Recombinant Mouse CUGBP Elav-like family member 6 (Celf6)

What is Celf6 and what are its primary functions?

Celf6 (CUGBP Elav-like family member 6) is an RNA-binding protein (RBP) belonging to the CUGBP Elav-Like Family. It functions primarily as a post-transcriptional regulator that binds to 3'UTRs of target mRNAs, particularly those coding for synaptic proteins . While initially characterized as a splicing factor, comprehensive functional validation studies have revealed that Celf6 acts as a repressor of translation by destabilizing target mRNAs containing UGU-rich sequence motifs . This repressive function appears to be shared across the CELF3-6 subfamily members . Celf6 expression is particularly high in monoaminergic neuronal populations, including serotonergic and dopaminergic cells, suggesting an important role in neurodevelopment and neuromodulatory functions .

What expression patterns does Celf6 show in the nervous system?

Celf6 shows a specific and restricted expression pattern within the nervous system:

Celf6 is enriched in both peptidergic and nonpeptidergic neuronal populations, with particularly high expression in cells involved in pain processing and neurogenic inflammation . Developmentally, Celf6 RNA and protein are present early in a subset of neurons during neurodevelopment and persist into adulthood, with peak expression around birth .

How is Celf6 mutation associated with neurological conditions?

Studies have identified potential connections between Celf6 mutations and neurodevelopmental disorders. Analyses of human polymorphisms in serotonin-expressed transcripts suggested an association between common variants of CELF6 and autism . Additionally, a rare inherited variant of CELF6 resulting in a premature stop codon was detected in a male proband with autism .

Functional studies using Celf6 knockout mouse models revealed behavioral phenotypes that may relate to aspects of neurodevelopmental disorders:

• Decreased ultrasonic vocalizations in pups when isolated from dams and littermates, potentially reflecting early social communicative deficits

• Resistance to change in exploratory behavior during reward conditioning

• Impaired conditioned learning responses to both reward and aversive stimuli

• Cognitive inflexibility in various behavioral tasks

These findings suggest Celf6's role in regulating mRNAs critical for normal neurodevelopment and neuronal function may contribute to behavioral regulation and adaptation.

What are the recommended methods for studying Celf6 protein expression?

Multiple validated approaches exist for detecting and quantifying Celf6 protein expression:

Antibody Selection and Validation:
Antibodies against specific Celf6 peptides have been developed and validated. Particularly effective are antibodies against the peptides QPGSDTLYNNGVSPC and AASEGRGEDRKC, which were selected based on:

• Cross-species conservation

• Relative uniqueness across the Celf family

• Hydrophobicity properties

Recommended Detection Methods:

• Western Blot: Antibodies against both peptides mentioned above demonstrate immunoreactivity by immunoblot when testing protein from Celf6-overexpressing cell lines .

• Immunofluorescence: For fixed-cell imaging, antibodies against the QPGSDTLYNNGVSPC peptide have proven most effective .

• Immunohistochemistry: For studying Celf6 expression in tissue sections, particularly in neuronal populations.

Controls and Validation:

• Use 3T3 cells transfected with GFP-tagged and untagged isoforms of Celf6 as positive controls

• Include Celf6 knockout tissue/cells as negative controls

• Confirm specificity by testing for cross-reactivity with other CELF family members

How can I generate and validate Celf6 knockout mice?

The generation of Celf6 knockout mice involves several critical steps:

Knockout Strategy:

• Design construct with LoxP sites flanking a critical exon (exon 4 of Celf6 has been successfully targeted)

• Include an Frt-flanked neomycin-resistance cassette gene for selection

• Electroporate construct into ES cells (B6(Cg)-Tyr c-2j/j-derived ES cells have been used successfully)

• Screen neomycin-resistant colonies by PCR and southern blot for proper integration

• Inject positive colonies into C57BL/6J mouse blastocysts

• Breed chimeric mice to germline Flpe expressing C57BL/6J mice to remove the neomycin selection cassette

• Cross with actin-Cre C57BL/6J mice to create germline deletions of Celf6

• Confirm recombination in progeny by PCR

Validation Approaches:

• Genomic Verification: PCR using primers flanking the deleted region

• Transcript Analysis: RT-PCR or qPCR to confirm absence of transcript

• Protein Verification: Western blot and immunostaining to confirm absence of protein

• Functional Validation: Assess derepression of known Celf6 target genes using expression microarrays or RNA-seq

What cell lines are appropriate for Celf6 functional studies?

Based on published research, several cell lines have been utilized successfully for Celf6 studies:

When conducting transfection experiments with Celf6, researchers should consider:

• Omitting antibiotics during transfection procedures

• Using appropriate controls (empty vector, mutant constructs)

• Verifying expression levels by western blot

• Allowing sufficient time (typically 24-48 hours) for expression and functional effects

How can I identify and validate in vivo RNA targets of Celf6?

CLIP-Seq (Cross-Linking Immunoprecipitation followed by sequencing) represents the gold standard for identifying direct RNA targets of Celf6:

CLIP-Seq Protocol Overview:

• Cross-link RNA-protein interactions in tissue/cells using UV light

• Immunoprecipitate Celf6 along with bound RNA fragments

• Process RNA for sequencing

• Analyze sequencing data to identify binding sites and motifs

Critical Analytical Considerations:

• Include both wild-type controls (representing non-specific pulldown) and input samples (accounting for differences in starting abundance of possible target mRNAs)

• Normalize for both total library size per sample and feature length

• Use differential expression analysis tools (such as edgeR) to make statistical inferences on enriched features in immunoprecipitated samples compared to controls

• Account for differences in starting abundance of possible target mRNAs to avoid bias toward highly expressed genes

Target Validation Approaches:

• Reporter Assays: Clone UTR elements found under CLIP-Seq peaks into reporter constructs

• Mutagenesis: Mutate predicted binding motifs (UGU-rich sequences) to confirm functional relevance

• Expression Analysis: Compare target mRNA levels between wild-type and Celf6 knockout tissues

• Ribosome Profiling: Assess changes in ribosome occupancy with and without Celf6

What binding motifs does Celf6 recognize and how do they affect function?

Celf6 predominantly recognizes UGU-rich sequence motifs within the 3'UTRs of target mRNAs:

Motif Characteristics:

• Primary sequence contains UGU trinucleotide core elements

• Predominantly located within 3'UTRs of target transcripts

• Similar binding preferences to other CELF family members

Functional Consequences of Binding:
When Celf6 binds to these motifs, it:

• Decreases RNA abundance of the target transcript

• Reduces ribosomal occupancy

• Subsequently decreases protein production

Experimental Validation Approach:
Comprehensive functional validation can be accomplished through massively parallel reporter assays:

• Clone >400 UTR elements containing CLIP-Seq peaks into reporter constructs

• Measure reporter expression with and without Celf6 overexpression

• Systematically mutate all potential binding motifs to assess their importance

• Measure both RNA abundance and ribosomal occupancy to distinguish mechanisms

Such approaches have demonstrated that Celf6-bound elements are generally repressive of translation, and Celf6 enhances this repression via decreasing RNA abundance through a process dependent on UGU-rich sequence motifs .

How does Celf6 function differ from other CELF family members?

Understanding the functional distinctions between CELF family members requires analysis at multiple levels:

Expression Pattern Distinctions:

CELF2 and CELF4 are restricted to peptidergic neurons, while CELF3 is found in tyrosine hydroxylase-expressing neurons, and CELF6 shows the broadest expression pattern in both peptidergic and non-peptidergic populations .

Functional Similarities:

• CELF3-6 appear to share the ability to function as repressors of translation through RNA destabilization

• All recognize similar UGU-rich binding motifs

Regulatory Distinctions:

• The distinct expression patterns suggest non-redundant roles in different neuronal populations

• CELF4 is known to control a regulon that coordinates the translation of mRNAs encoding components of the protein translation apparatus in nociceptors

• CELF4 has been specifically identified as a negative regulator of protein translation and neural excitability

What are the most informative behavioral assays for Celf6 knockout mice?

Based on published phenotypes, several behavioral assays are particularly informative for characterizing Celf6 knockout mice:

Communication and Social Behavior:

• Ultrasonic vocalization recording in pups separated from dams (shows robust decreases in Celf6-/- mice)

• Social interaction tests to assess sociability and social novelty preference

Learning and Reward Processing:

• Conditioned place preference (reveals impairments in reward-based learning)

• Fear conditioning paradigms (reveals deficits in aversive stimulus learning)

• Holeboard exploration test with familiarization to rewarding stimuli (shows failure to potentiate exploratory behavior)

Cognitive Flexibility:

• Reversal learning tasks

• T-maze or Y-maze alternation

Sensory Processing:

• Hot plate or tail flick tests (especially relevant given expression in sensory neurons)

• Von Frey filament testing (for mechanical sensitivity)

When designing behavioral experiments with Celf6 knockout mice, researchers should consider:

• Using heterozygous breeding pairs (Celf6+/- × Celf6+/-) to generate wild-type littermate controls

• Balancing for sex within experimental groups

• Controlling for age (phenotypes have been characterized between 3.5-9 months)

• Including multiple complementary assays to thoroughly characterize the behavioral domain of interest

How can I analyze differential gene expression in Celf6 knockout models?

To effectively analyze transcriptomic changes in Celf6 knockout models:

Sample Preparation Considerations:

• Use age-matched Celf6+/+ and Celf6-/- littermates

• Balance sex within experimental groups (e.g., 3-4 males and 4-5 females per genotype)

• Carefully dissect relevant brain regions based on known Celf6 expression patterns

• Process samples consistently to minimize technical variation

Analytical Approaches:

• Microarray Analysis:

  • Has been successfully used to identify derepressed targets in Celf6 knockout mice

  • Allows comparison to CLIP-Seq data to identify direct targets

• RNA-Sequencing:

  • Provides more comprehensive transcriptome analysis

  • Enables detection of alternative splicing events

  • Allows identification of low-abundance transcripts

• Targeted Validation:

  • qRT-PCR for key target genes identified in genome-wide analyses

  • Western blotting to confirm protein-level changes for select targets

Data Analysis Strategy:

• Focus analysis on known Celf6 CLIP targets to identify direct regulatory effects

• Perform pathway and gene ontology enrichment analysis to identify biological processes affected

• Compare results with other CELF family member knockouts to identify unique vs. shared functions

• Correlate transcriptomic changes with behavioral or physiological phenotypes

How is Celf6 implicated in neurodevelopmental disorders?

Evidence connecting Celf6 to neurodevelopmental disorders comes from both human genetic studies and animal models:

Human Genetic Evidence:

• Common variants in CELF6 have been associated with autism spectrum disorder

• A rare inherited variant resulting in a premature stop codon was identified in a male proband with autism

• CELF6 is expressed in serotonergic neurons, a system implicated in various neurodevelopmental disorders

Animal Model Evidence:
Celf6 knockout mice demonstrate several phenotypes relevant to neurodevelopmental disorders:

• Decreased ultrasonic vocalizations, potentially modeling communication deficits

• Resistance to change in behavioral patterns, potentially modeling behavioral inflexibility

• Impaired conditioned learning, suggesting altered neural plasticity

Molecular Mechanisms:
The role of Celf6 as a regulator of synaptic protein mRNAs suggests potential mechanisms:

• Altered synaptic development due to dysregulation of key synaptic proteins

• Abnormal neuronal excitability and circuit function due to imbalanced protein translation

• Disrupted monoamine signaling due to Celf6's expression in monoaminergic neurons

What approaches can be used to model Celf6 dysfunction in vitro?

Several complementary approaches can be employed to model Celf6 dysfunction in cellular systems:

Overexpression Systems:

• Transfect cells with Celf6 expression constructs to analyze gain-of-function effects

• Use GFP-tagged and untagged isoforms to track localization and function

• Include reporter constructs containing target 3'UTR elements to assess functional effects

Knockdown/Knockout Approaches:

• siRNA or shRNA to achieve transient knockdown

• CRISPR-Cas9 to generate stable knockout cell lines

• Compare effects on known target mRNAs identified by CLIP-Seq

Neuronal Models:

• Primary neuronal cultures from Celf6 knockout mice

• iPSC-derived neurons from humans with CELF6 variants

• SH-SY5Y neuroblastoma cells with CELF6 manipulation

Functional Readouts:

• Ribosome profiling to assess translational efficiency of target mRNAs

• RNA stability assays to measure decay rates of target transcripts

• Protein synthesis assays using puromycin incorporation

• Electrophysiology to assess neuronal excitability (particularly relevant given CELF4's known role)

What are the emerging approaches for studying Celf6 function?

Several cutting-edge approaches are being applied or could be applied to further understand Celf6 function:

Single-Cell Technologies:

• Single-cell RNA-Seq to define cell-type-specific expression patterns with higher resolution

• Single-cell CLIP techniques to identify cell-type-specific RNA targets

• Spatial transcriptomics to map Celf6 expression in intact tissue contexts

Advanced Protein Interaction Studies:

• Proximity labeling approaches (BioID, APEX) to identify Celf6 protein interaction partners

• Mass spectrometry to identify post-translational modifications regulating Celf6 function

• Structural biology approaches to understand Celf6-RNA interactions at atomic resolution

Conditional and Inducible Models:

• Cell-type-specific conditional knockout models to dissect the role of Celf6 in specific neuronal populations

• Temporally controlled expression systems to distinguish developmental vs. acute functions

• Circuit-specific manipulations using viral vectors

In Vivo RNA Dynamics:

• RNA live imaging to track target mRNA localization and stability

• Translating ribosome affinity purification (TRAP) to assess cell-type-specific translation of Celf6 targets

• In vivo CLIP approaches to identify activity-dependent changes in RNA binding

How might Celf6 function be targeted therapeutically?

While direct therapeutic applications remain speculative, several approaches warrant investigation:

Potential Therapeutic Strategies:

• Small molecules that modulate Celf6-RNA interactions

• Antisense oligonucleotides to modulate levels of Celf6 or its targets

• Gene therapy approaches to restore normal Celf6 function in cases of loss-of-function mutations

• Targeted modulation of downstream pathways affected by Celf6 dysregulation

Target Validation Approaches:

• Rescue experiments in Celf6 knockout models using viral delivery of Celf6

• Pharmacological manipulation of identified downstream pathways

• Temporally controlled rescue to identify critical periods for intervention

Biomarker Development:

• Expression profiles of Celf6 targets as potential biomarkers for related disorders

• Identification of accessible Celf6-regulated RNAs in peripheral tissues that might reflect CNS status