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Article

Discovery of Novel APOC3 Isoforms in Hepatic and Intestinal Cell Models Using Long-Read RNA Sequencing

by
Kara Farstad-O’Halloran
1,†,
Anuradha Sooda
1,†,
Tooba Iqbal
1,
Steve Wilton
1,2 and
May T. Aung-Htut
1,2,*
1
Personalised Medicine Centre, Health Futures Institute, Murdoch University, Murdoch, Perth, WA 6150, Australia
2
Perron Institute for Neurological and Translational Science, The University of Western Australia, Nedlands, Perth, WA 6009, Australia
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Genes 2025, 16(4), 412; https://doi.org/10.3390/genes16040412
Submission received: 14 March 2025 / Revised: 28 March 2025 / Accepted: 29 March 2025 / Published: 31 March 2025
(This article belongs to the Section Molecular Genetics and Genomics)
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Abstract

:
Background: Apolipoprotein C-III (APOC3) plays a crucial role in triglyceride metabolism and is closely associated with cardiovascular disease risk. Elevated APOC3 levels contribute to higher plasma triglycerides and increased risk of atherosclerosis, making APOC3 expression an attractive and logical therapeutic target. Methods: While studying various APOC3 transcript isoforms expressed in hepatoma cell lines (HepG2, Huh7) and healthy liver tissue using publicly available long-read RNA sequencing, we found three novel APOC3 isoforms. These isoforms were validated through RT-PCR and Sanger sequencing. Results: All three novel isoforms are splicing variants of the MANE transcript, APOC3-201. Isoforms 1 and 2 exhibit splicing patterns similar to APOC3-201 from exons 2–4; however, isoform 1 shares its exon 1 splicing pattern with APOC3-203, while isoform 2 features an extended exon 1 that includes exon 1a, the adjacent intronic region, and exon 1b. The third isoform closely resembles APOC3-201, but lacks exon 2, which contains the translation start codon. Remarkably, similar APOC3 splicing patterns and transcript variants were observed in Caco-2 cells, a model of the small intestine, indicating that these isoforms are not liver-specific. Conclusions: This study identifies three novel APOC3 isoforms and highlights their expression in both hepatic and intestinal cell models. Further studies are needed to elucidate the functional roles of these novel isoforms and their contribution to the regulation of APOC3 gene expression.

1. Introduction

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality worldwide. A well-established association exists between elevated triglyceride levels and an increased risk of CVD [1,2]. Apolipoprotein C-III (APOC3), a critical component of triglyceride-rich lipoproteins (TRLs), plays a central role in regulating lipid metabolism by inhibiting the uptake and clearance of circulating TRLs [3]. This leads to hypertriglyceridemia [4], a well-established risk factor for atherosclerosis and ischemic heart disease. Genetic studies have shown that loss-of-function mutations in the APOC3 gene are associated with significantly lower triglyceride levels, reduced remnant cholesterol, and less coronary artery calcification, a marker of atherosclerosis [5,6,7]. Additionally, transgenic and knockout mouse models have reinforced these findings, demonstrating that APOC3 overexpression induces hypertriglyceridemia, whereas its absence promotes efficient lipid clearance and a favourable cardiovascular profile [8,9]. In addition to its role in triglyceride metabolism, APOC3 has been implicated in type 2 diabetes mellitus (T2DM) pathophysiology through mechanisms such as promoting pancreatic β-cell apoptosis, dysregulation of glucose metabolism, and influencing insulin resistance [10,11,12,13]. Its role in inflammatory processes, including monocyte adhesion and endothelial dysfunction, further emphasizes its significance in the pathogenesis of cardiometabolic diseases [14]. Given these multifaceted roles, targeting APOC3 presents a promising therapeutic target for lowering cardiovascular risk.
To date, four protein-coding transcripts of APOC3 have been reported in the Ensembl genome database (release 113) [15]. The primary transcript, APOC3-201 (ENST00000227667.8), includes all four exons and encodes a 99 amino acid (aa) protein. Another full-length transcript, APOC3-202 (ENST00000375345.3), closely resembles APOC3-201 but contains a longer exon 2 sequence, resulting in a 117 aa protein. Two truncated isoforms, APOC3-203 and APOC3-205, have also been described. APOC3-203 (ENST00000433777.5) utilises an alternate splice donor site in exon 3, leading to the exclusion of exon 4 from the mature transcript. This is predicted to produce a truncated 55 aa protein lacking the C-terminal domain, though experimental evidence to confirm the presence of this peptide is currently lacking. Notably, APOC3-203 also uses an alternative upstream exon 1 (exon 1a) sequence distinct from the exon 1 (exon 1b) employed by the full-length transcripts. Finally, APOC3-205 (ENST00000630701.1) contains exons 2 to 4 and is reported to produce a functional 117 aa protein. We detected a novel isoform missing exon 2 in hepatoma cell lines (HepG2 and Huh7) when APOC3 transcripts were amplified using primers targeting exon 1a/b (forward) and 3 (reverse) and hence further investigated the presence of additional isoforms.
The advancement of high-throughput sequencing technologies and the growing adoption of long-read sequencing platforms have led to a rapid expansion of transcriptomic data. This has provided an opportunity to evaluate full-length transcripts and more accurately identify alternative splicing events, offering insights into previously underexplored areas of gene regulation [16,17]. Such findings are crucial for understanding the regulatory mechanisms governing APOC3 expression and for identifying additional isoforms that could be involved in lipid metabolism.
In this study, we utilised publicly available long-read RNA-seq data from commonly used hepatoma cell lines (HepG2 and Huh7), liver tissue, and Caco-2 cells, a model of the small intestine, to investigate the isoform diversity of APOC3. Through the analysis of these datasets, we identified novel isoforms of APOC3 and gained valuable insights into its splicing patterns. While lipid metabolism is not our primary area of expertise, our findings provide a foundation for further exploration into the regulation of lipid metabolism and APOC3 isoforms.

2. Materials and Methods

2.1. RNA Sequencing Data

The raw RNA sequencing data from Oxford Nanopore used in this study were obtained from the NCBI Sequence Read Archive (SRA) under the following accession numbers: PRJNA765908 (HepG2) [18], PRJNA893571 (Huh7) [19], PRJEB81685 (Liver) [20], and PRJNA850621 (Caco-2) [21] (Supplementary Table S1). Data acquisition was performed using the prefetch tool from the SRA Toolkit (v2.11.3), and raw sequencing reads in FASTQ format were extracted using fastq-dump (https://github.com/ncbi/sra-tools, accessed on 27 October 2023). The resulting FASTQ files were then processed and analysed using computational pipelines optimised for long-read RNA sequencing data.

2.2. Bioinformatics Analyses

To identify isoforms of the APOC3 gene, we applied the Full-Length Analysis of Mutations and Splicing in long-read RNA-seq data (FLAMES) pipeline (https://github.com/LuyiTian/FLAMES, accessed on 16 December 2024) with the default parameters, adjusting “strand_specific” to 1 for direct RNA sequencing and 0 for cDNA sequencing. Additionally, we filtered transcripts to include only those supported by at least 10 reads to minimise technical noise and sequencing errors.
Isoform classification and splicing analysis were performed using SQANTI3 (v5.2) (https://github.com/ConesaLab/SQANTI3, accessed on 16 December 2024) [22]. Gencode human hg38.v47 was used as the gene reference annotation. For the reference files of CAGE Peak data, and polyA motif list, we used the files provided in SQANTI3.
The bioinformatics workflow is summarised in Supplementary Figure S1.

2.3. Secondary Structure Prediction

To predict the secondary structures of the 5′ untranslated region (5′UTR) of Novel-2, we used the Vienna RNAfold web server (http://rna.tbi.univie.ac.at, accessed on 25 February 2025) [23]. The analysed region included exon 1a (116,829,706–116,829,886), intron 1 (116,829,887–116,829,906), exon 1b (116,829,907–116,829,940), and exon 2 (116,830,570–116,830,582), spanning a total of 248 bp. For comparison, we included the 5′UTRs of APOC3-201 (exon 1b: 116,829,907–116,829,940; exon 2: 116,830,570–116,830,582 (exon 2); 47 bp) and APOC3-203 (exon 1a: 116,829,706–116,829,886; exon 2: 116,830,570–116,830,582; 194 bp). The sequences were input into the RNAfold web interface, and the resulting minimum free energy (MFE) structures were analysed to assess structural stability and potential functional elements.

2.4. Phylogenetic Trees

Multiple sequence alignment and phylogenetic tree construction were performed using the MAFFT online server (https://mafft.cbrc.jp/alignment/server/index.html, accessed on 28 January 2025). Phylogenetic trees were generated using the Neighbour–Joining method with the Jukes–Cantor nucleotide substitution model and a bootstrap value of 1000.

2.5. Cell Lines and Culture

HepG2 (Cat. #85011430) and Huh-7 (Cat. #JCRB0403) cell lines were purchased from CellBank Australia (Sydney, Australia) and originally supplied by the European Collection of Cell Cultures (Salisbury, UK). The cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco, Thermo Fisher Scientific, Melbourne, Australia; Cat. #11995-065) supplemented with 10% fetal bovine serum (Fisher Biotec, Perth, Australia; Cat. # FBS-AU-015). Cultures were maintained in a humidified incubator at 37 °C with 5% CO2 (v/v).

2.6. RNA Extraction

Total RNA was extracted using the MagMAX™ Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. RNA concentration and purity were measured with an Implen NanoPhotometer® (Implen NanoPhotometer®, Westlake Village, CA, USA).

2.7. RT-PCR and Gel Electrophoresis

Reverse-transcription polymerase chain reaction (RT-PCR) was performed using the SuperScript® III One-Step RT-PCR System (Life Technologies, Melbourne, Australia) with 50 ng of RNA as the template. Primer sequences are detailed in Supplementary Table S2. RT-PCR products were separated on a 3% agarose gel in 1× TAE buffer, stained with 1× RedSafe™ (iNtRON Biotechnology, Burlington, MA, USA), and imaged using a Fusion-FX Gel Documentation System (Vilber Lourmat, Marne la Vallee, France).

2.8. Band Stab PCR and Sanger Sequencing

Target bands were excised under UV light using a 200 µL pipette tip and transferred into a PCR master mix containing AmpliTaq Gold DNA Polymerase (Applied Biosystems, Melbourne, Australia) [24]. PCR was performed with a 5 °C lower annealing temperature and an additional 5 cycles compared to the optimised conditions to isolate specific amplicons. The resulting PCR products were purified using Diffinity Rapid Tips (Diffinity Genomics, West Chester, PA, USA) and combined with the corresponding primer. Purified products were sent to the Australian Genome Research Facility (AGRF, Perth, Australia) for Sanger sequencing.

2.9. Data and Code Availability

The Sanger sequencing data have been submitted to NCBI and are available under the GenBank accession numbers OR575746, OR575747, and PQ732990. Code used to perform the novel isoform detection and data to generate the figures are available from https://github.com/anuradhareddi/APOC3, accessed on 13 March 2025.

3. Results

3.1. Long-Read Transcriptome Annotation Reveals Novel APOC3 Isoforms

Given that APOC3 is predominantly expressed in the liver, we analysed publicly available long-read transcriptomic datasets from immortalised human hepatoma cell lines, HepG2 and Huh7, as well as from healthy liver tissue (Supplementary Table S1) to explore isoform diversity. The FLAMES pipeline was employed for detecting alternative splicing isoforms, while SQANTI3 was used for quality control, classification, and functional annotation of the transcripts.
Our analysis identified three novel isoforms in both cell lines and liver tissue (Figure 1A). All three isoforms (Novel-1, Novel-2, and Novel-3) are splicing variants of the MANE APOC3-201 transcript. Novel-1 and Novel-2 share exons 2–4 with APOC3-201; however, Novel-1 shares exon 1a with APOC3-203 (Figure 1B), while Novel-2 exhibits an extension of exon 1a into the adjacent intronic region, which merges with exon 1b to form a single exon (Figure 1B). The third isoform, Novel-3, differs from APOC3-201 by skipping exon 2, which contains the canonical translation start site (Figure 1B). Additionally, we observed a transcript classified as novel in HepG2 that was not detected in Huh7. Several unique transcripts were also identified in liver tissue samples (Supplementary Figure S2); however, they were excluded from further analysis due to lack of reproducibility across datasets.
To explore the expression profiles of these isoforms, we evaluated their relative abundance in both hepatoma cell lines and liver tissue. Among the novel isoforms, Novel-2 was the most abundant, ranking second only to the MANE transcript, while Novel-1 and Novel-3 exhibited consistently lower expression levels across all sample types (Figure 1C). To further classify and functionally annotate these novel isoforms relative to existing annotations, we compared them to reference transcripts. Novel-1 was classified as “novel-in-catalog” and identified as a coding transcript, exhibiting a combination of known splice sites (Table 1). Novel-2 was classified as a “full splice match” with an alternative 5′ end, corresponding to the reference transcript ENST00000227667.8 (APOC3-201), and was also identified as a coding transcript with a combination of known junctions (Table 1). Novel 3, also classified as “novel-in-catalog”, was found to be a non-coding transcript with a combination of known splice sites (Table 1).

3.2. Extended 5′UTR in Novel-2 Is Associated with More Secondary Structures

Since Novel-2 has a longer 5′UTR, we investigated whether this region forms additional secondary structures using in silico prediction via the RNAfold web server. For comparison, we included the canonical 5′UTR of APOC3-201 and the alternative 5′UTR of APOC3-203. The 5′ UTR of APOC3-201 exhibited moderate stability with an MFE of −10.03 kcal/mol, indicating a simpler folding pattern (Figure 2A). In contrast, the 5′UTR sequence of APOC3-203 formed a more complex secondary structure with a lower MFE of −85.69 kcal/mol (Figure 2B). However, the 5′ UTR of Novel-2, which includes the 5′UTR of both APOC3-203 and APOC3-201 and the intronic region in between (Figure 1B) displayed the most intricate structure with a significantly lower MFE of −106.82 kcal/mol, suggesting much higher stability of secondary structures (Figure 2C).

3.3. Novel Isoforms Are Expressed in Caco-2 Cells

To determine whether the novel APOC3 isoforms identified in liver-derived samples are expressed in other tissues, we extended our analysis to the small intestine, where APOC3 is also expressed, albeit to a lesser extent. For this purpose, we analysed long-read sequencing data from intestinal epithelial Caco-2 cells. This analysis revealed the presence of Novel-2 and Novel-3 isoforms, while Novel-1 was not detected under the applied stringent cutoff of 10 counts (Supplementary Figure S2). The expression profiles of Novel-2 and Novel-3 were consistent with hepatic models, with Novel-2 ranking second in abundance after the MANE transcript and Novel-3 ranking third (Figure 1C).

3.4. RT-PCR and Sanger Sequencing Confirm Novel APOC3 Isoforms

To validate the expression of the novel isoforms identified through long-read transcriptomics in cells, we performed RT-PCR using exon-specific primers designed for Novel-1 to -3 (Supplementary Table S1; Figure 3A). Amplification products of the expected sizes were observed in both HepG2 and Huh7 cell lines, confirming the presence of the novel exons (Figure 3B).
Sanger sequencing of the RT-PCR products further validated the inclusion of the novel exons and confirmed the splice junctions predicted by the long-read data (Figure 3C). These results provide strong evidence for the existence of Novel-1 to -3 in hepatoma cell lines. Due to the lack of liver tissue samples, the validation process was limited to hepatoma cell lines.

3.5. Novel Isoforms Cluster Closely with Known Human APOC3 Transcripts

To assess the evolutionary conservation of the novel APOC3 isoforms, we performed a phylogenetic analysis based on sequence similarity (Supplementary Figure S3). The three novel isoforms (Novel-1, Novel-2, and Novel-3) cluster within the human clade, closely with previously annotated human APOC3 transcripts. Novel-1 is most similar to ENST00000433777.5 (APOC3-203), Novel-2 to ENST00000227667.8 (APOC3-201; MANE), while Novel-3 forms a separate branch within the human clade, indicating a more distant relationship to known APOC3 isoforms. None of the novel isoforms group directly with non-human orthologs, suggesting they are unique to humans or have significantly diverged.

4. Discussion

In this study, we identified three novel APOC3 isoforms, expanding the known splicing landscape of this gene. These novel isoforms, Novel-1, Novel-2, and Novel-3, exhibit distinct splicing patterns that may contribute to the regulation of APOC3 expression. Given the critical role of APOC3 in triglyceride metabolism and its emerging connection to cardiovascular diseases [1,2], the discovery of these isoforms presents new opportunities for understanding APOC3 regulation and exploring potential therapeutic interventions.
Our findings were supported by advancements in long-read sequencing technologies, which enable the resolution of complex transcript structures and the detection of previously overlooked or undetected isoforms [25]. Analysis of publicly available long-read sequencing data from hepatoma cell lines (HepG2 and Huh7) and healthy liver tissue revealed the three novel APOC3 isoforms. These isoforms were likely missed in earlier studies due to the limitations of short-read sequencing, such as insufficient read length or challenges in assembling fragmented reads [26]. The ability of long-read sequencing technologies to capture full-length transcripts, combined with advanced transcript analysis tools, demonstrates their potential to identify novel transcript variants.
The presence of all three novel APOC3 isoforms in both cancer cell lines and healthy liver tissue indicates that they are not a result of cancer-specific splicing alterations but rather part of the normal APOC3 transcript repertoire. Novel-1 and Novel-2 share splice junctions of exons 2–4 with the APOC3-201 transcript but are distinguished by the usage of a longer exon 1 (Figure 1B). This alternative splicing event located within the 5′UTR does not alter the protein coding sequence but may contribute to transcript diversity and regulation of APOC3 transcript levels.
Interestingly, Novel-1, which includes a longer exon 1a in the 5′UTR similar to that in APOC3-203, was either absent or expressed at lower levels than isoforms with the same coding regions (APOC3-201 and Novel-2) across all analysed samples. The stronger secondary structures predicted by in silico analysis in the 5′UTR region of Novel-1 (identical to APOC3-203; Figure 2B) could contribute to slow transcription or reduce transcript stability. These findings align with evidence that alternative splicing within UTRs influences gene expression in at least 13% of mammalian genes [27,28]. For instance, SERPINA1, which encodes α-1-antitrypsin, has multiple splicing isoforms that share the same coding sequence but differ in their 5′UTRs, affecting translation efficiency through upstream regulatory elements and secondary structures [29]. Similarly, BRCA1 expression in certain breast cancers is downregulated by a switch to a longer 5′UTR, which introduces secondary structures or upstream regulatory elements that modulate expression levels [30].
Novel-2, which contains exon 1a but extends farther downstream with the inclusion of adjacent intronic sequences and exon 1b, retains the same splice sites as the MANE transcript. Despite these structural differences, Novel-2 was the second most highly expressed isoform after the MANE transcript, likely due to the preservation of the essential splice architecture found in the MANE transcript. However, its longer 5′UTR may contribute to reduced expression compared to the MANE transcript, as longer 5′UTRs are often associated with increased secondary structures or additional miRNA binding sites, which can interfere with translation initiation or mRNA stability [31,32,33,34]. The secondary structure predictions indicate that the 5′UTR of Novel-2 exhibits greater structural complexity than those of APOC3-201 and APOC3-203 (Figure 2C); however, its expression levels remain high, suggesting that additional regulatory mechanisms may mitigate the structural constraints imposed by its extended 5′UTR.
The third isoform, Novel-3, lacks exon 2, which contains the translation initiation site (TIS), potentially disrupting the production of a functional APOC3 protein (Table 1). This alternative splicing event represents a mechanism of unproductive splicing, where the exclusion of critical exons leads to the generation of non-functional or unstable transcripts [35,36,37,38]. Consequently, the absence of TIS likely leads to early degradation of Novel-3, potentially explaining its lower abundance despite having a shorter exon 1 similar to the MANE transcript. Similar regulatory mechanisms have been observed in other genes, such as PTBP1, where exon 11 skipping generates an mRNA targeted for degradation by nonsense-mediated decay in a negative feedback loop [37]. Similarly, RNA-binding protein, RBM10, autoregulates its expression by promoting the skipping of essential exons [38], suggesting that unproductive splicing may serve as a broader regulatory strategy, potentially relevant to APOC3. The removal of exon 2 from the MANE transcript may represent a mechanism for regulating APOC3 levels.
Additionally, while exon 2 skipping removes the canonical TIS, Novel-3 retains an in-frame downstream ATG. However, since exon 2 encodes the signal peptide necessary for APOC3 secretion (Supplementary Figure S4), the loss of exon 2 eliminates this signal peptide, likely impairing protein translocation to the endoplasmic reticulum [39]. As a result, any translated protein would remain in the cells rather than being secreted. This further supports the idea that Novel-3 undergoes unproductive splicing, as the absence of both the TIS and the signal peptide disrupts normal protein synthesis and function, leading to degradation or mislocalisation.
Previous studies have shown that antisense oligonucleotide (ASO) inhibition of APOC3 expression leads to reduced triglyceride levels and improved hepatic clearance of TRLs [40,41,42]. By promoting the skipping of exon 2, we could increase the expression of Novel-3. Since Novel-3 is non-coding and unable to produce a protein (Table 1), its increased expression could lower functional APOC3 levels, providing a potential therapeutic strategy for conditions like hypertriglyceridemia, where reducing APOC3 expression has been shown to be advantageous [42].
The APOC3 is known to be expressed primarily in liver and to a lesser extent in the small intestine [43]. While our study mainly focused on hepatoma cell lines and healthy liver tissue, we also investigated the expression of APOC3 isoforms in small intestine models, specifically Caco-2 cells. Our analysis suggests that these isoforms are present in both the liver and small intestine. However, due to the unavailability of liver or small intestine tissue samples, RT-PCR validation was confined to hepatoma cell lines. Nonetheless, our long-read RNA-seq analysis confirmed the presence of these isoforms and supported their expression in both liver tissue and small intestinal models. Further studies involving additional pathological tissues or disease models could provide additional insights into whether these isoforms are more broadly expressed across various conditions. The new isoforms identified in this study were not previously reported and do not cluster with non-human orthologs, suggesting that these alternative splicing events may be unique to humans or have evolved recently in primates.
Although this study provides valuable insights into the novel APOC3 isoforms, there are some limitations. First, while RNA-seq identified these isoforms, RT-PCR validation was limited to hepatoma cell lines due to the unavailability of primary tissue samples. Further experiments using primary tissue samples from both healthy and disease contexts are necessary to validate the expression of these isoforms and evaluate their relevance across different conditions. Second, although we identified Novel-3 as a non-coding isoform, additional experimental validation is required to determine its role as a regulatory non-coding transcript or its susceptibility to nonsense-mediated decay. Lastly, while our study discovered novel isoforms with distinct splicing patterns, their functional roles in lipid metabolism and cardiovascular disease need to be experimentally verified, particularly in disease models and therapeutic contexts.

5. Conclusions

In conclusion, we identified three novel APOC3 isoforms with distinct splicing patterns that may influence APOC3 expression and lipid metabolism. The presence of alternative 5′UTRs and a non-coding isoform suggests potential mechanisms that could regulate transcript stability and translational efficiency. Since APOC3 is a key regulator of triglyceride metabolism and cardiovascular risk, these findings provide new opportunities for therapeutic strategies, particularly through ASO-mediated modulation of splicing. The discovery of a non-coding isoform (Novel-3) suggests that promoting exon 2 skipping could serve as a strategy to lower functional APOC3 levels, with potential benefits for hypertriglyceridemia treatment. Additionally, the presence of these isoforms in both hepatic and intestinal models suggests broader physiological relevance. While further research is required to fully understand their functional roles, these novel isoforms offer the potential to explore therapeutic strategies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes16040412/s1, Figure S1. Schematic representation of the bioinformatics workflow and validation process for identifying and analysing novel APOC3 isoforms. Figure S2. Transcript structure of APOC3 isoforms identified in hepatoma cell lines, liver tissue, and Caco-2 cells. Figure S3. Comparative analysis of human APOC3 isoforms and their orthologs. Figure S4. Open reading frames (ORFs) of the MANE transcript and Novel-3 isoform of APOC3. Table S1. RNA sequencing dataset used for analysis. Table S2. RT-PCR primers used for validation of novel APOC3 isoforms.

Author Contributions

Conceptualisation, M.T.A.-H.; methodology, K.F.-O. and A.S.; validation, K.F.-O., A.S. and T.I.; investigation, K.F.-O. and A.S.; writing—original draft preparation, K.F.-O. and A.S.; writing—review and editing, A.S., K.F.-O., T.I., S.W. and M.T.A.-H.; supervision, M.T.A.-H. and S.W. All authors have read and agreed to the published version of the manuscript.

Funding

Murdoch University and Perron Institute for Neurological and Translational Science.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The Sanger sequencing data has been submitted to NCBI and is available under the GenBank accession numbers OR575746, OR575747, and PQ732990. Code used to perform the novel isoform detection and data to generate the figures is available from https://github.com/anuradhareddi/APOC3, accessed on 13 March 2025.

Conflicts of Interest

The authors declare no conflict of interest.

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Genes 16 00412 g001
Figure 1. Long-read RNA-seq analysis identifies novel splice isoforms of the APOC3 gene. (A) Schematic representation of APOC3 splice isoforms, including known isoforms (red) and novel isoforms (blue). Untranslated regions (UTRs) are depicted as open boxes, while coding regions are shown as filled boxes. (B) Sequence alignment of the novel isoforms detected through long-read RNA-seq with the APOC3-203 and APOC3-201 isoforms. Exon and intron regions are highlighted with coloured rectangular boxes to illustrate structural differences. The dashed box represents the canonical splicing of exon 3. (C) Boxplots representing the expression levels of the detected isoforms across HepG2, Huh7, liver tissue, and Caco-2 samples. The y-axis displays the isoform names, which correspond to those shown in panel A and the x-axis represents counts per million (CPM) values.
Figure 1. Long-read RNA-seq analysis identifies novel splice isoforms of the APOC3 gene. (A) Schematic representation of APOC3 splice isoforms, including known isoforms (red) and novel isoforms (blue). Untranslated regions (UTRs) are depicted as open boxes, while coding regions are shown as filled boxes. (B) Sequence alignment of the novel isoforms detected through long-read RNA-seq with the APOC3-203 and APOC3-201 isoforms. Exon and intron regions are highlighted with coloured rectangular boxes to illustrate structural differences. The dashed box represents the canonical splicing of exon 3. (C) Boxplots representing the expression levels of the detected isoforms across HepG2, Huh7, liver tissue, and Caco-2 samples. The y-axis displays the isoform names, which correspond to those shown in panel A and the x-axis represents counts per million (CPM) values.
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Genes 16 00412 g002
Figure 2. In silico prediction of secondary structures for 5′UTR sequences. The secondary structures were generated using the Vienna RNAfold web server, with minimum free energy (MFE) values indicated for each structure. (A) The APOC3-201 structure forms a relatively simple hairpin loop with an MFE of −10.03 kcal/mol. (B) The APOC3-203 structure adopts a more complex configuration with multiple interior loops and an MFE of −85.69 kcal/mol. (C) The Novel-2 structure displays the most intricate configuration with multiple stem-loops and an MFE of −106.82 kcal/mol. The colour bar below represents the base-pairing probability.
Figure 2. In silico prediction of secondary structures for 5′UTR sequences. The secondary structures were generated using the Vienna RNAfold web server, with minimum free energy (MFE) values indicated for each structure. (A) The APOC3-201 structure forms a relatively simple hairpin loop with an MFE of −10.03 kcal/mol. (B) The APOC3-203 structure adopts a more complex configuration with multiple interior loops and an MFE of −85.69 kcal/mol. (C) The Novel-2 structure displays the most intricate configuration with multiple stem-loops and an MFE of −106.82 kcal/mol. The colour bar below represents the base-pairing probability.
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Figure 3. Validation of APOC3 transcript isoforms using RT-PCR amplification and Sanger sequencing. (A) Schematic representation showing the locations of primers (arrows) designed within the exons to detect novel APOC3 isoforms. (B) Gel electrophoresis of amplified APOC3 transcripts from HepG2 (*) and Huh7 (#) cell lines, with distinct bands corresponding to different transcript isoforms. (C) Sanger sequencing chromatograms of the identified APOC3 transcript isoforms. Black dashed vertical lines indicate exon–exon or exon–intron junctions. Sequencing was performed using both forward and reverse primers to fully resolve splicing junctions. Miscall errors in trace files were manually corrected where necessary. (M—100 bp marker; 1a-3 (exon 1a–exon 3); 1a-4 (exon 1a–exon 4); 1b-3 (exon 1b–exon 3); 1b-4 (exon 1b–exon 4).
Figure 3. Validation of APOC3 transcript isoforms using RT-PCR amplification and Sanger sequencing. (A) Schematic representation showing the locations of primers (arrows) designed within the exons to detect novel APOC3 isoforms. (B) Gel electrophoresis of amplified APOC3 transcripts from HepG2 (*) and Huh7 (#) cell lines, with distinct bands corresponding to different transcript isoforms. (C) Sanger sequencing chromatograms of the identified APOC3 transcript isoforms. Black dashed vertical lines indicate exon–exon or exon–intron junctions. Sequencing was performed using both forward and reverse primers to fully resolve splicing junctions. Miscall errors in trace files were manually corrected where necessary. (M—100 bp marker; 1a-3 (exon 1a–exon 3); 1a-4 (exon 1a–exon 4); 1b-3 (exon 1b–exon 3); 1b-4 (exon 1b–exon 4).
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Table 1. Classification and functional annotation of novel APOC3 isoforms using SQANTI3.
Table 1. Classification and functional annotation of novel APOC3 isoforms using SQANTI3.
Isoform Structural_Category Associated_Transcript Coding Subcategory
Novel-1novel_in_catalognovelcodingcombination_of_known_junctions
Novel-2full-splice_matchENST00000227667.8codingalternative_5end
Novel-3novel_in_catalognovelnon_codingcombination_of_known_splicesites
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Farstad-O’Halloran, K.; Sooda, A.; Iqbal, T.; Wilton, S.; Aung-Htut, M.T. Discovery of Novel APOC3 Isoforms in Hepatic and Intestinal Cell Models Using Long-Read RNA Sequencing. Genes 2025, 16, 412. https://doi.org/10.3390/genes16040412

AMA Style

Farstad-O’Halloran K, Sooda A, Iqbal T, Wilton S, Aung-Htut MT. Discovery of Novel APOC3 Isoforms in Hepatic and Intestinal Cell Models Using Long-Read RNA Sequencing. Genes. 2025; 16(4):412. https://doi.org/10.3390/genes16040412

Chicago/Turabian Style

Farstad-O’Halloran, Kara, Anuradha Sooda, Tooba Iqbal, Steve Wilton, and May T. Aung-Htut. 2025. "Discovery of Novel APOC3 Isoforms in Hepatic and Intestinal Cell Models Using Long-Read RNA Sequencing" Genes 16, no. 4: 412. https://doi.org/10.3390/genes16040412

APA Style

Farstad-O’Halloran, K., Sooda, A., Iqbal, T., Wilton, S., & Aung-Htut, M. T. (2025). Discovery of Novel APOC3 Isoforms in Hepatic and Intestinal Cell Models Using Long-Read RNA Sequencing. Genes, 16(4), 412. https://doi.org/10.3390/genes16040412

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