HNRNPC Human

What is the molecular structure and function of HNRNPC?

HNRNPC is an RNA-binding protein that exists in two isoforms (C1/C2) and forms the core of heterogeneous nuclear ribonucleoprotein particles that associate with nascent transcripts. It controls multiple aspects of RNA metabolism including:

• Alternative splicing regulation

• mRNA stabilization

• Translation control

The protein contains specific domains including a C2 domain in the second isoform. Of particular research interest is the C-terminal region, where a recurrent in-frame deletion of nine amino acids (p.Arg284_Asp292del for HNRNPC-iso1 and p.Arg297_Asp305del for HNRNPC-iso2) has been identified in individuals with neurodevelopmental disorders .

How is HNRNPC expression regulated at the transcriptional level?

Transcriptional regulation of HNRNPC involves specific signaling pathways and promoter elements:

• The IL-6/STAT3 signaling pathway has been shown to activate HNRNPC transcription, particularly in hepatocellular carcinoma

• The HNRNPC promoter sequence (-1984/+158) contains regulatory elements that respond to specific transcription factors

• Siltuximab, an IL-6 inhibitor, blocks IL-6/STAT3-mediated transcriptional activation of HNRNPC

This transcriptional control represents one layer of regulation that determines cellular HNRNPC levels and subsequent RNA processing events.

What other HNRNP family members are associated with neurodevelopmental disorders?

Six members of the HNRNP family have been associated with neurodevelopmental disorders:

• HNRNPH1

• HNRNPH2

• HNRNPK

• HNRNPR

• HNRNPU

• HNRNPC

The identification of HNRNPC-related neurodevelopmental disorder supports the inclusion of HNRNPC in the family of HNRNP-related neurodevelopmental disorders, suggesting potential shared pathogenic mechanisms across these conditions .

What is the subcellular localization of HNRNPC in normal and disease states?

• In cardiac pathologies, HNRNPC adopts a sarcomeric distribution and associates with the translation machinery upon pathological ECM remodeling

• Biomechanical stress generated by cardiac ECM remodeling can control HNRNPC intracellular localization

• Relocation of a fraction of the protein from the nucleus affects alternative splicing of transcripts coding for components of the mechanosensitive Hippo pathway

This dynamic localization suggests HNRNPC functions as a mechanosensitive switch affecting RNA metabolism in pathological conditions.

How does HNRNPC haploinsufficiency contribute to neurodevelopmental disorders?

Studies of 13 individuals with heterozygous HNRNPC germline variants have provided critical insights into the molecular mechanisms of HNRNPC-related neurodevelopmental disorders:

• While protein localization and oligomerization were unaffected by the recurrent C-terminal deletion variant, total HNRNPC levels were decreased

• This reduction in HNRNPC levels was associated with specific changes in alternative splicing patterns

• Meta-analysis of RNA-seq datasets identified a ubiquitous HNRNPC-dependent signature of alternatively spliced exons

• This signature showed significant enrichment for genes associated with intellectual disability

• Both decreased and increased levels of HNRNPC affect neuronal arborization and neuronal migration, suggesting the developing brain is sensitive to aberrant HNRNPC levels

These findings establish a mechanistic link between HNRNPC levels, alternative splicing of neurodevelopmental genes, and clinical phenotypes including global developmental delay, intellectual disability, and behavioral abnormalities.

What is the role of HNRNPC in cancer progression and metastasis?

HNRNPC has emerged as a potential biomarker and therapeutic target in cancer, particularly hepatocellular carcinoma (HCC):

These findings highlight HNRNPC as a potential biomarker for diagnosis, prognosis, and therapeutic targeting in HCC.

How do researchers distinguish between the effects of different HNRNPC isoforms?

Differentiating between HNRNPC isoforms requires specialized molecular approaches:

• Construction of isoform-specific expression vectors using PCR-based cloning with primers that target unique regions of each isoform

• For example, HNRNPC-iso2 can be generated by amplification of HNRNPC-iso1 with specific primers that enable insertion of the C2 domain

• Use of epitope tags such as FLAG, HA, or eGFP to track specific isoforms without interfering with function

• Isoform-specific knockdown strategies targeting unique exons or untranslated regions

• Analysis of isoform-specific RNA binding patterns and protein interactions

These approaches allow researchers to delineate the potentially distinct functions of HNRNPC isoforms in different cellular contexts.

What is the relationship between HNRNPC and mechanotransduction in cardiac pathology?

HNRNPC has been identified as a mechanosensitive regulator in cardiac tissue:

• Ischemic and chronic cardiac pathologies are accompanied by altered expression of HNRNPC

• The protein adopts a sarcomeric distribution and associates with the translation machinery upon pathological ECM remodeling

• HNRNPC intracellular localization can be controlled by increased biomechanical stress generated by cardiac ECM remodeling

• Relocation of a fraction of the protein from the nucleus affects the alternative splicing of transcripts coding for components of the mechanosensitive Hippo pathway, which is heavily involved in the progression of cardiac diseases

These findings suggest HNRNPC acts as a mechanosensitive switch affecting RNA metabolism in the pathological heart, potentially linking mechanical stimuli to gene expression changes in cardiac disease.

How do HNRNPC levels affect global splicing patterns and what are the downstream consequences?

Changes in HNRNPC levels have broad effects on RNA processing with important functional consequences:

• Meta-analysis of RNA-seq datasets from multiple cell types revealed a ubiquitous HNRNPC-dependent signature of alternatively spliced exons

• This signature showed significant enrichment for genes associated with intellectual disability, providing a molecular basis for neurodevelopmental phenotypes

• In cancer contexts, HNRNPC levels affect the stability and expression of specific mRNAs like HIF1A, impacting tumor progression

• Both decreased and increased levels of HNRNPC affect neuronal arborization and migration, suggesting precise regulation is critical for normal development

• In cardiac tissue, HNRNPC affects alternative splicing of transcripts coding for components of the mechanosensitive Hippo pathway

These findings highlight the importance of maintaining appropriate HNRNPC levels for normal cellular function across multiple tissues and cell types.

How can HNRNPC binding sites be mapped at nucleotide resolution?

The iCLIP (individual-nucleotide resolution UV-cross-linking and immunoprecipitation) technique has revolutionized the mapping of HNRNPC binding sites:

• Traditional CLIP methods had limitations in resolution (distances less than 30 nucleotides weren't resolved)

• iCLIP exploits the fact that cDNAs prematurely truncate immediately before the 'cross-link nucleotide'

• The technique captures truncated cDNAs through introduction of a second adapter after reverse transcription via self-circularization

• A random barcode is added to the DNA adapter to quantify cDNA molecules that truncate at the same nucleotide and discriminate between unique cDNA products and PCR duplicates

• This approach allows precise mapping of HNRNPC binding to pre-mRNAs at single-nucleotide resolution

When applied to HNRNPC, iCLIP demonstrated that the protein recognizes uridine tracts with defined long-range spacing and that the positioning of hnRNP particles determines their effect on inclusion of alternative exons.

What experimental models are most suitable for studying HNRNPC function?

Multiple experimental models have been developed to study HNRNPC:

For neurodevelopmental disorders, induced pluripotent stem cells (iPSCs) and fibroblasts from affected individuals have proven particularly valuable for confirming the effects of HNRNPC variants on alternative splicing and cellular phenotypes .

How can researchers identify and validate HNRNPC-dependent alternative splicing events?

A multi-layered approach is necessary to robustly identify and validate HNRNPC-regulated splicing events:

• Identification methods:

  • Meta-analysis of RNA-seq datasets from cells with normal versus reduced HNRNPC levels

  • Integration of iCLIP binding data with alternative splicing profiles to create an "RNA map"

• Validation approaches:

  • Confirmation of identified splicing changes in patient-derived cells

  • RT-PCR analysis of specific alternatively spliced exons

  • Minigene splicing assays to test direct regulation

• Functional assessment:

  • Evaluation of how altered splicing affects cellular phenotypes (e.g., neuronal arborization)

  • Investigation of the impact on disease-relevant processes like tumor invasion

This comprehensive strategy links molecular changes to cellular phenotypes and disease manifestations.

What approaches can be used to generate and characterize HNRNPC variants?

The search results describe several strategies for studying HNRNPC variants:

• Generation methods:

  • PCR-based cloning with primers that introduce specific mutations or deletions

  • For the recurrent variant (c.889_915del [p.Arg297_Asp305del]), primers were designed to introduce the 27 bp deletion

  • Site-directed mutagenesis of existing constructs

• Expression systems:

  • Cloning into expression vectors with different promoters (e.g., CAG promoter)

  • Addition of epitope tags (FLAG, HA, eGFP) for detection and purification

  • Generation of constructs for both major isoforms (iso1 and iso2)

• Functional characterization:

  • Assessment of protein localization, oligomerization, and expression levels

  • Analysis of RNA binding patterns and alternative splicing effects

  • Evaluation of cellular phenotypes in relevant model systems

These approaches allow for detailed characterization of how HNRNPC variants affect protein function and contribute to disease.

How can the effects of HNRNPC on the transcriptome be comprehensively analyzed?

Comprehensive transcriptomic analysis of HNRNPC effects requires integration of multiple techniques:

• Binding site identification:

  • iCLIP to map HNRNPC binding sites at nucleotide resolution

  • CLIP-seq or RIP-seq for broader identification of bound transcripts

• Splicing analysis:

  • RNA-seq with specialized analytical pipelines to detect alternative splicing events

  • Meta-analysis across multiple cell types to identify core HNRNPC-dependent splicing events

• Expression analysis:

  • Quantification of transcript and protein levels following HNRNPC manipulation

  • Assessment of mRNA stability for specific HNRNPC targets

• Functional genomics:

  • Integration with phenotypic data to correlate transcriptome changes with functional outcomes

  • Pathway analysis to identify biological processes affected by HNRNPC-mediated regulation

This integrated approach provides a comprehensive view of how HNRNPC shapes the transcriptome in health and disease.