Recombinant Candida glabrata Pre-mRNA-processing ATP-dependent RNA helicase PRP5 (PRP5), partial

What is the fundamental function of Candida glabrata PRP5 in pre-mRNA processing?

PRP5 in Candida glabrata functions as a critical DEAD-box RNA helicase that facilitates prespliceosome assembly through two primary mechanisms. First, it performs ATP-dependent remodeling of U2 snRNP, causing conformational changes that enable branch site recognition. Second, it provides ATP-independent stabilization by binding U2 snRNA near the branchpoint-interacting stem-loop (BSL), promoting proper spliceosome assembly. These dual functions are essential for the initial stages of the splicing reaction, particularly in the formation of the prespliceosome where U2 snRNP must interact with the pre-mRNA branch site. The protein's role in splicing fidelity makes it particularly important for understanding post-transcriptional regulation in this pathogenic yeast species.

How does PRP5 associate with the spliceosome during pre-mRNA processing?

PRP5 binds to the spliceosome specifically in association with U2 snRNP rather than binding independently to pre-mRNA. Experimental evidence from U2-depletion studies has demonstrated that when U2 snRNA is depleted by oligo-directed RNase H cleavage (>70% depletion), PRP5 association with the spliceosome is dramatically reduced . This indicates that PRP5 requires the presence of U2 snRNP for stable spliceosome binding. The mechanism involves PRP5 interacting with regions on or near the branchpoint-interacting stem-loop (BSL) structure in U2 snRNA, which helps stabilize U2 snRNP's association with pre-mRNA during the critical initial stages of the splicing process . This U2-dependent binding mechanism suggests that PRP5 acts on U2 snRNP to facilitate its proper interaction with the branch site sequence rather than independently recruiting U2 to the spliceosome.

What evidence supports PRP5's dual ATP-dependent and ATP-independent functions?

Multiple experimental approaches have confirmed PRP5's dual functionality. In vitro studies have demonstrated that PRP5 exhibits an ATP-dependent role in remodeling U2 snRNP structure, enabling proper branch site recognition . When ATP-deficient mutants of PRP5 are analyzed, they show impaired U2 snRNP remodeling and delayed spliceosome assembly. Concurrently, evidence for an ATP-independent function comes from binding studies showing that PRP5 directly interacts with U2 snRNA near the branchpoint-interacting stem-loop (BSL) even in the absence of ATP hydrolysis . This binding stabilizes the BSL during prespliceosome formation, facilitating proper U2-branch site interactions. The complementary nature of these two functions ensures both the dynamic restructuring and stable positioning of U2 snRNP needed for accurate splicing initiation.

What molecular interactions occur during PRP5-mediated U2 snRNP remodeling?

The ATP-dependent remodeling of U2 snRNP by PRP5 involves specific molecular rearrangements. PRP5 binds to regions on or near the branchpoint-interacting stem-loop (BSL) structure in U2 snRNA . This interaction triggers ATP-dependent conformational changes in the U2 snRNP structure that expose the branch site recognition sequence within U2 snRNA. During this process, PRP5 unwinds certain RNA structures to facilitate access to binding sites necessary for U2 snRNP's proper positioning at the branch site. These structural rearrangements enable the nucleotides in U2 snRNA to base-pair with the conserved branch site sequence in the pre-mRNA, particularly with the critical branch point adenosine . This precise molecular remodeling requires PRP5's ATPase activity, and mutations affecting this activity disrupt the remodeling process and impair prespliceosome formation .

How do PRP5 mutations affect branch site recognition and splicing efficiency?

PRP5 mutations create distinct effects on branch site recognition and splicing efficiency depending on which functional domain is affected. Mutations in PRP5 that suppress branch site defects typically operate by weakening PRP5's interaction with U2 snRNP . These suppressor mutations reduce the stable association of PRP5 with the spliceosome, which paradoxically allows more tri-snRNP to be recruited even in the presence of branch site mutations . Experimental evidence shows that the affinity of PRP5 mutants with the spliceosome negatively correlates with suppression efficiency, meaning weaker binding leads to better suppression of branch site defects . Importantly, this suppression mechanism is separate from PRP5's ATPase activity, as the suppression efficiency does not correlate with changes in ATPase function . These findings demonstrate that PRP5 mutations can bypass the normal proofreading function, allowing progression of defective spliceosomes that would typically be blocked, thus revealing the delicate balance between splicing fidelity and efficiency.

What experimental designs can effectively assess PRP5's role in prespliceosome formation?

To effectively study PRP5's role in prespliceosome formation, researchers should implement a multi-faceted experimental approach. In vitro splicing assays using depleted extracts provide a powerful methodology, as demonstrated in studies where Sad1-depleted extracts were used to isolate prespliceosome formation from later splicing steps . This approach revealed that PRP5 is not required after the prespliceosome stage, as adding Sad1 alone was sufficient to restore splicing activity in extracts depleted of both Sad1 and PRP5 . For analyzing PRP5's interaction with spliceosomes, immunoprecipitation studies using tagged PRP5 (such as Prp5-V5) can be employed together with radiolabeled pre-mRNA to track spliceosome assembly . U2 snRNA depletion experiments using oligo-directed RNase H cleavage provide valuable insights into the dependency of PRP5 on U2 for spliceosome association . For studying branch site proofreading, using pre-mRNAs with specific mutations in the branch site sequence (such as U257A/G/C) allows observation of how PRP5 responds to defective branch sites . Combined, these methodologies provide a comprehensive toolkit for dissecting PRP5's specific functions in prespliceosome formation and splicing fidelity.

Which techniques are most suitable for studying PRP5-U2 snRNA interactions?

For investigating PRP5-U2 snRNA interactions, several complementary techniques yield the most comprehensive results. RNA-protein crosslinking studies have proven particularly valuable, as they can capture the direct interaction between PRP5 and specific regions of U2 snRNA . In published studies, crosslinking revealed that PRP5 interacts with regions on or near the branchpoint-interacting stem-loop (BSL) of U2 snRNA . RNA immunoprecipitation (RIP) using tagged PRP5 followed by RNA analysis can identify which portions of U2 snRNA associate with PRP5 under various conditions . For more precise mapping of interaction sites, site-directed mutagenesis of suspected binding regions in U2 snRNA followed by binding assays provides detailed insights into which nucleotides are critical for PRP5 association . Structure-function analyses using recombinant PRP5 with mutations in predicted RNA-binding domains can complement these approaches by identifying which protein domains mediate U2 snRNA recognition. Additionally, in vitro reconstitution assays with purified components allow for controlled testing of how specific factors influence PRP5-U2 interactions under defined conditions .

How can the ATP-dependent and ATP-independent functions of PRP5 be separately assessed?

Distinguishing between PRP5's ATP-dependent and ATP-independent functions requires specific experimental strategies that isolate each activity. For the ATP-dependent function, in vitro assays using PRP5 mutants with impaired ATPase activity (such as mutations in the conserved DEAD-box motifs) allow researchers to specifically disrupt ATP-dependent remodeling while potentially preserving binding capabilities. These assays typically measure U2 snRNP remodeling efficiency and prespliceosome formation rates . Conversely, to study the ATP-independent function, binding assays performed in the absence of ATP or with non-hydrolyzable ATP analogs can reveal PRP5's ability to stabilize U2 snRNA structures without energy input . Additionally, designing experiments with staged ATP addition can temporally separate the two functions: pre-incubation without ATP followed by ATP addition helps determine which steps require ATP hydrolysis . The use of specific U2 snRNA mutants that alter the structure of binding regions but not remodeling targets can also help dissect these dual roles . Combining these approaches with quantitative measurements of spliceosome assembly kinetics provides a comprehensive understanding of how PRP5's two distinct functions contribute to splicing regulation.

How does the genetic diversity of PRP5 in clinical C. glabrata isolates impact antifungal susceptibility?

The genetic diversity within C. glabrata clinical isolates, including variations in RNA processing genes like PRP5, may significantly impact antifungal susceptibility through several mechanisms. Population genetics studies of C. glabrata have revealed substantial genetic diversity across clinical isolates, with at least 19 distinct sequence types identified in Scotland alone . This genomic variation includes evidence of recombination between geographically diverse strains, creating novel genetic combinations that could affect drug response profiles . Within infected patients, microevolution during recurrent candidiasis has been documented to specifically impact drug resistance genes, including the echinocandin target FKS1/2, which in some cases coincided with marked changes in fluconazole minimum inhibitory concentration (MIC) . Though not specifically studied for PRP5, variations in splicing machinery components like PRP5 could potentially affect the proper processing of transcripts encoding drug resistance factors, membrane transporters, or stress response proteins . Research examining correlations between PRP5 sequence variants and altered expression profiles of drug resistance genes would provide valuable insights into whether specific PRP5 alleles contribute to the concerning increases in antifungal resistance observed in clinical C. glabrata isolates .

How does PRP5 function relate to C. glabrata's adaptation during infection and stress response?

PRP5's function in pre-mRNA processing likely plays a significant but unexplored role in C. glabrata's adaptation during infection and stress response. As a pathogen that has evolved an infection strategy based on stealth and evasion, C. glabrata requires precise regulation of gene expression to respond to host environments . Population genetics studies have identified signatures of positive selection in clinical C. glabrata isolates, particularly in epithelial adhesin genes that facilitate fungal adhesion to human epithelial cells . Since proper splicing is essential for accurate gene expression, PRP5's role in prespliceosome formation could influence the expression of these virulence factors . Additionally, microevolution during recurrent infections has been shown to enrich for nonsynonymous and frameshift indels in cell surface proteins, including adhesins and drug resistance genes . The splicing machinery, including PRP5, would need to properly process these genes' transcripts under various stress conditions encountered during infection . Though direct evidence is limited, the hypervariability observed in C. glabrata genomes, including substantial mitochondrial genome diversity, suggests that RNA processing mechanisms may need to adapt to accommodate sequence variations while maintaining proper expression of critical genes under changing host conditions .

What are the critical factors for successful expression and purification of recombinant C. glabrata PRP5?

Successful expression and purification of recombinant C. glabrata PRP5 depends on several critical factors. First, codon optimization for the expression system is essential since C. glabrata uses a non-standard genetic code compared to common expression hosts . Expression constructs should include affinity tags (such as His6 or GST) positioned to minimize interference with both ATP-binding domains and RNA-interaction regions. Expression temperature and induction conditions require careful optimization, as DEAD-box helicases often show reduced solubility when overexpressed; lower temperatures (16-18°C) and extended, mild induction periods typically yield better results than standard conditions. Buffer composition during purification is crucial, with the inclusion of low concentrations of non-ionic detergents (0.01-0.05% NP-40 or Triton X-100) often improving solubility. ATP or non-hydrolyzable ATP analogs in purification buffers can stabilize protein conformation. For functional studies, it's essential to verify that the recombinant protein retains ATPase activity using standard colorimetric assays measuring phosphate release in the presence of RNA substrates . Storage conditions should include glycerol (10-20%) and reducing agents to maintain long-term stability and prevent oxidation of conserved cysteine residues that might be important for structure or function.

How can researchers overcome challenges in studying PRP5-mediated spliceosome dynamics?

Studying PRP5-mediated spliceosome dynamics presents significant technical challenges that researchers can overcome through several specialized approaches. The transient nature of PRP5's spliceosome association requires time-resolved techniques such as synchronized splicing reactions with staged addition of components, allowing observation of assembly intermediates . The difficulty in observing PRP5 release can be addressed using FRET-based assays with fluorescently labeled PRP5 and spliceosome components to directly monitor binding and dissociation events in real-time . To capture the dynamic ATP-dependent conformational changes, chemical crosslinking coupled with mass spectrometry (XL-MS) can map interaction surfaces during different stages of the splicing reaction . Challenges in recreating proper U2 snRNP complexes for in vitro studies can be overcome using partially purified native complexes supplemented with recombinant factors, preserving critical protein-RNA interactions . For studying branch site proofreading, creating pre-mRNAs with systematically altered branch site sequences and sensitive quantification methods for spliceosome progression allows for measuring the kinetic effects of PRP5 on different substrates . Additionally, single-molecule approaches such as optical tweezers or fluorescence microscopy can provide unprecedented insights into the conformational dynamics of individual PRP5-U2 snRNP complexes during the remodeling process .