Recombinant Saccharomyces cerevisiae Uncharacterized protein YBL059W (YBL059W)

What is known about the YBL059W gene and its encoded protein in S. cerevisiae?

YBL059W, also identified as IAI11 in the Saccharomyces Genome Database, is a yeast gene that contains a 69-nucleotide intron with a 51-nucleotide lariat loop . While the protein remains uncharacterized in terms of its precise biological function, its intron has been well-studied as a model for RNA splicing and lariat formation. The gene is present in the reference S. cerevisiae genome derived from laboratory strain S288C . Current research approaches focus on characterizing this gene through comparative genomics, expression analysis, and recombinant techniques to determine its functional role in yeast cellular processes.

How does YBL059W compare structurally to other uncharacterized yeast proteins?

YBL059W contains specific sequence features that distinguish it from other uncharacterized yeast proteins, particularly its intron structure which has been useful for studying RNA splicing mechanisms. Structural analysis suggests potential similarities with other proteins involved in fundamental cellular processes. When conducting comparative analyses, researchers typically align YBL059W sequences from different yeast strains to identify conserved domains that may indicate functional importance. Bioinformatic tools such as DAMBE can be useful for these sequence comparisons and phylogenetic analyses . This approach has revealed that while YBL059W lacks extensive functional annotation, its conservation across strains suggests biological significance.

What expression patterns does YBL059W exhibit under different conditions?

Expression data available through resources like the Gene Expression Omnibus (GEO) indicate that YBL059W shows variable expression patterns under different environmental conditions . To properly analyze these patterns, researchers should normalize expression data using log2 transformation, which allows for comparison across different experimental conditions. The expression visualization tools available in the Saccharomyces Genome Database can help identify genes with similar expression patterns, potentially revealing functional relationships through guilt-by-association approaches . This methodological approach provides insights into conditions that might upregulate or downregulate YBL059W, offering clues to its biological role.

What are the optimal methods for creating recombinant S. cerevisiae strains expressing modified YBL059W?

When designing recombinant strains expressing modified YBL059W, researchers should consider two primary approaches:

Table 1: Comparison of Methods for Creating Recombinant S. cerevisiae Strains

The S-type population approach is recommended for detailed YBL059W studies as it provides better genetic control. This method involves pairing haploid strains of opposite mating types, allowing diploid colonies to form, inducing sporulation, and then dissecting tetrads to collect meiotic products . For verification of proper strain construction, drug resistance markers (such as hygromycin resistance) should be used, followed by sequencing to confirm the presence of the desired modifications .

How can I optimize the expression of recombinant YBL059W protein for structural studies?

For optimal expression of recombinant YBL059W, consider these methodological approaches:

• Codon optimization: Analyze the codon usage bias in S. cerevisiae to optimize translation efficiency. Tools like DAMBE can assist in analyzing codon usage patterns and optimizing sequences for expression .

• Expression system selection: For structural studies, a sterol-optimized yeast platform can provide significant advantages when expressing membrane-associated proteins. Engineering the yeast strain by tuning the FPP pathway and optimizing sterol esterification can enhance protein expression and stability .

• Purification strategy: Implement affinity tags that minimally interfere with protein structure, followed by size exclusion chromatography to ensure protein homogeneity required for crystallography or cryo-EM studies.

When analyzing expression levels, quantitative PCR with appropriate reference genes should be employed, and western blotting with antibodies against the affinity tag can verify expression levels and integrity of the recombinant protein.

What are the recommended approaches for studying YBL059W intron splicing mechanisms?

To study the splicing mechanisms of the YBL059W intron, researchers should consider these methodological approaches:

• Lariat RNA synthesis: The YBL059W intron (69-nt with a 51-nt lariat loop) can be efficiently synthesized using the 6BX22 deoxyribozyme, which catalyzes one-step lariat formation. This approach offers high yield and specificity without requiring natural splicing enzymes that have strict sequence requirements .

• Experimental conditions: For optimal results, use assay conditions of 50 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM KCl, and 20 mM MnCl₂ at 37°C, with a substrate:deoxyribozyme ratio of 1:2 .

• Verification methods: The lariat structure can be verified using 10-23 deoxyribozyme cleavage and yeast debranching enzyme assays. Additionally, radiolabeling with α-³²P-CTP allows for visualization and quantification of reaction products .

This approach avoids the limitations of traditional splicing studies that rely on natural enzymes with restrictive sequence requirements, allowing for more flexible experimental designs to study the YBL059W intron's splicing behavior.

How can synthetic recombinant populations be designed to study YBL059W variation across yeast strains?

For studying YBL059W variation across yeast strains using synthetic recombinant populations, researchers should implement a structured crossing design followed by genomic analysis:

• Crossing design selection: Based on research objectives, choose between K-type (simple mass mating) or S-type (controlled pairing) population designs. S-type designs produce populations with more equal founder haplotype representation and higher levels of genetic variation, making them superior for detecting subtle functional effects of YBL059W variants .

• Founder strain selection: Include diverse S. cerevisiae strains (4, 8, or 12 founder strains depending on desired genetic diversity). Ensure founder strains carry appropriate mating type markers and drug resistance genes for validation .

• Outcrossing strategy: Implement multiple rounds of outcrossing (minimum 6-12 cycles) to generate sufficient recombination breakpoints for fine mapping of YBL059W functional variants .

• Genomic analysis: Sequence populations at defined intervals (e.g., initial, cycle 6, cycle 12) to track allele frequencies and haplotype structures. This approach allows for identification of selective pressures on specific YBL059W variants .

This methodological framework provides a powerful system for dissecting the genetic architecture of YBL059W function across diverse genetic backgrounds.

What approaches can be used to investigate potential protein-protein interactions involving YBL059W?

To investigate potential protein-protein interactions involving YBL059W, consider these methodological approaches:

• Yeast two-hybrid screening: Implement a modified yeast two-hybrid system using YBL059W as bait against a genomic library. For membrane-associated interactions, consider a split-ubiquitin system variant.

• Co-immunoprecipitation coupled with mass spectrometry: Express epitope-tagged YBL059W in yeast, perform immunoprecipitation under native conditions, and identify co-precipitating proteins through mass spectrometry. This approach has successfully identified interaction partners for other uncharacterized yeast proteins.

• Proximity-based labeling: Implement BioID or TurboID fusions with YBL059W to identify proximal proteins in living cells, which is particularly useful if YBL059W participates in transient interactions or membrane-associated complexes similar to those observed in brassinosteroid biosynthesis .

• Scaffold protein analysis: If YBL059W functions as a scaffold protein similar to MSBP1 in plants, investigate its role in potential metabolon formation using systematic protein complex isolation followed by functional reconstitution experiments .

These methodologies should be integrated with appropriate controls, including parallel analyses with known interaction partners and non-interacting proteins, to validate genuine interaction partners.

How can I design experiments to investigate the potential function of YBL059W in metabolic pathways?

To investigate YBL059W's potential role in metabolic pathways:

• Metabolic profiling comparison: Compare metabolite profiles between wild-type and YBL059W deletion strains using liquid chromatography-mass spectrometry (LC-MS) or gas chromatography-mass spectrometry (GC-MS). Focus on multiple metabolic states (exponential growth, diauxic shift, stationary phase) to capture condition-specific functions.

• Flux analysis: Implement 13C metabolic flux analysis by feeding cultures with 13C-labeled carbon sources and analyzing isotopomer distributions in downstream metabolites. This approach reveals changes in pathway utilization when YBL059W is absent or overexpressed.

• Genetic interaction mapping: Perform systematic genetic interaction screens by crossing YBL059W deletion strains with a genome-wide deletion collection. Synthetic lethal or synthetic sick interactions often reveal functional relationships in shared or compensatory pathways.

• Heterologous expression systems: If YBL059W is hypothesized to function in a specific pathway, reconstitute the pathway in a heterologous host with and without YBL059W to directly assess its contribution to pathway function, similar to approaches used for characterizing plant proteins in yeast systems .

These complementary approaches provide a comprehensive framework for uncovering the metabolic functions of uncharacterized proteins like YBL059W.

What bioinformatic approaches are most effective for predicting the function of YBL059W?

For effectively predicting YBL059W function using bioinformatic approaches:

• Sequence-based comparative genomics: Implement PSI-BLAST against diverse fungal genomes to identify distant homologs beyond standard BLAST detection. Follow with multiple sequence alignment using MUSCLE or MAFFT to identify conserved residues that may indicate functional sites.

• Structural prediction and analysis: Generate protein structure predictions using AlphaFold2 or RoseTTAFold, followed by structural comparison against the PDB database to identify proteins with similar folds despite low sequence similarity. This approach can reveal functional analogies not apparent from sequence alone.

• Gene neighborhood analysis: Analyze the genomic context of YBL059W across multiple yeast species to identify consistently co-localized genes, which may suggest functional relationships. Tools like DAMBE can assist in systematic comparative analyses .

• Co-expression network analysis: Mine public gene expression databases to construct co-expression networks, identifying genes consistently co-regulated with YBL059W across diverse conditions. The Saccharomyces Genome Database provides resources for such expression correlation analyses .

• Evolutionary rate analysis: Calculate site-specific evolutionary rates to identify conserved regions under purifying selection, which often correspond to functionally important domains. Accelerated evolution in specific lineages may indicate adaptive specialization.

By integrating these complementary approaches, researchers can develop testable hypotheses about YBL059W function based on computational predictions.

How should RNA-seq data be analyzed to understand YBL059W splicing patterns across different conditions?

For analyzing RNA-seq data to characterize YBL059W splicing patterns:

• Splicing-specific alignment: Implement splicing-aware aligners like STAR or HISAT2 that can map reads across exon-intron junctions. Configure aligners to detect novel splice junctions without requiring prior annotation, which is crucial for identifying condition-specific splicing events.

• Quantification of splicing events: Use tools like rMATS or MAJIQ to quantify different splicing events, including percent spliced in (PSI) values for the YBL059W intron across conditions. These metrics provide a quantitative measure of splicing efficiency.

• Intron retention analysis: Given YBL059W contains a 69-nt intron with a 51-nt lariat loop , pay special attention to intron retention events, which may indicate regulatory splicing rather than constitutive splicing.

• Splice site strength analysis: Evaluate the strength of YBL059W splice sites using position weight matrices and compare against consensus yeast splice sites to identify potential regulatory mechanisms for condition-specific splicing.

• Lariat detection: Implement specialized computational pipelines to detect and quantify lariat intermediates, which provide insights into splicing kinetics and efficiency. This is particularly relevant given the established protocols for synthesizing YBL059W lariat RNAs .

This methodological framework allows researchers to comprehensively characterize how YBL059W splicing patterns respond to different environmental conditions or genetic backgrounds.

What statistical approaches should be used when analyzing genetic variation in YBL059W across recombinant populations?

When analyzing genetic variation in YBL059W across recombinant populations:

Table 2: Statistical Methods for Analyzing YBL059W Genetic Variation

For recombinant populations, it's crucial to account for the crossing design (K-type vs. S-type) in statistical analyses, as these designs produce different patterns of genetic variation . K-type populations may show more uneven founder representation, requiring statistical corrections for sampling bias. Additionally, when performing genome sequencing of recombinant populations, sampling at multiple timepoints (e.g., cycle 0, cycle 6, cycle 12) allows for tracking allele frequency trajectories and more robust inference of selection on YBL059W variants .

What CRISPR-based approaches can be used to precisely modify YBL059W for functional studies?

For precise CRISPR-based modification of YBL059W:

• gRNA design optimization: Design at least 3-4 guide RNAs targeting YBL059W, prioritizing sites with minimal off-target effects as predicted by tools like Cas-OFFinder. For targeting the intronic region, which has been used in lariat RNA studies , ensure the gRNA does not disrupt critical splicing signals.

• HDR template construction: For precise modifications, design homology-directed repair (HDR) templates with 40-60bp homology arms flanking the desired modification. When introducing tags or reporters, position them to minimize interference with the 69-nt intron structure known to be important for lariat formation .

• Delivery method selection: In S. cerevisiae, plasmid-based delivery of Cas9 and gRNA components is typically most efficient. Implement a two-plasmid system with Cas9 and gRNA on separate vectors to allow for modular gRNA testing.

• Efficient screening strategy: Design a PCR-based screening approach to rapidly identify successful editing events, followed by sequencing validation to confirm precise modifications. For modifications affecting splicing, implement RT-PCR to verify resulting splicing patterns.

• Phenotypic validation: Compare growth rates and fitness of edited strains under various conditions, including stress conditions that might reveal functions not apparent under standard laboratory conditions.

This systematic approach allows for precise genetic engineering of YBL059W while minimizing unintended consequences that could confound functional studies.

How can advanced microscopy techniques be applied to study YBL059W localization and dynamics?

To study YBL059W localization and dynamics using advanced microscopy:

• Fluorescent protein tagging strategy: Create C- and N-terminal fluorescent protein fusions (e.g., mNeonGreen or mScarlet) with YBL059W, expressed from its native locus. Verify protein functionality post-tagging through complementation assays.

• Colocalization experiments: Perform multi-channel imaging with established organelle markers to determine subcellular localization. For yeast studies, markers for the endoplasmic reticulum, Golgi apparatus, and other organelles should be selected based on spectral compatibility with the YBL059W tag.

• Live-cell imaging approaches: Implement time-lapse microscopy with optimized acquisition parameters (interval timing, exposure, laser power) to minimize phototoxicity while capturing dynamic behaviors. For yeast cells, consider microfluidic platforms that allow for long-term imaging with precise environmental control.

• Advanced techniques for dynamic analysis: Apply fluorescence recovery after photobleaching (FRAP) or photoactivation to measure protein mobility and turnover rates. For protein-protein interactions, implement Förster resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) between YBL059W and candidate interacting proteins.

• Super-resolution approaches: For detailed localization studies, implement structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) to achieve resolution below the diffraction limit, particularly valuable for determining membrane association patterns.

These approaches provide comprehensive spatial and temporal information about YBL059W behavior in living cells, offering insights into its biological function.

What approaches are recommended for investigating YBL059W's potential role in RNA metabolism given its intronic features?

To investigate YBL059W's potential role in RNA metabolism:

• RNA immunoprecipitation (RIP) analysis: If YBL059W is hypothesized to interact with RNA, perform RIP using epitope-tagged YBL059W followed by sequencing (RIP-seq) to identify associated RNA species. Include appropriate controls such as untagged strains and non-RNA-binding proteins.

• Lariat RNA interaction studies: Given the established protocols for synthesizing YBL059W lariat RNAs , investigate whether the protein interacts with its own intronic lariat using in vitro binding assays. The 6BX22 deoxyribozyme system allows efficient production of YBL059W lariat RNA for such studies .

• Splicing efficiency analysis: Compare splicing kinetics and efficiency of the YBL059W intron between wild-type strains and strains with modified YBL059W protein expression. Utilize reverse transcription-quantitative PCR (RT-qPCR) with primers spanning exon-intron junctions.

• Global splicing impact assessment: Perform RNA-seq on strains with YBL059W deletions or overexpression to assess genome-wide impacts on splicing, particularly focusing on introns with structural similarities to the YBL059W intron.

• Debranching assays: Utilize yeast debranching enzyme assays to compare lariat turnover rates for the YBL059W intron versus other introns, potentially revealing specialized features of this intronic element .

This comprehensive approach integrates both protein-centric and RNA-centric methods to elucidate YBL059W's potential functions in RNA metabolism, with particular emphasis on leveraging the established YBL059W lariat RNA synthesis methodologies.