late steps of splicing .

What is the relationship between PRP18 and cell cycle regulation?

Research in fission yeast has revealed an unexpected connection between PRP18 function and cell cycle progression:

• SpPrp18 depletion causes cell cycle arrest specifically before S phase (G1 arrest)

• The mechanism involves compromised splicing of transcripts coding for key G1-S regulators, including:

  • Res2, a transcription factor involved in cell cycle progression

  • Skp1, a factor involved in regulated proteolysis

• This contrasts with other splicing factor mutations in S. pombe that typically affect the G2-M transition, suggesting a specific role for PRP18 in G1-S regulation

• The evidence supports a model where intron-specific effects of PRP18 on splicing efficiency lead to cumulative impacts on cell cycle regulatory networks

This relationship highlights how specificity in splicing factor function can translate into distinct cellular phenotypes and demonstrates the integration of splicing regulation with cell cycle control pathways.

How do exon sequences influence splicing efficiency in PRP18-deficient cells?

Exon sequences at splice junctions have profound effects on splicing efficiency in PRP18-deficient cells, revealing key insights into the normal function of PRP18:

• In prp18ΔCR yeast (lacking the conserved region), exonic bases near splice junctions critically determine splicing efficiency

• Position preferences in prp18ΔCR mutants include:

  • Strong preference for adenosine at position -1 (last base of exon 1)

  • Strong preference for adenosine at position +1 (first base of exon 2)

• These preferences correlate with the potential for base-pairing with U residues in loop 1 of U5 snRNA

• Experimental analysis using the ACT1-CUP1 reporter system and randomized exon libraries confirmed these sequence preferences

The data from these experiments supports a revised model of exon-U5 interactions where the exons are arranged in a continuous double helix that facilitates the second reaction of splicing .

What methods can be used to generate conditional PRP18 mutants for functional studies?

Several effective strategies have been developed for creating conditional PRP18 mutants:

• Promoter replacement approach:

  • Integration of regulatable promoters like nmt81 (no message in thiamine)

  • Expression can be controlled by adding or removing thiamine from the media

  • Example: S. pombe strains spprp18Δ leu1::Pnmt:prp18+ and spprp18Δ leu1::Pnmt:prp18V194R

• Temperature-sensitive alleles:

  • Exploitation of the natural temperature sensitivity of prp18Δ in S. cerevisiae

  • Creation of point mutations that confer temperature sensitivity

  • Example: S. pombe prp18-5 missense mutant with V194R mutation

• Selective mutation of functional domains:

  • Targeted mutagenesis of the conserved region (CR) between helices 4 and 5

  • Example: prp18ΔCR mutant lacking the conserved region

  • Triple alanine scanning mutants (e.g., G196A/V197A/T198A)

The table below summarizes results from genetic analyses using conditional PRP18 mutants in S. pombe:

What experimental evidence supports PRP18's role in the second step of splicing?

Multiple lines of experimental evidence firmly establish PRP18's role in the second step of pre-mRNA splicing:

• Accumulation of specific splicing intermediates:

  • In PRP18-deficient cells, primer extension analyses show accumulated pre-mRNA

  • Notably, lariat intron-exon 2 splicing intermediates become undetectable, indicating a block before or during the second catalytic step

• In vitro splicing assays:

  • Extracts from prp18Δ cells are defective in the second step of splicing

  • Similar results are seen when human PRP18 is immunodepleted from HeLa cell extracts

• Genetic interaction studies:

  • PRP18 mutations show genetic interactions with other second-step splicing factors

  • They also interact with some early-acting factors, suggesting coordination between splicing steps

• Substrate specificity experiments:

  • The exon sequence requirements in prp18ΔCR mutants specifically affect the second step

  • Similar sequence preferences affect the inefficient splicing of AT-AC pre-mRNAs by wild-type spliceosomes, indicating that these rules apply to the normal second-step mechanism

This body of evidence collectively demonstrates that PRP18 functions primarily in facilitating the second catalytic step of pre-mRNA splicing by stabilizing the interaction of exons with the spliceosome.

What expression systems are optimal for producing functional recombinant PRP18?

For producing functional recombinant PRP18, several expression systems have been successfully employed:

• Yeast expression systems:

  • Endogenous expression in S. cerevisiae using native or regulatable promoters

  • Expression in S. pombe using nmt-based promoters of varying strengths (nmt1, nmt41, nmt81)

  • Integration at specific loci (e.g., leu1) for stable expression

• Plasmid-based approaches:

  • Use of pREP series vectors for S. pombe expression (e.g., pREP42HA, pREP41MH)

  • Implementation of epitope tags (HA, MH) for detection and purification

• Truncated constructs:

  • Expression of functional N-terminally truncated versions (e.g., ScPrp18Δ79)

  • These constructs retain activity and are easier to work with for structural studies

Researchers should consider that wild-type and mutant Prp18 proteins show differences in post-translational modifications, as evidenced by the detection of a slower migrating species (~3-4 kDa increase) in immunoblotting of wild-type SpPrp18 protein .

What approaches can be used to identify intron-specific effects of PRP18 mutations?

To identify intron-specific effects of PRP18 mutations, researchers can employ several complementary approaches:

• Global splicing analysis:

  • RNA-seq of wild-type and PRP18-depleted or mutant cells

  • Comparison of splicing efficiency across different introns

  • Identification of characteristic features of affected introns

• Reporter-based assays:

  • ACT1-CUP1 reporter system where copper resistance indicates splicing efficiency

  • Construction of libraries with randomized exon sequences at splice junctions

  • Screening for sequences that enhance or reduce splicing in PRP18-deficient backgrounds

• Transcript-specific analyses:

  • RT-PCR of specific transcripts known to contain multiple introns

  • Primer extension assays to detect splicing intermediates

  • Analysis of splicing in cells lacking both PRP18 and Dbr1 (lariat debranching enzyme) to stabilize lariat intermediates

• Functional correlation:

  • Linking splicing defects to phenotypic outcomes

  • For example, correlating G1 arrest with compromised splicing of transcripts coding for G1-S regulators

These approaches have revealed that PRP18 depletion causes widespread but intron-specific splicing defects, with particular impact on transcripts involved in cell cycle regulation.

How do the functions of PRP18 differ between yeast and mammalian systems?

PRP18 shows both conserved and divergent functions between yeast and mammalian systems:

• Conserved functions:

  • Both yeast and human PRP18 function in the second step of pre-mRNA splicing

  • The C-terminal five-helix bundle structure is conserved across species

  • Both interact with components of the U5 snRNP

• Divergent aspects:

  • Human PRP18 (hPrp18) contains an N-terminal splicing factor motif that is absent in S. cerevisiae ScPrp18 but present in S. pombe SpPrp18

  • hPrp18 cannot rescue the temperature-sensitive phenotype of S. cerevisiae prp18Δ cells, suggesting differences in spliceosomal associations

  • Interaction networks may differ, as evidenced by the inability of human PRP18 to functionally replace yeast PRP18 despite their shared role in the second step of splicing

• Context-dependent requirements:

  • The essentiality of PRP18 may differ between organisms based on their splicing complexity

  • S. pombe, with its higher number of introns per gene and more degenerate splice sites, may rely more heavily on PRP18 function than S. cerevisiae

These differences reflect the evolutionary adaptation of the splicing machinery to the distinct splicing landscapes of different organisms.

What structural features of the PRP18 conserved region are critical for function?

The conserved region (CR) of PRP18, located between helices 4 and 5 of the C-terminal domain, contains several critical structural features that determine its function:

• Specific amino acid residues:

  • Targeted mutagenesis has identified key residues within the CR

  • The G196A/V197A/T198A triple mutation in S. pombe PRP18 eliminates function

  • The V194R mutation (prp18-5) creates a conditional allele with temperature sensitivity

• Conformational characteristics:

  • The CR forms a loop structure between helices 4 and 5

  • This conformation is likely important for proper positioning within the spliceosome

  • Changes that alter the loop structure disrupt function

• Surface exposure:

  • The conserved residues are likely exposed on the protein surface

  • This positioning facilitates interactions with other splicing components

  • The loop may directly contact the U5 snRNA or exonic sequences

• Helix 5 importance:

  • Studies have shown that helix 5, adjacent to the CR, also plays a critical role in function

  • The entire helical bundle provides a structural framework positioning the CR correctly

Structural models based on homology to ScPrp18 have been valuable for predicting functional consequences of mutations in the conserved region of PRP18 orthologs .

Can PRP18 be used as a tool for manipulating splicing in biotechnology applications?

PRP18's specific role in splicing suggests several potential biotechnology applications:

• Controlled gene expression systems:

  • Conditional PRP18 mutants could be used to regulate splicing of specific transcripts

  • This would allow temporal control of gene expression for transcripts with introns matching the sequence preferences of PRP18-dependent splicing

• Splicing enhancement for difficult-to-express genes:

  • Overexpression of PRP18 or engineered variants might improve splicing efficiency

  • This could be particularly valuable for heterologous expression of intron-containing genes

• Cell cycle synchronization tools:

  • Given PRP18's role in cell cycle regulation in S. pombe, conditional mutants could serve as tools for synchronizing cell populations at G1

  • This would provide an alternative to chemical synchronization methods

• Synthetic biology applications:

  • Understanding the sequence rules for PRP18-dependent splicing could inform the design of synthetic introns

  • These could serve as regulatable elements in engineered genetic circuits

• Structure-guided engineering:

  • The detailed structural understanding of PRP18 could allow engineering of variants with altered specificity

  • Such variants might be useful for manipulating alternative splicing patterns