Recombinant Schizosaccharomyces pombe DNA-directed RNA polymerase II subunit rpb7 (rpb7)

What is the role of Rpb7 in RNA polymerase II?

Rpb7 is one of the twelve subunits (Rpb1 to Rpb12) that constitute eukaryotic RNA polymerase II, the enzyme responsible for transcription of protein-coding genes. It forms a heterodimer with Rpb4 that protrudes from the 10-subunit core of the enzyme, creating a distinct structural module with specialized functions. This heterodimer is strategically positioned near the RNA exit channel, allowing it to interact with nascent RNA transcripts. Unlike some other subunits, Rpb7 is not shared among the different RNA polymerases (Pol I, Pol II, and Pol III), making it specific to the transcription of mRNAs from protein-coding genes .

How conserved is Rpb7 across different species?

The sequence of Rpb7 is highly conserved across organisms from yeast to humans, indicating its fundamental importance in transcription machinery. This conservation is demonstrated functionally, as human Rpb7 can completely rescue the growth sensitivity of S. pombe cells with low rpb7+ levels under DNA-damaging conditions. This remarkable interchangeability suggests that despite evolutionary distance, the core functions of Rpb7 remain preserved . Sequence analyses show varying degrees of conservation in different domains, with approximately 37% sequence identity between yeast and human Rpb7 . The strong conservation of specific surface-exposed residues, particularly in the RNA-binding regions, further highlights the evolutionary pressure to maintain Rpb7's critical functions.

What is the relative abundance of Rpb7 compared to other RNA polymerase II subunits?

Studies in S. pombe have revealed significant variation in the intracellular concentrations of the twelve RNA polymerase II subunits. Quantitative analyses using Western blot and competitive RT-PCR techniques show approximately 15-fold difference between the least abundant subunit (Rpb3) and the most abundant subunit (Rpb12). Rpb7 is among the subunits with relatively low mRNA abundance, similar to Rpb1, Rpb3, and Rpb9. This contrasts with other subunits like Rpb2, whose mRNA levels are approximately 40-fold higher than Rpb3 . The protein-to-mRNA ratio for different subunits also varies, indicating differential translation efficiency or protein stability among the various polymerase components.

What are effective strategies for depleting Rpb7 in experimental systems?

Researchers have employed several approaches to study Rpb7 function through controlled depletion:

• Degron Systems: Auxin-inducible degron technology has proven effective for rapid depletion of Rpb7. This system allows for temporal control of Rpb7 levels through the addition of auxin to cell cultures, enabling the study of immediate effects versus adaptive responses .

• shRNA Knockdown: Short hairpin RNA (shRNA) targeting of RPB7 has been successfully used in mammalian cells to reduce Rpb7 levels. This approach typically achieves partial depletion rather than complete elimination .

• Conditional Expression Systems: In S. pombe, regulated expression constructs that allow for controlled reduction of rpb7+ levels have been developed, enabling researchers to study the effects of different expression levels on cellular phenotypes .

The choice of method depends on the experimental questions, with degron systems offering advantages for studying immediate effects of protein loss, while conditional expression systems may be more suitable for analyzing dose-dependent phenotypes.

How can researchers purify recombinant Rpb7 or Rpb4/Rpb7 complex for in vitro studies?

Purification of functional recombinant Rpb7 or the Rpb4/Rpb7 heterodimer typically follows these methodological steps:

• Expression System Selection: E. coli expression systems have been successfully used for both individual Rpb7 and co-expression of Rpb4/Rpb7 complexes, though eukaryotic expression systems may better preserve post-translational modifications.

• Co-expression Strategy: For the Rpb4/Rpb7 heterodimer, co-expression of both subunits is preferable to separate expression and subsequent reconstitution, as it enhances complex formation and stability.

• Affinity Purification: Tagging strategies (typically His-tag or FLAG-tag) followed by affinity chromatography provide an effective initial purification step.

• Additional Purification Steps: Ion exchange chromatography and size exclusion chromatography are typically employed as secondary purification methods to achieve high purity.

• Quality Control: RNA binding assays can be used to confirm the functional integrity of the purified protein or complex .

Researchers should consider that mutation of certain conserved residues, particularly phenylalanine 98 in human Rpb7, can significantly impact protein solubility during recombinant expression .

How does Rpb7 depletion affect global transcription?

Depletion of Rpb7 leads to a global repression of gene expression, as demonstrated by comprehensive genomic analyses. RNA-seq, chromatin-associated RNA-seq (ChAR-seq), and RPB1 ChIP-seq experiments with spike-in controls after Rpb7 depletion consistently show widespread transcriptional downregulation . This global effect can be attributed to two primary mechanisms:

These effects differ somewhat from those observed in yeast, highlighting potential evolutionary divergence in Rpb7 functions between species.

Does Rpb7 play a role in specific transcriptional programs rather than just general transcription?

While Rpb7 is essential for general transcription, evidence suggests it may have preferential effects on specific gene programs. In S. pombe, genome-wide transcriptional analysis revealed that reduced rpb7+ expression particularly downregulated genes involved in various DNA repair pathways . This selective effect suggests Rpb7 may play a regulatory role in specific transcriptional programs beyond its structural role in RNA polymerase II.

The mechanism for this specificity remains to be fully elucidated but may involve:

• Differential sensitivity of certain promoters to reduced polymerase activity

• Specific interactions between Rpb7 and transcription factors involved in particular pathways

• RNA-binding capabilities of Rpb7 affecting post-transcriptional processing of specific transcripts

This selective transcriptional role represents an important area for further research, as it may reveal additional regulatory functions of this conserved subunit.

What evidence supports Rpb7's role in DNA damage response pathways?

Multiple lines of evidence establish Rpb7's significant role in DNA damage response:

• Transcriptional Analysis: Global transcriptional profiling in S. pombe revealed that genes belonging to different DNA repair pathways were specifically downregulated when rpb7+ expression was reduced .

• Phenotypic Testing: S. pombe cells with reduced rpb7+ levels showed compromised survival under various genotoxic stress conditions (e.g., UV radiation, hydroxyurea, methyl methanesulfonate) .

• Genetic Interaction Studies: Rpb7 exhibits genetic interactions with genes involved in diverse DNA repair pathways, providing functional evidence for its role in damage response .

• Functional Conservation: The growth sensitivity of S. pombe cells with low rpb7+ levels under DNA-damaging conditions was completely rescued by human Rpb7, indicating that this function is evolutionarily conserved .

These findings collectively suggest that Rpb7 plays a crucial role in facilitating proper cellular responses to DNA damage, potentially by ensuring adequate expression of repair pathway components.

How can researchers experimentally assess Rpb7's role in DNA damage response?

Methodological approaches to evaluate Rpb7's function in DNA damage response include:

• Survival Assays: Exposing cells with normal versus reduced Rpb7 levels to various DNA-damaging agents (UV radiation, radiomimetic drugs, alkylating agents) and quantifying survival rates provides direct functional assessment.

• Repair Kinetics Measurement: Monitoring the repair of specific DNA lesions over time in cells with different Rpb7 levels can reveal defects in repair efficiency or pathway choice.

• Genetic Interaction Analysis: Combining rpb7 mutations with mutations in known DNA repair genes can reveal synthetic phenotypes that indicate pathway relationships. This can be done systematically using genetic screens or targeted approaches .

• Chromatin Immunoprecipitation: ChIP assays examining Rpb7 recruitment to sites of DNA damage can determine if Rpb7 directly participates in the damage response beyond its role in transcription.

• Expression Rescue Experiments: Testing whether overexpression of specific DNA repair factors can rescue the DNA damage sensitivity of rpb7-deficient cells helps identify which repair pathways are most affected .

These approaches collectively allow researchers to dissect both the transcriptional and potential non-transcriptional roles of Rpb7 in DNA damage response.

How functionally conserved is Rpb7 between yeast and humans?

• Protein Stabilization Role: In mammalian cells, Rpb7 is required for the stability of Rpb1 proteins, whereas this function appears to differ in yeast, suggesting evolutionary divergence in some aspects of Rpb7 function .

• RNA-Binding Domain Conservation: The RNA-binding domains and critical residues involved in RNA interaction show strong conservation between species, though with some differences in the specific patterns of conserved surface residues between archaeal and eukaryotic homologs .

This combination of functional conservation and species-specific adaptations provides valuable insights into the core essential functions of Rpb7 versus roles that may have evolved more recently.

What are the key structural differences between Rpb7 in different species?

Comparative structural analysis reveals both conserved features and species-specific differences in Rpb7:

These structural differences may underlie some of the functional distinctions observed between Rpb7 from different species.

Which specific residues of Rpb7 are critical for RNA binding?

Mutational analysis has identified specific residues in human Rpb7 that are critical for RNA binding, with varying degrees of impact:

• Moderate Impact Residues: Mutations of Thr90, Asn93, Lys94, and Phe107 decrease RNA binding by approximately 50% .

• Strong Impact Residues: Other residues, including His14, Glu33, Lys41 (in the N-terminal domain) and Ser109, His111, Arg151, Asp153, and Phe158 (in the C-terminal domain), have a more pronounced effect on RNA binding when mutated .

• Critical Residues: Phenylalanine 98 appears to be particularly crucial, as mutations at this position severely affect protein solubility, preventing functional analysis. This residue is highly conserved across species .

The pattern of RNA-binding residues spans both domains of Rpb7, suggesting that RNA interacts with Rpb7 along an extended surface. This binding may be important for guiding nascent RNA as it exits the polymerase complex.

How can researchers experimentally characterize the RNA-binding properties of Rpb7?

Methodological approaches for studying Rpb7-RNA interactions include:

• Mutational Analysis: Site-directed mutagenesis of conserved surface residues followed by RNA binding assays can identify critical binding determinants, as demonstrated in studies of both archaeal and human Rpb4/Rpb7 complexes .

• RNA Binding Assays: Electrophoretic mobility shift assays (EMSAs) have been effectively used to quantify the RNA binding capabilities of wild-type and mutant Rpb7 proteins, allowing comparison of relative binding affinities .

• Structural Studies: X-ray crystallography of Rpb4/Rpb7 complexes provides insights into the structural basis for RNA binding. These structures can be determined at high resolution (e.g., 2.0 Å), revealing detailed molecular interactions .

• Cross-linking and Immunoprecipitation: Techniques such as CLIP-seq can identify RNA sequences that interact with Rpb7 in vivo, providing insights into binding preferences and potential regulatory roles.

• Proximity Labeling Approaches: Methods like TurboID can identify proteins that interact with Rpb7 in an RNA-dependent manner, helping to characterize the broader functional context of Rpb7-RNA interactions .

These complementary approaches allow researchers to develop a comprehensive understanding of how Rpb7 interacts with RNA and how these interactions contribute to its functions in transcription and RNA processing.

How does Rpb7 interact with Rpb4 and what is the functional significance of this heterodimer?

The Rpb4/Rpb7 heterodimer forms a distinct structural module that protrudes from the 10-subunit core of RNA polymerase II. Key aspects of this interaction include:

• Structural Basis: Crystal structures reveal that Rpb4 and Rpb7 form a tight heterodimer with extensive interaction surfaces. In human Rpb4/Rpb7, specific regions (including residues 154-172 of Rpb7) mediate these interactions .

• Functional Interdependence: The formation of the Rpb4/Rpb7 heterodimer is critical for the stability and function of both proteins. The heterodimer as a unit then associates with the core polymerase complex.

• RNA Polymerase II Assembly: The Rpb4/Rpb7 heterodimer appears to play a role in the assembly or stability of the complete RNA polymerase II complex, particularly in mammalian cells where Rpb7 is required for Rpb1 stability .

• RNA Processing Functions: The Rpb4/Rpb7 heterodimer is positioned near the RNA exit channel and may coordinate transcription with RNA processing events such as splicing and 3' end formation .

• Regulatory Interactions: The heterodimer can serve as a platform for interaction with other regulatory factors, as evidenced by its association with proteins like CTDP1, a CTD phosphatase .

The Rpb4/Rpb7 heterodimer thus serves both structural and functional roles in the context of RNA polymerase II activity.

What other protein factors interact with Rpb7 during transcription?

Rpb7 interacts with several protein factors during transcription, forming a network of functional interactions:

• CTDP1 Interaction: Rpb7 interacts with CTDP1, a CTD phosphatase, with the 154-172 region of Rpb7 being necessary for this interaction. This interaction appears to be dependent on the presence of Rpb1, suggesting a tripartite relationship .

• RNA Processing Factors: Proximity labeling studies using TurboID-tagged Rpb1 have identified numerous RNA processing factors that interact with the RNA polymerase II complex in an Rpb7-dependent manner. These include factors involved in RNA splicing and 3' end processing .

• Chromatin Modifiers: Rpb7 depletion affects interactions between Rpb1 and various chromatin modification factors, suggesting a role for Rpb7 in coordinating transcription with chromatin state .

The interaction network of Rpb7 thus extends beyond the core transcription machinery to include factors involved in various aspects of gene expression regulation, highlighting its multifunctional role in eukaryotic cells.

How does Rpb7 contribute to transcription termination and RNA processing?

Recent research has begun to uncover Rpb7's role in post-initiation processes including termination and RNA processing:

• Transcription Termination: ChIP-seq analysis following Rpb7 depletion has revealed increased polymerase read-through at termination sites, suggesting Rpb7 may contribute to efficient termination. The read-through index calculated from pSer2 ChIP-Seq data significantly increases following Rpb7 degradation, even after normalizing by the total Rpb1 read-through index .

• RNA Splicing: Analysis of intron retention events following Rpb7 depletion indicates that Rpb7 plays a role in RNA splicing. Interestingly, the splicing events affected by Rpb7 and CTDP1 showed limited overlap, suggesting they may have distinct impacts on RNA processing .

• Mechanistic Questions: Key unresolved questions include whether Rpb7's effects on RNA processing are direct consequences of its RNA-binding activity or indirect results of its role in transcription, and whether these functions can be separated experimentally.

Future research using approaches that can distinguish direct from indirect effects and temporally resolve the sequence of events will be critical for understanding these complex functions.

What is the relationship between Rpb7 and RNA polymerase II phosphorylation?

The interplay between Rpb7 and RNA polymerase II phosphorylation represents an important area for further investigation:

• CTDP1 Interaction: Rpb7 interacts with CTDP1, a phosphatase that dephosphorylates the C-terminal domain (CTD) of Rpb1. This interaction suggests Rpb7 may influence the phosphorylation state of the polymerase .

• Phosphorylation Dynamics: Rpb7 depletion affects the relative levels of Ser2 and Ser5 phosphorylation on the Rpb1 CTD, potentially through altered recruitment or activity of CTD kinases and phosphatases .

• Functional Consequences: Changes in CTD phosphorylation patterns following Rpb7 depletion may contribute to observed defects in transcription reinitiation, elongation, and RNA processing .

Understanding the molecular mechanisms by which Rpb7 influences polymerase phosphorylation and how these effects translate to functional outcomes remains an important frontier in transcription research.

How do different expression levels of Rpb7 affect cellular phenotypes?

Research in S. pombe has revealed that the relative abundance of different RNA polymerase II subunits varies significantly, raising questions about the functional significance of these differences:

This variation suggests potential regulatory mechanisms through differential expression or stability of subunits. Future research directions could include:

• Systematically altering Rpb7 expression levels to determine dose-dependent effects on transcription of different gene classes

• Investigating whether Rpb7 abundance changes in response to different cellular stresses or developmental stages

• Determining whether excess unassembled Rpb7 has functions independent of its role in RNA polymerase II

These approaches would help clarify whether relative subunit abundance serves as a regulatory mechanism for transcription or reflects other aspects of cellular economy.

What are the key technical challenges in studying Rpb7 function in vivo?

Researchers face several technical challenges when investigating Rpb7 function:

• Essential Gene Status: Rpb7 is essential for viability in most eukaryotes, requiring the use of conditional systems rather than simple knockout approaches.

• Distinguishing Direct from Indirect Effects: Because Rpb7 depletion affects global transcription, distinguishing specific functions from general transcriptional defects requires carefully designed experiments and controls.

• Temporal Resolution: Many phenotypes observed following Rpb7 depletion could represent either immediate effects or downstream consequences of prolonged transcriptional defects. Time-resolved approaches are needed to distinguish these possibilities.

• Separating RNA Polymerase II-Dependent and Independent Functions: Determining whether certain Rpb7 functions occur in the context of the polymerase complex or independently requires specialized approaches.

• Quantitative Analysis of Multiprotein Complexes: Accurately measuring the stoichiometry and dynamics of Rpb7 within different complexes presents technical challenges.

Addressing these challenges requires combining multiple complementary approaches and careful experimental design.

How can researchers distinguish between transcriptional and post-transcriptional roles of Rpb7?

Separating transcriptional from post-transcriptional functions of Rpb7 requires sophisticated experimental strategies:

• Mutational Separation of Function: Developing Rpb7 mutants that maintain polymerase association but lose specific functions (or vice versa) can help distinguish different roles.

• Rapid Depletion Approaches: Using fast-acting degron systems followed by time-course analysis can help identify primary versus secondary effects.

• In Vitro Reconstitution: Biochemical systems using purified components can test whether Rpb7 directly affects RNA processing events independently of its role in transcription.

• Structural Studies: High-resolution structures of Rpb7 in different functional contexts can provide insights into the molecular basis for different activities.

• Genome-Wide Correlation Analysis: Comparing patterns of transcriptional changes with RNA processing defects following Rpb7 perturbation can help distinguish direct from indirect effects.

The combination of these approaches will be necessary to fully elucidate the multifaceted functions of this conserved polymerase subunit.

What are the most significant unanswered questions about Rpb7 function?

Despite significant advances in understanding Rpb7, several important questions remain:

• Mechanistic Basis for Differential Gene Regulation: How does Rpb7 preferentially affect certain gene programs, such as DNA repair pathways, and what is the molecular basis for this specificity?

• Species-Specific Functions: What accounts for the functional differences observed between yeast and mammalian Rpb7, particularly regarding Rpb1 stability?

• RNA Processing Role: Does Rpb7 directly participate in RNA processing events beyond its role in transcription, and if so, through what mechanisms?

• Regulatory Modifications: Are there post-translational modifications of Rpb7 that regulate its various functions, and how are these controlled?

• Stress Response Functions: How does Rpb7 contribute to cellular responses to different types of stress, and are these functions conserved across species?

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, genetics, and systems biology.

What emerging technologies might advance Rpb7 research?

Several emerging technologies show promise for advancing our understanding of Rpb7:

• Cryo-EM: High-resolution cryo-electron microscopy can capture RNA polymerase II complexes in different functional states, providing insights into the dynamic roles of Rpb7.

• Single-Molecule Techniques: Methods that track individual polymerase molecules during transcription can reveal how Rpb7 influences polymerase dynamics and processivity.

• Genome Editing with Base-Specific Precision: CRISPR-based approaches that allow precise mutation of specific residues in endogenous genes will facilitate structure-function studies of Rpb7 in its native context.

• Proximity Labeling Technologies: Advanced proximity labeling methods can map the changing interaction partners of Rpb7 under different conditions and in different subcellular locations.

• Integrative Multi-Omics Approaches: Combining transcriptomics, proteomics, and structural biology data can provide a comprehensive view of Rpb7 function in different cellular contexts.