RPRD1B Human

What is RPRD1B and what is its basic function in human cells?

RPRD1B is a protein that interacts with the C-Terminal Domain (CTD) of RNA Polymerase II (Pol II). It plays several critical roles in cellular processes, particularly in transcriptional regulation. RPRD1B contains a CTD-interacting domain (CID) that specifically recognizes phosphorylated serine residues in the RNA Polymerase II CTD repeats . Recent research has identified RPRD1B as an important component in:

• The heat shock response (HSR) pathway, where cells depleted of RPRD1B show decreased expression of heat shock proteins and reduced survival after heat shock

• Maintaining RNA Polymerase II occupancy on long cell cycle genes

• Transcriptional regulation, particularly affecting gene elongation processes

RPRD1B belongs to a family that includes RPRD1A and RPRD2, which share structural similarities but have distinct binding preferences to the CTD of RNA Polymerase II, suggesting specialized functions within the transcriptional machinery.

How does RPRD1B structurally interact with RNA Polymerase II?

RPRD1B directly interacts with the C-Terminal Domain (CTD) of RNA Polymerase II through its CID (CTD-Interacting Domain). Structural and biophysical studies have revealed several key characteristics of this interaction:

• RPRD1B binds with different affinities to CTD peptides depending on their phosphorylation status, with higher affinity for phosphorylated peptides

• The protein contains highly conserved residues that form specific contacts with the CTD, including R106 which forms two hydrogen bonds with the S2P phosphate group

• RPRD1B can form domain-swapped structures that are likely stabilized by a disulfide bond involving C100 residues

The crystal structure of RPRD1B CID bound to S2P CTD peptide has been solved at 1.85Å resolution, revealing that the folding of the RPRD1B CID and its contact residues with the S2P CTD peptide are highly similar to those observed in the RPRD1A CID-S7P CTD complex .

Crystallography Data for RPRD1B Structures:

What experimental approaches are most effective for studying RPRD1B function?

Researchers have employed several complementary techniques to elucidate RPRD1B functions, each providing unique insights:

For structural studies:

• X-ray crystallography to determine the three-dimensional structure of RPRD1B CID in complex with various CTD peptides

• Isothermal Titration Calorimetry (ITC) to measure binding affinities between RPRD1B CID and CTD peptides with different modifications

• Site-directed mutagenesis of conserved residues (e.g., R114, D65, R106) to verify the interaction surface and critical binding residues

For functional studies:

• siRNA and shRNA knockdown approaches to deplete RPRD1B (shRNA has been shown to reduce RPRD1B to 19% of its total protein level)

• CRISPR-Cas9 screening to identify RPRD1B's role in the heat shock response

• ChIP-seq analysis to assess RNA Polymerase II distribution changes when RPRD1B is depleted

• mRNA-seq and TT-Seq (Transient Transcriptome Sequencing) for differential gene expression analysis and nascent RNA profiling

These approaches collectively provide a comprehensive view of both the structural interactions and functional impacts of RPRD1B in cellular processes.

How does RPRD1B depletion affect the heat shock response pathway?

RPRD1B has been recently identified as a critical factor in the cellular heat shock response. Proteomic and genomic screening revealed that RPRD1B depletion significantly impacts this essential cellular stress response:

• Cells depleted for RPRD1B show significant reduction in heat shock survival, demonstrated through colony formation assays

• RPRD1B knockdown results in reduced expression of key heat shock proteins, including:

  • Up to 65% reduction in HSPH1 levels

  • Up to 45% reduction in DNAJB1 levels

• Transcriptomic analysis identified approximately 280 genes upregulated during heat shock, with 111 of these showing downregulation when RPRD1B is depleted

• Interestingly, RPRD1B depletion affects both HSF1-dependent and -independent heat shock-induced genes, suggesting a broad role in the heat shock transcriptional program

The heat shock factor HSF1 appears to be activated normally in the absence of RPRD1B, as indicated by its phosphorylation status and the formation of characteristic nuclear foci, suggesting that RPRD1B functions downstream of HSF1 activation or in parallel pathways .

What is the role of RPRD1B in transcriptional elongation during heat shock?

Analysis of nascent transcription in RPRD1B-depleted cells reveals specific effects on transcriptional elongation during heat shock:

• Under normal conditions, RPRD1B depletion has little or no effect on the expression of heat shock genes, suggesting it is largely dispensable for their basal expression

• After heat shock, a subtle but easily detectable downregulation across gene bodies becomes apparent, which becomes more pronounced toward the 3'-end of genes

• This pattern suggests RPRD1B plays a specific role in aspects of transcript elongation during the heat shock response

• The effect is observed specifically during heat shock conditions and not under normal growth temperatures, indicating a stress-specific function

These findings suggest that RPRD1B has a specialized role in facilitating proper transcriptional elongation specifically during cellular stress responses such as heat shock.

How does RPRD1B affect RNA Polymerase II distribution on genes?

ChIP-seq analysis of RNA Polymerase II in RPRD1B knockdown cells has provided detailed insights into how this protein affects Pol II distribution:

• Across all expressed genes, there is no global difference in the state of promoter-paused Pol II, indicating that RPRD1B's effects are gene-specific rather than universal

• When analyzed using unsupervised clustering, specific gene clusters show significant decreases in pausing index when RPRD1B is knocked down:

  • Cluster 2 (n = 3405 genes) shows significant reduction (p < 2.2 × 10⁻¹⁶)

  • Cluster 3 (n = 1499 genes) also shows significant reduction (p < 2.2 × 10⁻¹⁶)

• Similar patterns were observed when measuring the traveling ratio (TR) to quantify RPB1 distribution

• RPRD1B knockdown specifically reduces Pol II occupancy at the promoters of long genes with higher numbers of exons/introns (approximately 5000 genes)

These findings suggest RPRD1B plays a crucial role in maintaining proper RNA Polymerase II distribution, particularly on complex, long genes with multiple exons/introns.

What is the relationship between RPRD1B, HSF1, and heat shock gene regulation?

The relationship between RPRD1B and HSF1 (Heat Shock Factor 1, the master regulator of heat shock response) is complex:

These findings indicate that RPRD1B affects HSF1-dependent and -independent heat shock-induced genes alike, suggesting it functions broadly in the heat shock transcriptional response rather than specifically in the HSF1 pathway.

What methods are recommended for analyzing transcriptional effects of RPRD1B?

To comprehensively analyze the impact of RPRD1B on transcription, researchers have successfully employed multiple complementary approaches:

Genomic and Transcriptomic Methods:

• mRNA-seq for differential gene expression analysis comparing control and RPRD1B-depleted cells under various conditions

• TT-Seq (Transient Transcriptome Sequencing) to analyze nascent RNA profiles, which provides insight into active transcription rather than steady-state mRNA levels

• ChIP-seq of RNA Polymerase II to assess genomic distribution changes when RPRD1B is depleted

Quantitative Measures:

• Calculation of pausing index (ratio of Pol II signal near promoter to the gene body and after TES) to assess promoter-proximal pausing

• Traveling ratio (TR) analysis to quantify Pol II distribution between promoter region and gene body

• Unsupervised clustering approaches to identify gene groups with similar responses to RPRD1B depletion

Validation Methods:

• qPCR analysis of nascent transcripts for individual genes of interest

• Western blot analysis to confirm changes in protein expression of target genes

• Heat shock survival assays to correlate transcriptional changes with phenotypic effects

This multi-faceted approach provides insights into how RPRD1B affects different stages of transcription, from initiation to elongation and termination.

How does RPRD1B contribute to the regulation of non-heat shock genes?

Beyond its role in heat shock response, RPRD1B has been implicated in regulating other gene sets:

• Differential gene expression analysis showed approximately 400 genes were upregulated by RPRD1B knockdown under both normal and heat shock conditions

• Intriguingly, interferon-stimulated genes (ISGs) were among the most upregulated genes when RPRD1B was depleted

• Heat shock appeared to repress, and in some instances completely abolish, the ISG induction observed in RPRD1B-depleted cells, as evident from the nascent RNA profile

• RPRD1B has been shown to maintain Pol II occupancy on long cell cycle genes with higher numbers of exons/introns

These findings suggest RPRD1B has broader roles in transcriptional regulation beyond heat shock response, potentially serving as a checkpoint or regulatory factor for multiple gene expression programs, including those involved in interferon response and cell cycle regulation.

What structural features of RPRD1B are critical for its function?

Structural studies have identified several key features of RPRD1B that are essential for its function:

• The CTD-interacting domain (CID) of RPRD1B forms a specific structure that allows it to recognize and bind to the CTD of RNA Polymerase II

• Highly conserved residues within the CID are critical for binding:

  • R106 forms two hydrogen bonds with the S2P phosphate group, explaining the enhanced affinity for phosphorylated CTD peptides

  • R114 is essential for binding, as R114A mutation abolishes binding to both S2P and S7P CTDs

  • Other residues like N69 form water-mediated hydrogen bonds with specific CTD residues

• RPRD1B can form domain-swapped structures that are likely stabilized by a disulfide bond involving C100 residues, though whether this occurs in vivo remains unclear

• The protein contains multiple functional domains beyond the CID, including a coiled-coil domain (residues 171-304) that has been structurally characterized

Understanding these structural features provides insights into how RPRD1B selectively recognizes specific forms of the RNA Polymerase II CTD and potentially how it influences transcriptional processes.

What are the best methods for depleting RPRD1B in experimental models?

Researchers have successfully employed several approaches to deplete RPRD1B in cellular models, each with specific advantages:

RNA Interference Approaches:

• siRNA pools for transient knockdown: Efficiently reduces RPRD1B levels while not affecting its paralogue RPRD1A

• Different single siRNAs have been used to rule out off-target effects

• shRNA for stable knockdown: Demonstrated to reduce RPRD1B to 19% of its total protein level in HEK293 cells

Genetic Approaches:

• CRISPR-Cas9 screening has been used for genomic depletion and to identify RPRD1B's role in heat shock response

Validation Methods:

• Western blot analysis to confirm protein depletion both in whole cell extracts and in isolated chromatin fractions

• qPCR to measure changes in target gene expression following RPRD1B depletion

• Biological replicates showing high correlation and reproducibility in downstream analyses such as ChIP-seq

These approaches provide researchers with multiple options for studying RPRD1B function, allowing selection of the most appropriate method based on experimental requirements and cellular context.