Recombinant Drosophila melanogaster 60S ribosomal protein L22 (RpL22)

What is the structural organization of Drosophila melanogaster RpL22?

Drosophila melanogaster RpL22 is a member of the conserved RpL22e family specific to eukaryotes. Unlike many other ribosomal proteins, RpL22 contains a unique N-terminal extension that is homologous to the C-terminal end of histone H1 . This N-terminal domain is rich in alanine, lysine, and proline residues, which structurally resembles the carboxy-terminal portion of histone H1 and histone H5 . The protein has two distinct domains: the N-terminal histone H1/H5-like domain and the C-terminal ribosomal domain (L22e). This structural organization suggests that RpL22 may have dual functionality - one role in ribosome organization and another potentially involving DNA binding similar to histone H1 .

How does RpL22 differ from its paralogue RpL22-like in Drosophila melanogaster?

Despite being paralogues, RpL22 and RpL22-like share only 37% amino acid identity, suggesting considerable divergence in their functions . The structural divergence is most prominent within the N-terminal extension. Their expression patterns are also strikingly different:

RpL22-like mRNA is highly enriched in adult testes compared to ovaries, and the corresponding protein is exclusively found within testes and not within seminal vesicles or accessory glands .

What methods are used to detect and quantify RpL22 expression in different tissues?

To analyze RpL22 and RpL22-like expression, researchers commonly employ:

• Quantitative RT-PCR (qRT-PCR) for mRNA quantification across tissues

• Western blotting with paralogue-specific antibodies to detect protein expression

• Immunohistochemistry (IHC) for spatial localization within tissues

• Polysome fractionation followed by western blotting to confirm ribosomal incorporation

For quantitative analysis, qRT-PCR is particularly valuable as it allows precise measurement of transcript levels. Western blotting can detect the predicted 34 kDa RpL22-like protein in testes and potentially higher molecular weight immunoreactive species in other tissues like fly heads . IHC analysis is essential for determining the exact cellular localization, having confirmed that RpL22-like is exclusively found within testes and not within other reproductive tract tissues .

How can recombinant RpL22 protein be produced and purified for functional studies?

Production of recombinant Drosophila RpL22 involves:

• Amplification of the full-length cDNA using high-fidelity PCR

• Cloning into an appropriate expression vector (e.g., pET-200)

• Transformation into E. coli expression hosts (such as BL21 Star)

• Induction of protein expression

• Purification under native conditions

The detailed methodology includes:

• Amplifying the RpL22 gene using specific primers (e.g., pETup/pETlow) from cDNA

• Cloning the purified PCR product into the pET-200 expression vector

• Transforming the resulting plasmid into E. coli BL21 Star expression host

• Expressing the gene following manufacturer's protocols

• Purifying the His6-tagged protein using Ni-NTA affinity chromatography under native conditions

• Confirming protein size by SDS-PAGE and determining concentration by Bradford method

For specific domain studies, separate amplification and cloning of the histone-like domain and ribosomal domain can be performed using domain-specific primers (e.g., pETup/H5low and pETlow/L22up respectively) .

What techniques are effective for studying RpL22 interaction with nucleic acids?

For investigating RpL22's interaction with DNA or RNA:

• Electrophoretic Mobility Shift Assay (EMSA) is highly effective for studying direct interactions between RpL22 and nucleic acids. The interaction specificity can be validated through competition experiments using:

  • Radiolabeled target sequence (e.g., TERM sequence)

  • Increasing amounts of unlabeled specific competitor

  • Non-specific competitor (e.g., sonicated λ-DNA)

• Yeast One-Hybrid assay for detecting protein-DNA interactions in vivo

• RNA immunoprecipitation (RIP) followed by sequencing (RIP-seq) to identify RNAs associated with RpL22 in vivo

For example, EMSA experiments have demonstrated that purified RpL22 specifically binds to TERM3 DNA, with the interaction mediated by the H1/H5-like domain rather than the ribosomal domain . Adding small quantities (5x) of specific unlabeled competitor can disrupt the complex, while up to 500-fold excess of non-specific competitor does not affect binding, confirming interaction specificity .

How can researchers differentiate between ribosomal and extra-ribosomal functions of RpL22?

Distinguishing between ribosomal and extra-ribosomal functions requires:

• Subcellular fractionation to separate cytoplasmic and nuclear compartments

• Polysome profiling to identify RpL22 association with actively translating ribosomes

• Immunofluorescence to visualize protein localization

• Domain-specific mutagenesis to disrupt specific functions

• RNA-seq analysis of polysome-associated mRNAs

Nuclear localization of RpL22, demonstrated by immunofluorescence, supports potential extra-ribosomal roles . Meanwhile, detection in 80S/polysome fractions confirms its role as a ribosomal component . Domain-specific studies have shown that the H1/H5-like N-terminal domain is responsible for DNA binding activities, while the C-terminal domain maintains ribosomal function . Creating domain-specific mutants can help determine which cellular processes are affected by each function.

What evidence supports the existence of specialized ribosomes containing RpL22 or RpL22-like in Drosophila?

Several lines of evidence support the existence of specialized ribosomes:

• RNA-seq analysis has identified 12,051 transcripts with approximately 50% being enriched on specific polysome types (either RpL22 or RpL22-like containing ribosomes)

• Analysis of the most abundant mRNAs suggests ribosome specialization for translating groups of mRNAs expressed at specific stages of spermatogenesis

• Rescue experiments show that eRpL22-like cannot fully substitute for eRpL22 function, confirming functionally distinct roles

• The differential expression patterns, with RpL22 being ubiquitous and RpL22-like being testis-specific, suggest tissue-specific roles for each paralogue

This evidence collectively indicates that RpL22 and RpL22-like contribute to ribosome heterogeneity with functional consequences for specific mRNA translation, particularly during germline differentiation .

How do RpL22 and RpL22-like contribute to spermatogenesis in Drosophila?

Both RpL22 and RpL22-like are essential for proper spermatogenesis, but their roles are not completely interchangeable:

• Both proteins are required for sperm maturation and fertility

• Flies depleted of eRpL22 and rescued by eRpL22-like overexpression have reduced fertility

• Specific mRNAs preferentially associate with either eRpL22 or eRpL22-like containing ribosomes

Research shows that approximately 10% of the most abundant testis mRNAs are translated by specialized ribosomes containing specific RpL22 paralogues at different stages of spermatogenesis . Enrichment of "model" eRpL22-like polysome-associated testis mRNAs can occur outside the germline within S2 cells transfected with eRpL22-like, indicating that germline-specific factors are not required for selective translation . This suggests that the ribosome composition itself directly influences which mRNAs are translated, potentially through structural differences in the ribosomes or through specific interactions between mRNAs and the ribosomal proteins.

What methodologies are recommended for studying paralogue-specific translation?

To study paralogue-specific translation:

• Polysome profiling combined with RNA-seq:

  • Isolate polysomes containing specific RpL22 paralogues (using epitope-tagged versions)

  • Extract and sequence associated mRNAs

  • Compare transcripts associated with different paralogue-containing ribosomes

• Ribosome profiling (Ribo-seq) to analyze the exact positions of ribosomes on mRNAs

• Reporter assays:

  • Create reporter constructs with candidate mRNA regulatory elements

  • Express in cells with different ribosome compositions

  • Measure translation efficiency

• Genetic approaches:

  • Paralogue-specific knockdowns or knockouts

  • Rescue experiments with wild-type or mutant versions

  • Analysis of resulting phenotypes, particularly in tissues like testes

• In vitro translation systems reconstituted with specific ribosome compositions

These approaches can reveal which mRNAs are preferentially translated by ribosomes containing specific RpL22 paralogues and identify the mRNA features that mediate selective translation.

What is known about the DNA-binding capabilities of RpL22's histone H1-like domain?

The histone H1-like N-terminal domain of Drosophila RpL22 has been experimentally demonstrated to bind DNA:

• EMSA experiments have shown that RpL22 can directly and specifically bind to DNA sequences, particularly the Transposable Element Redundant Motif (TERM)

• When the H1/H5-like domain and the ribosomal domain are separately expressed and purified, only the H1/H5-like domain shows DNA binding activity

• Competition assays confirm the specificity of this interaction, as it can be disrupted by small amounts of specific competitor DNA but remains unaffected by large excesses of non-specific DNA

• The binding appears to target specific sequence motifs rather than binding DNA indiscriminately

This DNA-binding capability suggests potential roles in transcriptional regulation or control of transposable elements, though these functions require further investigation.

How does RpL22 interact with transposable elements in Drosophila?

RpL22 interacts with transposable elements through:

• Direct binding to a specific 13 bp motif called the Transposable Element Redundant Motif (TERM), identified in the 5'-UTR of LTR-retrotransposons

• This interaction occurs through the H1/H5-like N-terminal domain of RpL22

• The nuclear localization of RpL22 supports its potential role as a controller of the activity of retrotransposons carrying the TERM sequence

The functional significance of this interaction may involve:

• Regulation of retrotransposon mobility to prevent excessive mutational load

• Control of retrotransposon transcription through mechanisms similar to histone H1-mediated repression

• Contribution to genome stability by suppressing transposable element activity

These interactions suggest that RpL22 may have evolved extra-ribosomal functions to protect the genome from potentially harmful transposon activity.

What approaches can be used to investigate potential extra-ribosomal functions of RpL22?

To investigate extra-ribosomal functions:

• Chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-seq):

  • Identify genomic regions bound by RpL22 in vivo

  • Analyze for enrichment of specific sequences or genomic features

• Transcriptome analysis in RpL22 mutants or knockdowns:

  • RNA-seq to identify differentially expressed genes

  • Focus on transposable elements or other potential targets

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify interaction partners

  • Yeast two-hybrid screens to discover novel interactions

  • Mass spectrometry of immunoprecipitated complexes

• Subcellular localization studies:

  • Immunofluorescence to track RpL22 localization under different conditions

  • Cell fractionation followed by western blotting

• Domain-specific mutations:

  • Create mutations specifically in the H1/H5-like domain

  • Assess effects on DNA binding, transposon activity, and ribosomal function

The Drosophila Interactions Database has cataloged several potential protein-protein interactions for RpL22, including interactions with transcriptional repressor complexes and nuclear enzymes like poly-ADP ribose polymerase (mediated through the N-terminal histone H1-like domain) . These interactions provide starting points for investigating extra-ribosomal functions.

How does Drosophila RpL22 compare with its orthologs in other species?

Drosophila RpL22 shows several unique features compared to its orthologs in other species:

• The N-terminal extension of Drosophila RpL22 that is homologous to histone H1 appears to be a distinctive feature not widely found in other species

• The presence of a duplicated gene (RpL22-like) with specialized expression is characteristic of Drosophila but not universal across species

• In contrast to yeast, where most ribosomal protein genes are duplicated, most mammalian ribosomal proteins are thought to be encoded by a single gene copy

This suggests that different evolutionary strategies for ribosomal protein diversity exist across species, with gene duplication and specialization being one mechanism in Drosophila. The acquisition of the histone H1-like domain in Drosophila RpL22 may represent an evolutionary innovation that allowed for additional regulatory functions beyond the protein's role in the ribosome.

What methodology would best determine if the DNA-binding properties of Drosophila RpL22 are conserved in other species?

To assess conservation of DNA-binding properties:

• Comparative sequence analysis:

  • Align RpL22 sequences from multiple species

  • Identify conservation of the histone H1-like domain

  • Predict DNA-binding potential based on structural features

• Recombinant protein studies:

  • Express and purify RpL22 from diverse species

  • Perform EMSA experiments with TERM or other DNA sequences

  • Compare binding affinities and specificities

• Domain swap experiments:

  • Create chimeric proteins with domains from different species

  • Test DNA-binding capabilities of these chimeras

  • Identify which regions are necessary and sufficient for binding

• Functional complementation:

  • Express RpL22 from different species in Drosophila

  • Assess rescue of RpL22 mutant phenotypes

  • Determine if DNA-binding functions are complemented

• Structural studies:

  • Solve structures of RpL22 from multiple species

  • Compare DNA-binding domains

  • Perform docking simulations with DNA

These approaches would provide insights into whether the DNA-binding capacity is a conserved ancestral trait or a derived feature specific to certain lineages.