Long-Read MDM4 Sequencing Reveals Aberrant Isoform Landscape in Metastatic Melanomas

Abstract

MDM4 is upregulated in the majority of melanoma cases and has been described as a "key therapeutic target in cutaneous melanoma". Numerous isoforms of MDM4 exist, with few studies examining their specific expression in human tissues. The changes in splicing of MDM4 during human melanomagenesis are critical to p53 activity and represent potential therapeutic targets. Compounding this, studies relying on short reads lose "connectivity" data, so full transcripts are frequently only inferred from the presence of splice junction reads. To address this problem, long-read nanopore sequencing was utilized to read the entire length of transcripts. Here, MDM4 transcripts, both alternative and canonical, are characterized in a pilot cohort of human melanoma specimens. RT-PCR was first used to identify the presence of novel splice junctions in these specimens. RT-qPCR then quantified the expression of major MDM4 isoforms observed during sequencing. The current study both identifies and quantifies MDM4 isoforms present in melanoma tumor samples. In the current study, we observed high expression levels of MDM4-S, MDM4-FL, MDM4-A, and the previously undescribed Ensembl transcript MDM4-209. A novel transcript lacking both exons 6 and 9 is observed and named MDM4-A/S for its resemblance to both MDM4-A and MDM4-S isoforms.

Keywords: MDM4; alternative splicing; isoforms; long-read nanopore sequencing; melanoma.

Figure 1

Illustration of the MDM2/MDM4 functional domains in parallel. In MDM4, from N-terminus to C-terminus (left to right), there is a p53 binding domain, the WWW autoinhibitory element that blends into the acidic domain, the zinc finger domain, and the inactive RING finger domain containing the nucleolar location signal (NoLS). In MDM2, from N-terminus to C-terminus (left to right), there is a p53 binding domain, a nuclear localization signal (NLS), a nuclear export signal (NES), the acidic domain, the zinc finger domain, and the active RING finger domain containing the nucleolar location signal (NoLS). Amino acid locations are denoted under each functional domain. Created using previous literature as references for amino acid locations of MDM4/MDM2 functional domains [19,20]. Created with BioRender.com.

Figure 2

The mRNA of the MDM4 alternative transcripts focused upon in this paper are illustrated here. Exon transparency indicates exon skipping and/or truncated exons. The functional domains are parallel to each isoform and scaled to the missing/retained exons with amino acid locations of each domain. Start codons are represented by AUG, and stop codons are represented by STOP signs. Figure created using Ensembl database and the previous literature as references [25,29]. Created with BioRender.com.

Figure 3

Summary of RT-PCR results in melanoma tumor samples. Red indicates expression of the isoform detected. The tumor site of the melanoma sample is also listed.

Figure 4

(A) Sample alignment coverage along human genome GRCh38.14. All samples were aligned to the FASTA reference of GRCh38.14 from Ensembl database. The x-axis corresponds to the nucleotide position in the reference genome, and the y-axis corresponds to the sequencing depth at that locus. Barcodes 1–9 correspond to samples 1–9. The largest spike in sequencing depth for all samples is observed in chromosome 1 around the 2 Mb position, where MDM4 is located within the genomic reference. (B) Sample alignment coverage along human chromosome 1. The FASTA reference of GRCh38.14 from Ensembl was trimmed down to a chromosome 1 reference and used for alignment. A single large peak in sequencing depth for all samples is again observed around the 2 Mb position, where MDM4 is located within the genomic reference. (C) Visualization of sample alignment depth along human chromosome 1, with representative reads aligned to the exonic sequences of MDM4. The generated BAM and BAM.bai files from the alignment were used for read and coverage track visualization through IGV. The Ensembl MDM4-FL (ENST00000367182) transcript was loaded onto IGV, with its position within chromosome 1 (q32.1) parallel to the sequencing data. The coverage tracks (gray) are loaded on top of representative reads, with sense reads in blue and anti-sense reads in red. Spikes in coverage are observed at exonic sequences of MDM4. The full 9008 bp 3′ UTR in exon 11 of MDM4 is not represented. Figures were created using EPI2ME Labs wf-alignment workflow (Oxford Nanopore Technologies), and images were created using IGV (https://igv.org), version 2.16.0; accessed on 27 March 2024.

Figure 5

Coverage tracks of sample alignment to the amplicon-specific MDM4-FL cDNA, with corresponding parallel exons. Representative images of coverage tracks from samples 1–9 (top to bottom) using BAM, BAM.bai alignment files, and the trimmed MDM4-FL FASTA reference file used in the EPI2ME Labs wf-alignment workflow (Oxford Nanopore Technologies). The total number of bases in the reference (1420 bp) is located above the coverage tracks, and the corresponding sequence is a colorful bar above the coverage tracks, where each color indicates a unique base: green = adenosine, blue = cytosine, red = thymine, and orange = guanine. The colorful bars within the tracks indicate significant nucleotide variations from the reference sequence within ≥20% of quality weighted reads. Small, bracketed numbers at the top left-hand side of each coverage track represent the number of reads used to create the track. Exons are parallel under the coverage tracks for a rough visual estimate of the position in the reference. Images were created using IGV (https://igv.org), version 2.16.0; accessed on 25 March 2024.

Figure 6

(A) The MDM4 isoform identity per melanoma sample. A stacked bar graph representing the percentage of reads aligned to each isoform per sample. The x-axis contains the sample numbers, and the y-axis represents the percentage of reads aligned, normalized to 100%. The graph was created in Excel using alignment statistics and reads aligned to each isoform averaged to the total reads mapped per sample, obtained from the wf-alignment pipeline using a custom MDM4 transcriptome FASTA reference (Oxford Nanopore Technologies). (B) Overall sample alignment to the amplicon-specific MDM4 transcriptome. A 3-D pie chart representing overall isoform identity of sample alignment results from the wf-alignment pipeline using a custom MDM4 transcriptome FASTA reference. Chart was created in Excel using alignment statistics of overall total reads aligned to each isoform, averaged to the overall total reads mapped for creation of percentage of total reads aligned to each isoform (Oxford Nanopore Technologies).

Figure 7

MDM4 isoform quantification via RT-qPCR. A stacked bar graph displaying the relative expression of MDM4-FL, MDM4-A, MDM4-S, and MDM4-209 isoforms presented as 2 ^(−ΔCT) and normalized to the overall isoform expression in the sample. The x-axis contains the sample numbers, and the y-axis represents the relative expression of the isoform.

Figure 8

Representation of the novel MDM4-A/S transcript observed in alignment results. The hybrid MDM4-A and MDM4-S transcript with concurrent exon 6 and exon 9 deletions. Representative reads from sample 1 are roughly parallel to an illustration of the isoform. A 68 bp deletion corresponds to the exon 6 deletion, and the 150 bp deletion corresponds to the exon 9 deletion. Small insertions of 2 bps are presented as blue ‘I’s within the read. Image of reads was created using IGV (https://igv.org), version 2.16.0; accessed on 27 March 2024. Exon transparency in the illustration below the reads indicates exon skipping, and stop codon is represented by STOP sign. Created with BioRender.com.

Figure 9

(A) The MDM4 isoform identity per melanoma sample, including the novel MDM4-A/S transcript. A stacked bar graph representing the percentage of reads aligned to each isoform per sample. The x-axis contains the sample numbers, and the y-axis represents the percentage of reads aligned, normalized to 100%. The graph was created in Excel using alignment statistics and reads aligned to each isoform averaged to the total reads mapped per sample, obtained from the wf-alignment pipeline using a custom MDM4 transcriptome FASTA reference with a novel MDM4-A/S sequence (Oxford Nanopore Technologies). (B) Overall sample alignment to the amplicon-specific MDM4 transcriptome, including the novel MDM4-A/S transcript. A 3-D pie chart representing the overall isoform identity of sample alignment results from the wf-alignment pipeline using a custom MDM4 transcriptome FASTA reference with a novel MDMD-A/S sequence. Chart was created in Excel using alignment statistics of overall total reads aligned to each isoform, averaged to the overall total reads mapped for creation of percentage of total reads aligned to each isoform (Oxford Nanopore Technologies).