HAfTs are novel lncRNA transcripts from aflatoxin exposure

Abstract

The transcriptome can reveal insights into precancer biology. We recently conducted RNA-Seq analysis on liver RNA from male rats exposed to the carcinogen, aflatoxin B1 (AFB1), for 90 days prior to liver tumor onset. Among >1,000 differentially expressed transcripts, several novel, unannotated Cufflinks-assembled transcripts, or HAfTs (Hepatic Aflatoxin Transcripts) were found. We hypothesized PCR-cloning and RACE (rapid amplification of cDNA ends) could further HAfT identification. Sanger data was obtained for 6 transcripts by PCR and 16 transcripts by 5'- and 3'-RACE. BLAST alignments showed, with two exceptions, HAfT transcripts were lncRNAs, >200nt without apparent long open reading frames. Six rat HAfT transcripts were classified as 'novel' without RefSeq annotation. Sequence alignment and genomic synteny showed each rat lncRNA had a homologous locus in the mouse genome and over half had homologous loci in the human genome, including at least two loci (and possibly three others) that were previously unannotated. While HAfT functions are not yet clear, coregulatory roles may be possible from their adjacent orientation to known coding genes with altered expression that include 8 HAfT-gene pairs. For example, a unique rat HAfT, homologous to Pvt1, was adjacent to known genes controlling cell proliferation. Additionally, PCR and RACE Sanger sequencing showed many alternative splice variants and refinements of exon sequences compared to Cufflinks assembled transcripts and gene prediction algorithms. Presence of multiple splice variants and short tandem repeats found in some HAfTs may be consequential for secondary structure, transcriptional regulation, and function. In summary, we report novel, differentially expressed lncRNAs after exposure to the genotoxicant, AFB1, prior to neoplastic lesions. Complete cloning and sequencing of such transcripts could pave the way for a new set of sensitive and early prediction markers for chemical hepatocarcinogens.

Fig 1. HAfT fold changes from AFB1 exposure.

RNA-Seq was performed on liver RNA from male rats exposed for 90 days to 1 ppm AFB1. HAfTs were originally assembled as unannotated transcripts from this RNA-Seq data as previously described [26]. Shown here, HAfTs met a two-fold change threshold at p<0.05 (mean ± rSEM). NCBI databases now list HAfT26 and HAfT27 as protein-coding genes, Rps27l and Cyp2c24. HAfT-6, -11, -23 and -24 were placed on a separate scale (see inset) due to the large magnitude of fold change.

Fig 2. Workflow for PCR and RACE, cloning and sequencing.

See Methods for further details.

Fig 3. Novel HAfTs.

Four novel, unannotated HAfTs and variants are shown in the UCSC genome browser. AFB1 treatment resulted in an increased number of RNA-Seq reads at specific genomic loci that enabled assembly of Cufflinks transcripts. Primers were designed from Cufflinks transcripts and PCR produced amplicons for portions of HAfT19 and 20 in Panel A, and HAfT22 and 24 in Panel B. Additional variants may exist for these novel HAfT transcripts.

Fig 4. MFE clover plots.

Minimum free energy (MFE) cloverleaf models were generated for HAfT19 variants. Each circle represents a base, and color-coding indicates the base-pairing probability, with 0 (blue) to 1 (red) representing low to high pairing probabilities. The adjusted MFE (AMFE) normalizes for differing transcript length. Brackets show predicted differences in secondary structure when comparing the shortest HAfT19_VariantX3 after addition of one exon (HAfT19_VariantX2) or two exons (HAfT19_VariantX1). See text for further details.

Fig 5. PCR cloning clarifies HAfT6 transcript sequence and structure.

The structure of HAfT 6 was studied by PCR cloning and Sanger sequencing. The overlap of Cufflinks transcripts (Cufflinks_00024116 and 00024274) suggested a longer, more complex transcript. Primer sets were designed using sequences from both Cufflinks transcripts to test if they comprised a longer single transcript. In Panel A, several primer sets spanned different portions of the two Cufflinks transcripts at this locus. Individual PCR products shown in the agarose gel were excised separately, cloned and Sanger sequenced. Primer Set#7 was amplified twice to clearly show a PCR product (far right lane). In Panel B, the combined consensus Sanger sequences from all primer sets showed two variants, X1 and X2, containing either four or five exons, respectively. Note that red bands in black exons indicate Sanger sequence base variants that differ from alignment with Rn6. See text for further details.

Fig 6. Antisense lncRNA HAfT3 overlaps Tex36.

Primers to transcript Cufflinks_00005778 on Chr1 show only 2 exons (solid arrows) were transcribed on the opposite strand in an antisense direction to the rat Tex36 homolog (also known as LOC499279) as shown by open arrows for each of 4 exons. A new predicted start site (TSS) for Tex36 that overlapped HAfT3 was evident from RNA-Seq reads that aligned with Cufflinks_00005445 and was missing from the predicted Tex36 transcript (NM001024288) for rat.

Fig 7. HAfT homologies to mouse and human genomes.

All transcripts from Table 1 were searched for homologous mouse or human chromosomal regions. Each HAfT locus was grouped and then sorted by the highest percent coverage at a chromosomal locus for mouse (MoHomolChrRegions tab) and for human (HuHomolChrRegions tab) as shown S5 Table. Results for a representative transcript at each HAfT locus are graphed in Fig 7. A 30% sequence coverage (dotted line) was set as the criterion for rat transcript homology to a mouse or human chromosomal locus. All rat HAfTs aligned with mouse homologous loci including 6 novel lncRNAs (*) without RefSeq annotation in rat or mouse. For human chromosomal loci, there were 17 rat HAfTs aligning above the 30% coverage criterion from which 2 were known genes (Cyp2c24, Rps27l) and 2 had potentially novel human homologs (*). Another 5 human homologs that were below the 30% coverage cutoff that included 3 potentially novel (?*) human homologous loci (HAfT 19, 20 and 21) and 2 loci (e.g. HAfT1 and HAfT12) that aligned to annotated rat lncRNAs (?H) based on genomic synteny (see Table 2).