Recombinant Dictyostelium discoideum Ribosome biogenesis regulatory protein homolog (rrs1)

Basic Research Questions

• What is RRS1 and what is its role in Dictyostelium discoideum?

  RRS1 (Regulator of Ribosome Synthesis 1) is a protein primarily localized in the nucleolus and endoplasmic reticulum that plays a critical role in ribosome biogenesis. In Dictyostelium discoideum, as in other eukaryotes, RRS1 regulates 25S rRNA maturation and 60S ribosomal subunit assembly . It forms complexes with other proteins, including RPF2, to recruit 5S rRNA, RPL5, and RPL11 during ribosome biosynthesis . Studies have shown that RRS1 is involved in multiple cellular processes beyond ribosome assembly, including protein secretion and cell cycle regulation .

• Why is Dictyostelium discoideum a useful model organism for studying RRS1?

  Dictyostelium discoideum offers several advantages as a model organism for studying proteins like RRS1:

  • It possesses a fully sequenced, haploid genome with low redundancy, facilitating genetic manipulations

  • It has a unique life cycle with both unicellular and multicellular phases, allowing researchers to study protein function in different cellular contexts

  • Most ribosomal protein genes exist as single copies, unlike the multiple paralogs found in many higher eukaryotes

  • The organism contains orthologs of many genes found in higher eukaryotes, making findings potentially translatable to human biology

  • Its rapid growth and development cycle (24 hours for complete development) enables efficient experimental timelines

• What methods are commonly used to express recombinant RRS1 in Dictyostelium discoideum?

  For expressing recombinant RRS1 in Dictyostelium discoideum, researchers typically employ:

  • Extrachromosomal plasmid vectors containing appropriate Dictyostelium promoters (such as the actin 15 promoter) for constitutive expression

  • Inducible expression systems using the tetracycline-responsive element

  • Expression constructs with fluorescent protein tags (GFP, mCherry) for protein localization studies

  • CRISPR-Cas9 genome editing for endogenous gene modification

  The expression protocol typically involves:

  1. Cloning the RRS1 gene into an appropriate Dictyostelium expression vector

  2. Transformation into Dictyostelium cells using electroporation

  3. Selection of transformants using appropriate antibiotics

  4. Confirmation of expression using Western blotting or fluorescence microscopy

Advanced Research Questions

• How does RRS1 interact with other proteins during ribosome biogenesis in Dictyostelium discoideum?

  RRS1 functions within a complex network of protein interactions during ribosome biogenesis:

  Interacting Partner	Function in Complex	Consequence of Disruption
  RPF2	Forms complex with RRS1 to recruit 5S rRNA, RPL5, and RPL11	Impaired 60S subunit assembly
  RPL11	Recruited by RRS1 for 60S subunit assembly	Reduced ribosome synthesis and p53 pathway activation
  EBP2	Forms complex with RRS1 to stabilize pre-ribosomal subunit	Decreased ribosome stability

  In Dictyostelium, the RRS1-RPF2 complex is particularly important for ribosome maturation. When this interaction is disrupted, pre-rRNA is incorrectly exported from the nucleolus to the nucleoplasm but fails to reach the cytoplasm, significantly affecting 60S subunit biosynthesis . Additionally, the binding of RRS1 to RPL11 appears to prevent RPL11 from interacting with MDM2, which would otherwise inhibit p53 ubiquitination .

• What are the experimental approaches to study RRS1's role in rRNA maturation in Dictyostelium discoideum?

  To investigate RRS1's role in rRNA maturation in Dictyostelium discoideum, researchers employ several methodologies:

  1. Northern blotting analysis:

     • To detect precursor and mature rRNA species following RRS1 knockdown or overexpression

     • Specific probes can distinguish between 28S, 18S rRNAs and their precursors

  2. Polyribosome profiling:

     • Sucrose gradient centrifugation to separate monosomes and polysomes

     • Analysis of ribosome subunit assembly and translational efficiency

  3. Immunofluorescence microscopy:

     • Localization of RRS1 within nucleolar compartments

     • Co-localization with nucleolar markers like NPM (nucleophosmin)

  4. Pulse-chase experiments with labeled nucleotides:

     • Track the kinetics of rRNA processing in the presence or absence of RRS1

  5. RNA-Seq:

     • Genome-wide analysis of transcriptional changes following RRS1 manipulation

• How does disruption of RRS1 affect genome stability in Dictyostelium discoideum?

  Ribosome biogenesis dysregulation through RRS1 disruption may impact genome stability in Dictyostelium through several mechanisms:

  • Defects in ribosome assembly trigger nucleolar stress, which can activate DNA damage response pathways

  • In the absence of proper RRS1 function, RPL11 may be released from the nucleolus and bind to MDM2, activating p53-dependent cell cycle checkpoint pathways

  • Dictyostelium cells are predominantly in G2 phase with a very short or undetectable G1 phase, which affects their response to genomic stress

  • Studies in Dictyostelium have shown that disruption of ribosome biogenesis can affect DNA repair pathway choice between non-homologous end joining (NHEJ) and homologous recombination (HR)

  Research indicates that Dictyostelium discoideum has remarkable resistance to DNA damaging agents and possesses orthologs of numerous DNA repair proteins previously thought to be restricted to vertebrates . This makes it an excellent model to study the interplay between ribosome biogenesis stress and genome stability.

• What is the role of RRS1 in the lifecycle of Dictyostelium discoideum and how can it be studied?

  To study RRS1's role throughout Dictyostelium's lifecycle:

  • During vegetative growth:

    • Create RRS1 knockdown or overexpression strains and measure growth rates

    • Analyze ribosome profiles and protein synthesis rates

    • Assess cell cycle distribution using flow cytometry

  • During development:

    • Induce development through starvation

    • Monitor developmental progression at key timepoints (0, 6, 12, 18, 24 hours)

    • Perform transcriptomic analysis to identify stage-specific changes in RRS1 expression

    • Use fluorescently labeled strains to track RRS1-manipulated cells during development

  • In spore formation and germination:

    • Examine the efficiency of spore formation in RRS1-modified strains

    • Test spore viability and germination rates

    • Analyze the role of NHEJ versus HR in germinating spores with altered RRS1 expression

  Dictyostelium's developmental cycle allows researchers to study how RRS1-dependent ribosome biogenesis might be regulated during different stages of multicellular development. Research has shown that hatching spores have an increased dependence on NHEJ for tolerance to DNA double-strand breaks, suggesting that repair pathway choice may change in differentiated cells .

• How does RRS1 contribute to translational regulation in Dictyostelium discoideum during stress conditions?

  During stress conditions, RRS1 may play a crucial role in translational reprogramming in Dictyostelium:

  • Under nutrient deprivation, which triggers Dictyostelium development, RRS1 may help coordinate ribosome production with cellular needs

  • In response to DNA damage, RRS1 retention of RPL11 in the nucleolus can influence p53 pathway activation and cell cycle progression

  • Stress-induced changes in ribosome composition might alter translational preferences for specific mRNAs

  Experimental approaches to study this include:

  1. Ribosome profiling under stress conditions with and without RRS1 manipulation

  2. Analysis of translational efficiency of specific mRNAs during stress

  3. Investigation of stress granule formation and composition in RRS1-depleted cells

  4. Examination of how the developmental program is affected by RRS1 alterations

• What are the technical challenges in purifying active recombinant RRS1 from Dictyostelium discoideum?

  Several technical challenges exist when purifying active recombinant RRS1:

  1. Protein solubility: RRS1 tends to form complexes with other nucleolar proteins, making isolation of the soluble protein challenging

  2. Maintaining native conformation: Nuclear/nucleolar proteins often require specific buffer conditions to maintain their functional structure

  3. Co-purification of binding partners: RRS1 strongly interacts with proteins like RPF2 and RPL11, which may co-purify

  4. Post-translational modifications: Ensuring proper phosphorylation or other modifications that may be essential for activity

  To address these challenges, researchers can:

  • Use mild detergents and optimized buffer conditions

  • Employ affinity tags that minimally impact protein function

  • Consider purifying functional complexes rather than isolated RRS1

  • Validate purified protein activity through in vitro ribosome assembly assays

• How can in situ structural biology techniques be applied to study RRS1 function in Dictyostelium ribosomes?

  Recent advances in in situ structural biology offer powerful approaches to study RRS1 in its native context:

  1. Cryo-electron tomography (cryo-ET) with subtomogram averaging:

     • Can resolve ribosome structures up to 3Å resolution in cryo-FIB milled Dictyostelium cells

     • Enables visualization of different translational states within the cellular environment

     • Can potentially capture RRS1-ribosome interactions in their native context

  2. Proximity labeling approaches:

     • BioID or APEX2 fused to RRS1 to identify neighboring proteins in vivo

     • Helps map the dynamic interactome of RRS1 during ribosome assembly

  3. Correlative light and electron microscopy (CLEM):

     • Combines fluorescence imaging of tagged RRS1 with electron microscopy

     • Provides context for RRS1 localization within cellular ultrastructure

  4. Super-resolution microscopy:

     • Techniques like PALM or STORM can visualize RRS1 distribution in nucleoli

     • Can track dynamic changes in RRS1 localization during cell cycle or stress

  These techniques can reveal how RRS1 contributes to the unique arrangement of rRNA expansion segments observed in Dictyostelium ribosomes , potentially uncovering species-specific adaptations in ribosome assembly.

• How does RRS1 expression change during Dictyostelium development and what are the implications?

  RRS1 expression may vary throughout Dictyostelium's developmental cycle:

  Developmental Stage	RRS1 Expression Pattern	Potential Function
  Vegetative growth	High expression	Active ribosome biogenesis for proliferation
  Early aggregation	Likely decreased	Adaptation to starvation conditions
  Slug formation	Possibly differential in pre-stalk vs. pre-spore cells	Cell-type specific protein synthesis
  Culmination	May increase in spore cells	Preparation for dormancy and future germination

  These expression changes likely impact:

  • The rate of ribosome biogenesis during different developmental stages

  • Cell-type specific translational programs

  • Stress responses during development

  • DNA repair pathway choice in different cell types

  To study these changes, researchers can use:

  1. Stage-specific RT-qPCR analysis of RRS1 expression

  2. Reporter constructs with the RRS1 promoter

  3. Cell-type specific transcriptomics

  4. Conditional RRS1 knockdown during specific developmental stages

• What is the potential of Dictyostelium RRS1 as a target for studying anti-cancer mechanisms?

  Dictyostelium RRS1 offers several advantages for studying anti-cancer mechanisms:

  1. Studies have shown that RRS1 overexpression promotes tumor growth and metastasis in various cancers, including retinoblastoma

  2. RRS1 activates the AKT/mTOR signaling pathway , which is frequently dysregulated in cancer

  3. RRS1 can prevent the activation of the RPL11-MDM2-p53 pathway , potentially allowing cancer cells to evade apoptosis

  4. Dictyostelium provides a simplified system to study these mechanisms:

     • The AKT/mTOR pathway components are conserved but with less redundancy

     • The p53 pathway can be studied in a more tractable system

     • High-throughput screening methods are established in Dictyostelium

  Experimental approaches could include:

  • Testing compounds that disrupt RRS1-RPL11 interaction

  • Screening for molecules that selectively inhibit cells with RRS1 overexpression

  • Using the Dictyostelium-Mycobacterium marinum infection model to identify compounds with both anti-infective and anti-cancer potential