DENR Human

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

DENR is a noncanonical translation factor that forms a heterodimeric complex with MCTS1 to facilitate translation re-initiation. This process occurs after ribosomes terminate translation at upstream open reading frames (uORFs), allowing the 40S ribosomal subunit to resume scanning and initiate another translation event downstream . This mechanism is distinct from canonical translation initiation and represents a specialized pathway for regulating protein biosynthesis. DENR appears to have tissue-specific effects, with particularly important roles demonstrated in neuronal development, making it a significant factor in human cellular function beyond general translation processes.

How does DENR interact with MCTS1 in translation re-initiation?

DENR forms a stable heterodimeric complex with MCTS1, creating a functional unit that specifically promotes re-initiation at certain uORFs. Research indicates that DENR is an obligatory component of this heterodimer, while MCTS1 can be substituted by its paralog MCTS2 in some contexts . The interaction between these proteins has been validated through both in vivo and in vitro approaches, including ribosome profiling and cell-free translation systems, which demonstrate the necessity of this complex for efficient re-initiation at specific mRNA sequences. When ribosomes scanning from the mRNA 5′ cap encounter uORFs, they may initiate translation, which typically reduces translation of the downstream main coding sequence (CDS) .

What methods can researchers use to detect DENR protein expression?

Multiple experimental methods are effective for detecting and analyzing DENR protein expression:

• Immunoblotting (Western blotting): Standard method for detecting DENR protein levels, as demonstrated in validation studies of degron system functionality .

• CRISPR gene editing for protein tagging: The endogenous DENR locus can be modified to express DENR protein with a C-terminal tag (e.g., 3×FLAG) suitable for immunoprecipitation and protein detection .

• Immunoprecipitation coupled with mass spectrometry: This approach enables purification and identification of DENR and its binding partners, including MCTS1 and MCTS2 .

• Ribosome profiling combined with RNA-seq: While not directly measuring DENR protein, this approach assesses DENR functionality by measuring translation efficiency changes when DENR is knocked down .

Each method provides complementary information about DENR expression and function at both the protein and functional levels, allowing researchers to develop a comprehensive understanding of DENR's role in human cells.

What is the mechanism of MCTS1-DENR-dependent re-initiation?

The mechanism of MCTS1-DENR-dependent re-initiation involves a specialized process that diverges from canonical translation principles. After translation termination at a uORF, the MCTS1-DENR complex facilitates the resumption of scanning by the 40S ribosomal subunit instead of complete ribosomal recycling . This allows the 40S subunit to reinitiate translation at a downstream start codon.

MCTS1-DENR appears to function specifically for re-initiation rather than general translation, making it distinct from other factors like components of eIF3 or eIF4 that also influence re-initiation but have broader roles in translation . For these general eIFs, analysis of specific re-initiation effects is challenging, as they cannot easily be separated from global translational alterations and their secondary effects. The development of cell-free re-initiation assays has enabled more detailed investigation of this mechanism, allowing researchers to dissect the requirements for specific uORF features and trans-acting factors under controlled conditions .

How does the MCTS2-DENR heterodimer differ functionally from MCTS1-DENR?

Recent research has revealed that MCTS2, a paralog and retrogene copy of MCTS1, can also form a heterodimer with DENR . Immunoprecipitation experiments identified MCTS2 as one of the most strongly enriched co-purifying proteins with DENR, indicating that DENR assembles into two distinct heterodimeric complexes: MCTS1-DENR and MCTS2-DENR .

While previous studies had suggested that MCTS2 might be largely non-functional, interaction data from endogenous proteins suggests that both MCTS paralogs form bona fide heterodimers with DENR . Expression analysis has indicated higher levels of MCTS2 than MCTS1 in certain cell types, suggesting MCTS2 may be a relevant interaction partner for DENR alongside MCTS1 . This finding provides a potential explanation for the clinical differences observed between DENR and MCTS1 mutations in humans, as MCTS2 may compensate for MCTS1 loss but not for DENR deficiency.

How can ribosome profiling be used to identify DENR-dependent translation events?

Ribosome profiling (Ribo-seq) combined with RNA-seq provides a powerful approach for identifying DENR-dependent translation events. The methodology involves:

• Preparing cells with DENR knockdown (e.g., via shRNA) and appropriate controls

• Performing Ribo-seq to capture the position of translating ribosomes on mRNAs by sequencing ribosome-protected fragments

• Conducting parallel RNA-seq to quantify mRNA abundance

• Calculating translation efficiency (TE) as the ratio of CDS ribosome footprints to mRNA levels for each transcript

• Identifying transcripts with significantly altered TE upon DENR depletion through statistical analysis

In published research, this approach revealed 223 transcripts with significantly reduced TE and 6 with increased TE after DENR knockdown . The transcripts with reduced TE had significantly longer 5' UTRs than the transcriptome average, consistent with increased uORF content . Further annotation of translated uORFs from the footprint data confirmed enrichment of uORFs among DENR-responsive transcripts, providing a genome-wide view of DENR-dependent translation events.

What are the clinical implications of DENR mutations in humans?

Research indicates notable clinical differences associated with DENR versus MCTS1 mutations in humans . While specific clinical presentations are not fully detailed in the current literature, it has been observed that phenotypes in mouse neuronal migration are stronger upon DENR loss than MCTS1 loss . This suggests that DENR deficiency has more severe consequences than loss of either interaction partner alone.

The identification of MCTS2 as an alternative DENR partner provides "a plausible explanation for the striking clinical differences associated with DENR vs. MCTS1 mutations in humans" . This is consistent with the finding that DENR can form functional heterodimers with either MCTS1 or MCTS2, making it an obligatory component while its partners may have some functional redundancy. These observations indicate that DENR mutations likely have significant clinical implications, particularly in neuronal development.

How can in vitro translation systems be used to study DENR function?

In vitro translation systems provide a powerful approach to study DENR function, particularly in the context of translation re-initiation. Researchers have developed a cell-free re-initiation assay using HeLa cell lysates that accurately recapitulates MCTS1-DENR-dependent re-initiation observed in vivo . This system offers several methodological advantages:

• Controlled conditions: The cell-free environment allows for precise manipulation of components and conditions to isolate specific effects.

• Quantitative measurement: The assay enables calculation of ribosomal fluxes and quantification of re-initiation efficiency under various conditions.

• Component manipulation: The system permits depletion of specific factors (e.g., using knockout lysates) and complementation with recombinant proteins to test necessity and sufficiency.

• Mechanistic dissection: It allows testing of specific uORF features and trans-acting factors under defined conditions that would be difficult to achieve in vivo.

The system has been validated by comparing in vivo and in vitro re-initiation activities on uORF-containing model reporters and demonstrating the dependence on MCTS1-DENR through knockout experiments and rescue with recombinant proteins . This approach enables researchers to query the requirement for specific factors in re-initiation independently of their roles in general translation.

What reporter assays are suitable for measuring DENR-dependent re-initiation?

Several reporter assay approaches have proven effective for measuring DENR-dependent re-initiation:

• Dual reporter systems: These typically involve a uORF followed by a reporter gene (e.g., luciferase), allowing quantification of re-initiation efficiency under various conditions .

• Cell-based reporter assays: In vivo reporter assays can be used in combination with ribosome profiling and cell-free translation systems to investigate MCTS1-DENR-dependent re-initiation in cellular contexts .

• In vitro re-initiation assay: Cell-free re-initiation assays using HeLa cell lysates can be validated with different reporter constructs and variants to measure re-initiation under controlled conditions .

• Gene-specific reporters: Specific uORF-containing transcripts (e.g., Klhdc8a and Asb8) that are regulated by DENR in vivo can be adapted for model reporter design to study physiologically relevant re-initiation events .

These reporters allow researchers to quantify re-initiation efficiency under various conditions, test the impact of mutations in uORF sequences or interactor proteins, and compare DENR-dependent re-initiation across different cellular contexts or experimental manipulations.

How can CRISPR-Cas9 gene editing be used to study DENR function?

CRISPR-Cas9 gene editing offers several powerful approaches for studying DENR function:

• Endogenous tagging: The endogenous DENR locus can be modified to express DENR protein with a C-terminal tag (e.g., 3×FLAG) suitable for immunoprecipitation while preserving the natural expression levels and regulation .

• Inducible degradation systems: CRISPR modification can include a degron tag (e.g., dTAG system) that allows for controlled depletion of DENR protein upon addition of a specific degrader compound (dTAG-13), providing temporal control over DENR levels .

• Knockout generation: CRISPR can be used to generate knockout cell lines by introducing frameshift mutations or larger deletions, allowing study of complete DENR loss .

• Domain-specific mutations: Specific mutations in functional domains of DENR can be introduced to dissect their roles in protein-protein interactions or ribosome binding.

These CRISPR-based approaches enable precise manipulation of DENR and its interacting partners in their endogenous context, providing insights into function that may not be achievable through overexpression or RNAi-based knockdown approaches.

What proteomics approaches can identify DENR-interacting proteins?

Immunoprecipitation coupled with mass spectrometry has proven effective for identifying DENR-interacting proteins. A methodological approach includes:

• Endogenous tagging of DENR: Using CRISPR gene editing to modify the endogenous DENR locus to express DENR with a C-terminal 3×FLAG tag suitable for immunoprecipitation .

• Inducible degradation control: Including a dTAG degron system, allowing for controlled depletion of DENR upon treatment with dTAG-13 degrader compound to create a negative control for the immunoprecipitation experiment .

• Differential immunoprecipitation: Performing FLAG-IP on extracts from control-treated versus dTAG-13-treated cells to identify proteins that specifically co-purify with DENR rather than binding non-specifically .

• Mass spectrometry analysis: Identifying proteins enriched in the control versus dTAG-13 samples by mass spectrometry .

This approach successfully identified MCTS1 and MCTS2 as the two most strongly enriched co-purifying proteins with DENR, revealing that DENR assembles into two distinct heterodimeric complexes . This finding had significant implications for understanding DENR function and the clinical differences between DENR and MCTS1 mutations.

How should translation efficiency changes be calculated in DENR knockdown experiments?

Translation efficiency (TE) changes in DENR knockdown experiments should be calculated using a combination of ribosome profiling (Ribo-seq) and RNA-seq data through the following methodology:

• Obtain ribosome footprint counts: Perform Ribo-seq to capture the position of translating ribosomes on mRNAs and quantify ribosome footprints mapping to the coding sequence (CDS) of each transcript .

• Measure mRNA abundance: Conduct parallel RNA-seq to quantify the abundance of each mRNA in the same samples .

• Calculate translation efficiency: For each transcript, compute the ratio of CDS ribosome footprints to mRNA abundance (TE = ribosome footprints / RNA) .

• Compare conditions: Calculate TE for both DENR knockdown and control conditions to identify differential translation .

• Identify significant changes: Apply statistical analysis to identify transcripts with significantly altered TE between conditions. In published research, an FDR-corrected p < 0.1 threshold has been applied for significance .

This approach allows for genome-wide assessment of translational changes specifically attributable to DENR depletion, separate from effects on mRNA abundance or stability, revealing the direct targets of DENR-dependent translation regulation.

How can uORF features be correlated with DENR dependency?

Several analytical approaches can be used to correlate uORF features with DENR dependency:

• Identify DENR-responsive transcripts: First, establish which transcripts show significant changes in translation efficiency (TE) upon DENR depletion through ribosome profiling and RNA-seq .

• Annotate uORF features: For all transcripts, particularly those responsive to DENR, annotate uORF features such as:

  • Number of uORFs per transcript

  • uORF length

  • uORF position relative to the cap and main CDS

  • Sequence context around start and stop codons

  • Secondary structure elements

  • Conservation across species

• Compare feature distributions: Analyze whether particular uORF features are enriched among DENR-responsive transcripts compared to non-responsive transcripts. In published research, transcripts with reduced TE upon DENR knockdown had significantly longer 5' UTRs, compatible with increased uORF content .

• Experimental validation: Test the impact of specific uORF features using reporter constructs where these features are systematically varied in in vitro and in vivo assays .

This multi-faceted approach can reveal the specific determinants that make certain uORFs dependent on DENR for efficient re-initiation, providing insights into the mechanism of DENR function.

What statistical approaches are appropriate for identifying DENR-responsive genes?

Appropriate statistical methods for identifying DENR-responsive genes include:

• Differential translation efficiency analysis: Statistical testing to identify significant differences in translation efficiency (TE) between DENR knockdown and control conditions .

• Multiple testing correction: Application of false discovery rate (FDR) correction to account for multiple hypothesis testing when analyzing transcriptome-wide data. Published research has applied FDR-corrected p < 0.1 as a significance threshold .

• Feature enrichment analysis: Statistical assessment of whether certain features (e.g., 5' UTR length) are significantly enriched among DENR-responsive transcripts compared to the transcriptome-wide distribution .

• Correlation analysis: Examination of the relationship between uORF features and the magnitude of TE change upon DENR depletion.

• Rigorous and accelerated data analysis techniques: For qualitative data analysis, the RADaR technique (rigorous and accelerated data reduction) can be applied to systematically analyze transcripts .

These statistical approaches enable robust identification of DENR-responsive genes while controlling for false positives and revealing patterns that provide insights into the determinants of DENR dependency.

How might DENR function be targeted for therapeutic applications?

While therapeutic targeting of DENR is still in early exploratory stages, several potential approaches can be considered:

• Small molecule modulators: The development of in vitro re-initiation assays enables compound screens for specific inhibitors or enhancers of re-initiation that could potentially modulate DENR function in a therapeutic context .

• Gene therapy approaches: Given the clinical differences associated with DENR mutations, gene therapy approaches to correct such mutations or supplement DENR function might be considered for associated conditions .

• Targeting specific mRNA targets: The identification of DENR-responsive transcripts through ribosome profiling provides potential downstream targets that might be modulated for therapeutic benefit in contexts where DENR function is disrupted .

• Exploiting paralog redundancy: The discovery that MCTS2 can partner with DENR alongside MCTS1 suggests potential approaches to enhance the function of one paralog when the other is deficient .

These potential therapeutic avenues require further research to determine their feasibility and efficacy, but they represent promising directions for translating basic DENR research into clinical applications.

What high-throughput screening approaches could identify DENR modulators?

Several high-throughput screening approaches could be developed to identify DENR modulators:

• Reporter-based screening platform: The in vitro re-initiation assay could be adapted for high-throughput screening by incorporating luminescent or fluorescent reporters downstream of DENR-dependent uORFs .

• Compound libraries: The cell-free assay system is suitable for screening chemical libraries to identify specific inhibitors of re-initiation .

• siRNA/CRISPR screens: Genome-wide screens could identify genes that modify DENR activity when depleted or overexpressed, revealing potential regulatory pathways.

• Structure-based virtual screening: Structural biology approaches, including cryo-EM on re-initiation complexes, could enable virtual screening for compounds that might modulate DENR-ribosome interactions .

The cell-free nature of the re-initiation assay makes it particularly suitable for high-throughput applications, as it eliminates many of the complexities associated with cell-based assays while still faithfully recapitulating DENR-dependent re-initiation .

How can researchers integrate DENR studies with broader translational regulation research?

Integration of DENR research with broader translational regulation studies can be achieved through several approaches:

• Comparative analysis: Investigating how DENR-dependent re-initiation intersects with other translation regulatory mechanisms such as canonical initiation, internal ribosome entry, and non-canonical translation .

• Global translation studies: Incorporating DENR analysis into global studies of translation regulation under various physiological and pathological conditions.

• Systems biology approaches: Using network analysis to place DENR within the broader context of translation regulation pathways and identify key nodes of interaction.

• Developmental and tissue-specific studies: Examining how DENR function varies across different developmental stages and tissues, particularly in neuronal contexts where DENR deficiency has strong phenotypes .

• Computational integration: Applying the RADaR technique (rigorous and accelerated data reduction) for qualitative data analysis to systematically analyze and integrate findings from various experimental approaches .

By connecting DENR-specific research with the broader field of translational regulation, researchers can develop a more comprehensive understanding of how this specialized factor contributes to the complex landscape of protein synthesis control in human cells.