The heterogenous nuclear ribonucleoprotein (hnRNP) family are multifunctional complexes of RNA and protein present in the cell nucleus that serve critical roles in gene regulation. Multiple lines of evidence have linked hnRNP abnormalities as key pathobiological drivers of cancer and neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although hnRNPs are known to impact various aspects of RNA metabolism, their direct effects on mRNA biogenesis and metabolic control remains largely unexplored.

Exploring the Varied Biopathology of hnRNP Raly

The RALY gene (a.k.a. heterogenous nuclear ribonucleoprotein C-like 2, HNRPCL2, autoantigen P542) encodes a member of the hnRNP gene family, complexes of RNA and protein present in the cell nucleus. In humans, the RALY gene encodes the RNA-binding protein Raly, which may play a role in pre-mRNA splicing and embryonic development. Multiple transcript variants are made possible with alternate splicing. In addition to its roles in nucleic acid binding and RNA binding, RALY is associated with a wide variety of diseases, including infectious mononucleosis, skin carcinoma in situ, and dermatosis papulosa nigra.1 Several genome-wide association studies have connected variants at RALY with cardiometabolic traits, including coronary artery disease and total cholesterol.

Role of Raly in Biosynthesizing Cholesterol

RNA-binding protein Raly also acts a transcriptional cofactor for cholesterol biosynthesis genes in the liver and is required for LeXis-mediated effect on cholesterogenesis. Liver X receptor (LXR) is a sterol-sensing nuclear receptor transcription factor that directly targets LeXis and can trigger a response to a lipid overload state. Activation of LXR induces the expression of genes involved in limiting lipid uptake, cholesterol efflux, and promoting triglyceride-rich lipoprotein formation (which includes SREBP1C).

As master regulators of sterol metabolism, sterol regulatory element-binding proteins (SREBPs) directly activate the expression of genes involved in cholesterol and fatty acid biosynthesis. Among the SREBP isoforms, which influence many genes, it is well established that SREBP1c preferentially activates genes involved in fatty acid biosynthesis, while SREBP2 influences cholesterol biosynthetic machinery. Although both isoforms have unique activation signatures, they appear to bind similar DNA response elements and to partner with common transcriptional coactivators, including nuclear transcription factor Y (NFY) and specificity protein 1 (SP1). A recent study, reviewed in greater detail below, has elucidated the connection between the genetic deletion of Raly on Srebp2 gene expression and shown that RALY interacts with the SREBP coactivator NFY.

Applications of Knockout Mouse in Drug Development

Since mice and humans share about 99% of the same genes across species, mouse models have served as accurate analogues for most human biological processes. The knockout (KO) mouse has been a valuable tool for geneticists to elucidate the biological roles of a genetic allele (and/or encoded protein) in embryonic development, physiological homeostasis, metabolic processes, or development of disease. Additionally, KO mice serve as valuable animal models for human genetic diseases in which a mutation is found to disable a protein – corresponding KO mice can be used to study the underlying pathophysiology and even develop therapeutics. Additionally, compared to other experimental animals, mice are small, prolific, and have relatively short life spans – allowing for faster in vivo modeling of geriatric diseases.

For early in vivo evaluation of protein inhibition, pharmaceutical companies often look at the phenotype of a knockout (KO) mouse to understand potential consequences of such a drug development approach. One potential drawback of this approach is that 15% of all KO mice have mutations that result in developmental lethality.2 In this case, studies would be limited to the gene’s role in embryonic development. To circumvent this, conditional knockout (cKO) mice have been developed using Cre/loxP technology for tissue-specific and/or temporal control of gene deletion.

Readily Available Raly Knockout Mice

Mice homozygous for a gene trap allele of Raly are viable, although there are limited studies which have utilized knockout (KO) mouse models for in vivo research of Raly. Cyagen Knockout Catalog Models offers the only ready-to-use Raly knockout mouse - C57BL/6J-Ralyem1cyagen – delivered in as fast as 3 months. All our catalog models provide ≥3 live mice as standard deliverables, with both HET and HOMO breeding and expansion options available.

Raly Conditional Knockout Mouse for Metabolic Research

Building on previous studies which have shown that RALY interacts with LeXis, a recent publication in Nature Communications has outlined a role for hnRNPs in regulatory circuits which control sterol homeostasis. This work expands the implications of hnRNPs in health and disease by showing that a conserved hnRNP can help direct the fundamental metabolic regulatory circuits. Given that RALY is one of the few hnRNPs connected to human lipid traits, researchers investigated the effect of genetic deletion of Raly on Srebp2 gene expression.

To study how hnRNPs contribute to metabolic disease, Ralyflox/flox mice - with LoxP sites flanking exons 3 and 4 of Raly - were generated by Cyagen according to the strategy outlined in Figure 1, below. The administration of Cre or control adenovirus to Ralyflox/flox primary murine hepatocytes resulted in ablation of RALY transcript and protein levels.3​

 

 

Figure 1: Schematic of Raly conditional knockout strategy3

Figure 1: Schematic of Raly conditional knockout strategy3

 

Tissue-Specific Raly Deletion – Effects on Cholesterogenesis

The liver-specific deletion of hnRNP Raly was shown to lower serum cholesterol and hepatic lipid content in the described mouse model. Further in vivo genome-wide mapping of RALY-binding pattern and interrogation of chromatin architecture have provided insights into the cooperative interactions of RALY, as well as its preferential binding patterns at gene promoter regions. DNA binding results suggest that RALY may be influencing cholesterogenesis by modulating collaborative interactions with transcriptional coactivators at the Srebp2 promoter.

Additional Findings About RALY:

  • Binds at the Srebp2 (but not Srebp1) promoter region
  • Interacts directly with NFY to influence transcription of cholesterogenic genes
  • Has multiple isoforms - it is unclear whether different isoforms perform different functions
  • Required for NFY-dependent transcription of SREBP2
  • Works cooperatively with NFY to influence gene expression

This work offers insights into the selective promoter activities of different SREBPs, some of the precise molecular mechanisms that link hnRNP abnormalities with pathologic states, and a model by which hnRNPs can impact metabolic disease states. This research shows that hnRNPs proactively participate in transcriptional control mechanisms regulating cholesterol homeostasis and that loss of a single hnRNP (RALY) influences hepatic lipid stores. RALY binding at the Srebp2 promoter region suggests that spatial collaborative interactions may favor a specific gene activation signature. Despite the newfound characteristics of RALY, this work did not exclude the possibility that it may play other functional roles or influence gene regulation though multiple mechanisms.

Several questions have been raised considering the newly discovered interactions of hnRNP RALY with SREBP coactivator NFY and its binding of the SREBP2 promoter region. It is possible that RALY can directly collaborate with transcriptional regulators other than NFY, which can be explored with additional binding association studies.

It remains unclear how generic collaborative partners of SREBPs (such as NFY and SP1) can turn on some but not all their target genes in response to specific environmental cues. Further investigation is needed to identify the biochemical basis for hnRNP interactions and understand how they selectively influence genes.

Additional Proteins Involved in hnRNP Complexes

There are a broad range of proteins involved in hnRNP complexes, and at least 16 genes have been identified in hnRNP gene family so far. In addition to our custom model generation capabilities, Cyagen Knockout Catalog Models offers a large variety of ready-to-use hnRNP knockout mice that can be delivered in as few as 3 months. Search for your gene of interest from our online database of over 10k gene knockout mice, provided on a 100% pure B6 background for the quickest time from model generation to research study.

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References:

  1. https://www.genecards.org/cgi-bin/carddisp.pl?gene=RALY
  2. genome.gov/12514551
  3. Zhang, Z., Feng, A., Salisbury, D. et al.Collaborative interactions of heterogenous ribonucleoproteins contribute to transcriptional regulation of sterol metabolism in mice. Nat Commun 11, 984 (2020). https://doi.org/10.1038/s41467-020-14711-4