PURB Human

What is PURB and what is its primary function in human cells?

PURB (purine rich element binding protein B) is a sequence-specific, single-stranded DNA-binding protein that belongs to the PUR family of proteins. It preferentially binds to purine-rich elements (termed PUR) present at replication origins and in gene flanking regions across various eukaryotes from yeasts to humans. Its primary functions involve the regulation of both DNA replication and transcription through its interaction with specific nucleic acid sequences . PURB is also known by the synonym PURBETA and is classified as a protein-coding gene in humans with Entrez Gene ID 5814 . Unlike its paralog PURA, which has been more extensively studied, PURB's specific cellular functions are still being elucidated in ongoing research.

How is PURB structurally organized and what domains contribute to its function?

PURB is structurally organized with conserved PUR repeats that correlate with its folded entities, similar to other members of the PUR protein family. Based on structural studies of the related PURA protein, PURB likely contains three PUR repeats that form stable PUR domains through specific interactions . Each PUR repeat typically exhibits a β-β-β-β-α topology, where four-stranded beta sheets are followed by a single alpha helix .

The PUR domains can form in two types:

• Type I: formed from a single peptide chain with a β-β-β-β-α-linker-β-β-β-β-α topology

• Type II: assembled from two identical peptides with β-β-β-β-α fold, creating an inter-molecular homodimer

These structural elements are critical for PURB's ability to bind to single-stranded DNA and RNA, and mutations affecting these domains would likely impair binding function and protein integrity, as has been observed with PURA syndrome-causing mutations .

How does PURB expression vary across different human tissues?

PURB expression patterns can be analyzed using resources such as the Allen Brain Atlas and other tissue expression databases. According to the Harmonizome database, PURB has 4,595 functional associations spanning 8 biological categories extracted from 89 datasets, suggesting widespread expression and function . The Allen Brain Atlas provides detailed information on PURB expression in brain tissues, indicating both high and low expression patterns relative to other tissues .

For researchers interested in tissue-specific expression:

• The Allen Brain Atlas Adult Human Brain Tissue Gene Expression Profiles show region-specific expression patterns

• The Allen Brain Atlas Developing Human Brain Tissue Gene Expression Profiles (by both Microarray and RNA-seq) can reveal temporal expression patterns during development

• Comparative analysis with mouse brain expression can provide evolutionary insights

These expression patterns suggest that PURB may have both tissue-specific and developmental stage-specific functions that warrant further investigation.

What is known about PURB's DNA and RNA binding specificity?

PURB binds preferentially to single-stranded purine-rich DNA elements (PUR elements), which are present at origins of replication and in gene flanking regions across eukaryotes . While specific binding studies of PURB are somewhat limited compared to PURA, the high conservation within the PUR family suggests similar binding preferences.

Based on studies of the PUR family, binding typically involves:

• Recognition of guanine-rich sequences

• Interaction with both DNA and RNA molecules containing PUR elements

• Formation of stable protein-nucleic acid complexes through its PUR domains

For experimental validation of binding, researchers typically employ:

• Electrophoretic mobility shift assays (EMSAs)

• Surface plasmon resonance

• RNA immunoprecipitation followed by sequencing (RIP-seq)

• CLIP-seq methods for genome-wide binding site identification

These approaches can help determine the specific binding motifs and identify genomic loci where PURB exerts its regulatory functions.

How does PURB interact with other cellular proteins and pathways?

While PURB-specific protein interactions are less characterized than those of PURA, evidence suggests that PUR family proteins participate in various molecular pathways. PURA, for example, has been shown to interact with the motor protein KIF-5 and co-localize with processing bodies . By extension and due to structural similarities, PURB likely engages in protein-protein interactions that mediate its functions in transcription and replication.

Potential pathways and processes involving PURB include:

• Transcriptional regulation through interaction with promoter regions

• DNA replication initiation and progression

• RNA processing and metabolism

• Potential roles in cellular stress responses

• Developmental processes, particularly in the nervous system

For studying PURB protein interactions, recommended methodologies include co-immunoprecipitation, proximity labeling approaches (BioID, APEX), and yeast two-hybrid screening followed by validation with direct binding assays.

What transcriptional targets are regulated by PURB?

While PURB-specific transcriptional targets are not extensively documented in the provided search results, studies on the related PURA protein provide insights into potential PURB targets. PURA has been shown to regulate various genes including:

• CD43 gene promoter

• FE65 promoter

• Gata2 promoter region

• MB1 regulatory region of the MBP gene

• Mhc promoter

• Ovine placental lactogen promoter

• TGF-β1

• VSM-alpha actin promoter

Given the sequence and structural similarities between PURA and PURB, there may be overlap in their target genes, though PURB-specific targeting is likely to exist. Researchers investigating PURB transcriptional regulation should consider:

• Performing ChIP-seq to identify genome-wide binding sites

• Coupling PURB depletion with RNA-seq to identify differentially expressed genes

• Validating direct regulation through reporter assays with wild-type and mutated binding sites

• Analyzing binding site conservation across species to identify evolutionarily conserved targets

These approaches would help establish a comprehensive map of PURB's transcriptional regulatory network.

What are effective CRISPR-Cas9 strategies for studying PURB function?

CRISPR-Cas9 technology offers powerful approaches to study PURB function through targeted genome editing. According to the search results, several gRNA sequences designed by Feng Zhang's laboratory at the Broad Institute are available for targeting the PURB gene with minimal off-target effects .

For effective CRISPR-Cas9 studies of PURB:

• Guide RNA selection:

  • Multiple validated gRNAs are available targeting the PURB gene

  • At least two gRNA constructs are recommended per targeting experiment to increase success rates

  • Researchers should verify gRNA sequences against their specific target sequence, especially when targeting specific splice variants or exons

• Delivery considerations:

  • The gRNA constructs come in plasmids containing all elements required for expression: U6 promoter, spacer sequence, gRNA scaffold, and terminator

  • Selection markers can be chosen based on the experimental system

• Experimental designs for functional studies:

  • Complete knockout to assess loss-of-function phenotypes

  • Knock-in of specific mutations to model disease variants or tag endogenous protein

  • CRISPRi or CRISPRa approaches for reversible modulation of gene expression

  • Base editing for introducing specific point mutations without double-strand breaks

When analyzing results, researchers should be aware that complex phenotypes may emerge from PURB manipulation, potentially affecting multiple cellular pathways given its role in transcriptional regulation .

How can RNA-binding properties of PURB be experimentally characterized?

Characterizing PURB's RNA-binding properties requires specialized techniques to identify RNA targets and binding motifs. Drawing from studies on PURA, which binds thousands of transcripts predominantly in the cytoplasm , similar approaches can be applied to PURB:

• Transcriptome-wide binding site identification:

  • CLIP-seq (Cross-linking immunoprecipitation followed by sequencing)

  • PAR-CLIP (Photoactivatable ribonucleoside-enhanced CLIP)

  • iCLIP (individual-nucleotide resolution CLIP)

  • eCLIP (enhanced CLIP)

• Motif analysis and validation:

  • Computational analysis of binding sites to identify consensus motifs

  • In vitro binding assays with synthetic RNA oligonucleotides

  • Mutagenesis of putative binding motifs to confirm specificity

• Functional impact assessment:

  • RNA stability assays following PURB depletion or overexpression

  • Polysome profiling to assess translational impacts

  • Subcellular localization studies to determine where PURB-RNA interactions occur

• Structural studies:

  • In vitro reconstitution of PURB-RNA complexes

  • Structural determination using X-ray crystallography or cryo-EM

  • NMR spectroscopy for dynamics of PURB-RNA interactions

The findings from PURA studies suggest that PURB might similarly bind multiple RNA targets and potentially impact RNA stability, localization, or translation efficiency .

What methods are most effective for studying PURB's role in disease contexts?

Given PURB's association with myelodysplastic syndrome and acute myelogenous leukemia , several methodological approaches are particularly useful for investigating its role in disease contexts:

• Patient sample analysis:

  • Genomic sequencing to identify PURB mutations or deletions

  • Expression analysis in patient tissues compared to controls

  • Correlation of PURB alterations with clinical parameters and outcomes

• Disease modeling:

  • Generation of cell line models with PURB mutations using CRISPR-Cas9

  • Patient-derived xenografts to study PURB alterations in vivo

  • Transgenic mouse models with tissue-specific PURB manipulation

• Molecular pathway analysis:

  • RNA-seq and proteomics following PURB perturbation to identify affected pathways

  • Phosphoproteomics to detect signaling changes

  • Metabolomics to identify downstream metabolic alterations

• Therapeutic targeting strategies:

  • Compound screening to identify modulators of PURB function

  • Rescue experiments to validate molecular mechanisms

  • Combination approaches targeting PURB-related pathways

By integrating these approaches, researchers can develop a comprehensive understanding of how PURB alterations contribute to disease pathogenesis and potential therapeutic vulnerabilities.

How does PURB functionally differ from PURA and PURG?

While PURB shares significant structural similarity with other PUR family members, its functional differences are important for understanding its specific biological roles:

• Structural comparisons:

  • All PUR proteins contain conserved PUR repeats that form PUR domains

  • PURA, PURB, and PURG show high sequence conservation, particularly in their DNA/RNA binding regions

  • Subtle structural differences likely contribute to their distinct functions

• Functional distinctions:

  • PURA has been extensively studied in neurodevelopment, with mutations causing PURA Syndrome

  • PURA co-localizes with processing bodies and impacts RNA metabolism

  • PURB's specific functions are less characterized but may complement or antagonize PURA functions

  • PURG is the least studied of the three paralogs

• Expression patterns:

  • Different tissue expression profiles suggest tissue-specific functions

  • Developmental regulation varies among PUR family members

  • Co-expression in certain tissues suggests potential functional redundancy or cooperation

• Disease associations:

  • PURA mutations cause PURA Syndrome, a neurodevelopmental disorder

  • PURB deletions are associated with myelodysplastic syndrome and acute myelogenous leukemia

  • These distinct disease associations suggest non-redundant functions

Researchers should consider these distinctions when designing experiments and interpreting results involving PURB, as findings from PURA studies may not directly translate to PURB functions.

What is known about evolutionary conservation of PURB across species?

The PUR family of proteins, including PURB, is highly conserved from plants to humans, suggesting essential biological functions . This evolutionary conservation provides valuable insights for researchers:

• Sequence conservation:

  • The core PUR domains show remarkable conservation across vertebrates

  • Human PURB shares high sequence identity with mouse PURB (~99%)

  • Conserved functional motifs can be identified through comparative genomics

• Structural conservation:

  • PUR domain architecture is maintained across species

  • Crystal structures from model organisms (e.g., D. melanogaster) provide insights applicable to human PURB

  • The β-β-β-β-α topology of PUR repeats is evolutionarily conserved

• Functional conservation:

  • DNA/RNA binding preferences appear conserved across species

  • Regulatory mechanisms may vary while core functions remain conserved

  • Cross-species rescue experiments can test functional equivalence

• Experimental implications:

  • Model organisms can provide valuable insights into PURB function

  • Zebrafish models have been used to study PUR protein function (e.g., Gata2 regulation)

  • Cross-species comparisons can highlight essential vs. species-specific functions

This evolutionary conservation underscores the fundamental importance of PURB in cellular processes and suggests that insights from model organisms can inform human PURB research.

How does PURB contribute to RNA processing and P-body dynamics?

While direct evidence for PURB's role in RNA processing bodies (P-bodies) is limited in the search results, studies on PURA provide important insights that may extend to PURB given their structural similarities:

• P-body association:

  • PURA co-localizes with P-bodies, which are cytoplasmic RNA processing centers

  • PURA depletion impairs P-body formation and composition

  • PURA syndrome-causing mutations impair its co-localization with P-bodies

• RNA stability regulation:

  • PURA depletion leads to stabilization of P-body-associated transcripts while decreasing other mRNAs

  • This suggests a role in determining which RNAs are processed in P-bodies versus other pathways

• P-body component regulation:

  • PURA regulates the expression of integral P-body components like LSM14A

  • Reduced PURA levels strongly impair P-bodies in human cells

• Research methodology for PURB:

  • Immunofluorescence co-localization studies with P-body markers

  • RNA-IP followed by sequencing to identify PURB-bound RNAs in P-bodies

  • Quantitative analysis of P-body dynamics following PURB manipulation

  • Comparative analysis of PURB and PURA effects on P-body structure and function

Given the structural similarities between PURA and PURB, investigating whether PURB shows similar or complementary roles in P-body regulation represents an important research direction with implications for understanding RNA metabolism in both normal and disease states.

What are current challenges in understanding PURB's role in transcriptional regulation?

Understanding PURB's role in transcriptional regulation faces several key challenges that researchers should consider:

• Target identification challenges:

  • Distinguishing direct vs. indirect transcriptional effects

  • Identifying tissue-specific transcriptional targets

  • Differentiating PURB targets from those of other PUR proteins

  • Determining whether PURB acts as an activator or repressor in different contexts

• Mechanistic uncertainties:

  • How PURB recruitment to specific genomic loci is regulated

  • Whether PURB functions primarily as a DNA-binding protein or also regulates RNA post-transcriptionally

  • The composition of PURB-containing transcriptional complexes

  • The interplay between PURB and other transcription factors

• Technological limitations:

  • Specificity of antibodies for distinguishing between PUR family members

  • Challenges in generating complete knockout models due to potential redundancy

  • Difficulties in studying dynamic interactions in physiologically relevant contexts

• Research strategies to address these challenges:

  • Development of highly specific antibodies or tagged endogenous PURB

  • Sequential ChIP approaches to distinguish PURA vs. PURB binding

  • Tissue-specific and inducible knockout models

  • Single-cell approaches to capture cell-type specific functions

  • Temporal resolution studies to capture dynamic regulatory events

Addressing these challenges will require integrated approaches combining genomics, proteomics, and functional studies in relevant biological contexts.

How might PURB function be targeted for therapeutic applications?

While direct therapeutic targeting of PURB is not extensively discussed in the search results, several potential approaches can be considered based on its molecular function and disease associations:

• Potential therapeutic strategies:

  • Small molecule modulators of PURB-DNA/RNA binding

  • Peptide inhibitors targeting PURB protein-protein interactions

  • Antisense oligonucleotides to modulate PURB expression

  • Gene therapy approaches to restore PURB expression in deficiency contexts

• Disease-specific considerations:

  • For myelodysplastic syndrome and acute myelogenous leukemia associated with PURB deletion, restoration approaches might be beneficial

  • In contexts where PURB might be overexpressed, inhibition strategies could be explored

  • Targeting downstream effectors rather than PURB directly may provide therapeutic windows

• Experimental approaches for therapeutic development:

  • High-throughput screening for compounds that modulate PURB function

  • Structure-based drug design targeting PUR domains

  • PROTAC approaches for controlled degradation

  • Identification of synthetic lethal interactions with PURB alterations

• Challenges in therapeutic targeting:

  • Potential redundancy with other PUR family members

  • Achieving tissue specificity to avoid off-target effects

  • Determining appropriate biomarkers for patient selection

  • Developing effective delivery systems for nucleic acid therapeutics

Given PURB's fundamental roles in transcription and replication, therapeutic targeting would require careful consideration of tissue-specific effects and potential toxicities. Combination approaches targeting multiple nodes in PURB-regulated pathways may provide more effective therapeutic strategies.