SPBC29A10.10c Antibody

Basic Research Questions

• What is SPBC29A10.10c and what is its function in Schizosaccharomyces pombe?

SPBC29A10.10c (also known as Dbl8) is an uncharacterized ATP-dependent helicase in fission yeast that functions as a tRNA-splicing endonuclease positive effector. Research indicates that Dbl8 is involved in RNA processing pathways, particularly in relation to splicing mechanisms. According to proteomic analysis, Dbl8 interacts with multiple components of the spliceosomal complexes . Its predicted helicase activity suggests a role in unwinding RNA secondary structures during splicing events, though direct biochemical confirmation of this activity requires further investigation.

• What experimental applications are SPBC29A10.10c antibodies suitable for?

According to available data, polyclonal SPBC29A10.10c antibodies have been validated for:

• ELISA (Enzyme-Linked Immunosorbent Assay) applications

• Western Blot analysis for identifying the protein in cell lysates

When performing Western blots, the antibody enables detection of the native protein in Schizosaccharomyces pombe extracts. The recommended experimental approach involves antigen-affinity purified rabbit polyclonal antibodies against specific epitopes of the protein .

• How is SPBC29A10.10c protein expressed and localized in fission yeast cells?

SPBC29A10.10c predominantly localizes to the nucleus, consistent with its role in RNA processing. Specific localization studies have shown that while not directly part of the nuclear pore complex itself, Dbl8 associates with nuclear components involved in RNA metabolism. To visualize this localization:

• Use indirect immunofluorescence with specific antibodies

• Alternatively, construct GFP-tagged SPBC29A10.10c expressing strains via homologous recombination techniques similar to those used for other nuclear proteins in S. pombe

The expression level of SPBC29A10.10c appears to be tightly regulated under normal growth conditions, which sometimes makes detection challenging without enrichment techniques.

• What is the relationship between SPBC29A10.10c (Dbl8) and RNA splicing machinery?

Dbl8 has been identified as a component that interacts with core splicing machinery in S. pombe. According to proteomic analysis outlined in Table 1 of reference , Dbl8 associates with several splicing factors including:

These interactions suggest that Dbl8 facilitates specific aspects of the splicing process, potentially in pre-mRNA recognition or spliceosome assembly .

Advanced Research Questions

• How does SPBC29A10.10c contribute to heterochromatin formation in S. pombe?

Recent findings suggest that SPBC29A10.10c/Dbl8 may be involved in heterochromatin assembly through its interaction with the Clr6 histone deacetylase (HDAC) complex. The protein appears to function in a mechanism similar to Rbm10, which facilitates heterochromatin assembly by:

• Serving as a platform to recruit the Clr6 complex to heterochromatic regions

• Associating with nascent heterochromatic transcripts

• Interacting with chromatin remodelers important for heterochromatin silencing

Experimental approaches to study this function include chromatin immunoprecipitation (ChIP) assays to examine the association of Dbl8 with heterochromatic regions, and reporter gene silencing assays (using ura4+ or his3+ reporters) to measure heterochromatin integrity in cells where SPBC29A10.10c is deleted or mutated .

• What are the technical challenges in detecting SPBC29A10.10c protein in S. pombe extracts?

Researchers face several challenges when attempting to detect SPBC29A10.10c:

• Low endogenous expression levels, similar to observations with Rbm10

• Potential degradation during extraction procedures

• Complex formation with other proteins that may mask epitopes

To overcome these challenges, recommended methods include:

• Use of denaturation buffers containing SDS and heat treatment (95°C for 5 minutes)

• Implementing a two-step affinity purification strategy similar to TAP-tag methods described for Rbm10

• Enrichment by immunoprecipitation before Western blot analysis

• Including phosphatase inhibitors in extraction buffers, as the protein may be subject to phosphorylation-dependent regulation

• How can researchers validate SPBC29A10.10c antibody specificity in their experiments?

To ensure antibody specificity, implement these validation strategies:

• Genetic controls: Compare Western blot signals between wild-type and SPBC29A10.10c deletion strains

• Blocking peptide competition assays: Pre-incubate the antibody with the immunizing peptide before Western blotting

• Immunoprecipitation followed by mass spectrometry: Confirm that the precipitated protein is indeed SPBC29A10.10c

• Epitope-tagged controls: Create strains expressing tagged versions (HA or FLAG tags) of SPBC29A10.10c and perform parallel detection with both tag-specific antibodies and SPBC29A10.10c antibodies

This approach has been successful for validating other S. pombe protein antibodies with similar technical characteristics .

• What experimental approaches can be used to investigate SPBC29A10.10c interactions with the splicing machinery?

To characterize SPBC29A10.10c interactions with splicing machinery components:

• Co-immunoprecipitation (Co-IP): Use SPBC29A10.10c antibodies to pull down protein complexes, followed by Western blot analysis for splicing factors. This approach successfully identified interactions between Rbm10 and splicing factors .

• Tandem Affinity Purification (TAP):

  • Create a strain expressing TAP-tagged SPBC29A10.10c

  • Perform two-step affinity purification

  • Analyze by mass spectrometry to identify interacting proteins

• Yeast two-hybrid screening: Use SPBC29A10.10c as bait to screen for interacting partners

• RNA-immunoprecipitation (RIP): To identify RNAs associated with SPBC29A10.10c, similar to methods that demonstrated Rbm10's association with heterochromatic transcripts .

• How does deletion or mutation of SPBC29A10.10c affect global RNA splicing patterns in S. pombe?

Based on studies with functionally related proteins in S. pombe:

• RNA-seq analysis methodology:

  • Prepare poly(A) RNA from wild-type and SPBC29A10.10c mutant strains

  • Perform high-throughput sequencing (Illumina platform)

  • Align reads to the S. pombe genome using TopHat

  • Analyze differential intron retention using DESeq package

  • Use Fisher's exact test to compare splicing patterns with adjusted p-value < 0.05 and |ΔPIR| > 10% as thresholds for significant splicing differences

Studies of related proteins suggest that deletion might affect specific subsets of transcripts rather than causing global splicing defects. Particular attention should be paid to intron retention events and exon skipping patterns, which can be quantified using the Percent Intron Retained (PIR) metric .

• What is the role of SPBC29A10.10c in relation to the nonsense-mediated mRNA decay pathway?

Evidence suggests SPBC29A10.10c/Dbl8 may function in conjunction with the nonsense-mediated mRNA decay (NMD) pathway:

• Research on functionally related helicase proteins in S. pombe has shown connections between RNA processing factors and NMD machinery, particularly through interactions with Upf1 .

• Experimental approach to investigate this function:

  • Generate double mutants of SPBC29A10.10c and NMD components (upf1Δ, upf2Δ)

  • Measure stability of model NMD substrates (e.g., ade6-M26 transcripts)

  • Perform RNA immunoprecipitation to identify common RNA targets

  • Analyze transcriptome-wide effects using microarray or RNA-seq approaches

• Expected outcomes: If SPBC29A10.10c functions in the NMD pathway, its deletion would stabilize transcripts containing premature termination codons, similar to effects observed with upf1Δ mutations .

• How does the ATP-dependent helicase activity of SPBC29A10.10c contribute to its cellular functions?

Although biochemical confirmation of SPBC29A10.10c helicase activity is still needed, researchers can investigate this function through:

• In vitro helicase assays:

  • Express and purify recombinant SPBC29A10.10c protein

  • Prepare labeled RNA substrates with secondary structures

  • Measure unwinding activity in the presence of ATP

  • Test helicase-dead mutations (e.g., K→A in Walker A motif) as controls

• Structure-function analysis:

  • Create point mutations in conserved helicase motifs

  • Assess effects on splicing and heterochromatin formation

  • Determine if the helicase activity is separable from other functions

• ATP-binding studies: Use techniques such as fluorescence spectroscopy with fluorescent ATP analogs to measure binding affinity and kinetics

This systematic approach will help determine whether the predicted helicase activity is essential for all functions or specifically required for certain processes like RNA splicing or heterochromatin assembly.

• What is the evolutionary conservation of SPBC29A10.10c across different yeast species and other eukaryotes?

SPBC29A10.10c shows evolutionary conservation across fungi and potentially other eukaryotes:

• Comparative genomic analysis reveals:

  • Homologs exist in various yeast species including Saccharomyces cerevisiae

  • The protein contains conserved functional domains including RNA recognition motifs (RRMs) and helicase domains

  • Higher eukaryotes contain related proteins with similar domain architecture

• Functional conservation studies approach:

  • Test complementation of S. pombe SPBC29A10.10c deletion with orthologs from other species

  • Compare protein interaction networks across species

  • Analyze conservation of regulatory mechanisms

• Significance for researchers: Understanding evolutionary conservation helps identify functionally critical domains and predict potential roles in higher eukaryotes, including humans, where related proteins might be implicated in disease processes.