Epitranscriptomics: Correlation of N6-methyladenosine RNA methylation and pathway dysregulation in the hippocampus of HIV transgenic rats

Abstract

Internal RNA modifications have been known for decades, however their roles in mRNA regulation have only recently started to be elucidated. Here we investigated the most abundant mRNA modification, N6-methyladenosine (m6A) in transcripts from the hippocampus of HIV transgenic (Tg) rats. The distribution of m6A peaks within HIV transcripts in HIV Tg rats largely corresponded to the ones observed for HIV transcripts in cell lines and T cells. Host transcripts were found to be differentially m6A methylated in HIV Tg rats. The functional roles of the differentially m6A methylated pathways in HIV Tg rats is consistent with a key role of RNA methylation in the regulation of the brain transcriptome in chronic HIV disease. In particular, host transcripts show significant differential m6A methylation of genes involved in several pathways related to neural function, suggestive of synaptodendritic injury and neurodegeneration, inflammation and immune response, as well as RNA processing and metabolism, such as splicing. Changes in m6A methylation were usually positively correlated with differential expression, while differential m6A methylation of pathways involved in RNA processing were more likely to be negatively correlated with gene expression changes. Thus, sets of differentially m6A methylated, functionally-related transcripts appear to be involved in coordinated transcriptional responses in the context of chronic HIV. Altogether, our results support that m6A methylation represents an additional layer of regulation of HIV and host gene expression in vivo that contributes significantly to the transcriptional effects of chronic HIV.

Fig 1. Distribution of genes with differential m⁶A RNA methylation in the hippocampus of HIV Tg rats.

A) A larger number of genes with down-regulation of m⁶A methylation was observed in HIV Tg rats than the number of genes with up-regulated methylation, in particular, 201 genes with significant down-regulated methylation (p-value<0.05) while 172 genes were found with significant up-regulated methylation (p-value<0.05). Inset: Distribution of m⁶A peaks within hippocampal mRNAs of HIV Tg and control rat. Transcript architecture is shown underneath: 5’ untranslated region (UTR); coding sequence (CDS); and 3’ UTR. The density of m⁶A peaks was greater on the distal CDS and 3’UTR and showed the greatest enrichment in the immediate vicinity of the stop codon at the CDS-3’UTR boundary. B) The distribution of log2 fold change (log2FC) of genes with up-regulated and down-regulated methylation C,D) Correlation between the methylation and expression changes for genes with up-regulated and down-regulated methylation (Pearson correlation = 0.67 and 0.58 respectively). Among the 201 genes with down-regulated methylation, the expression of 127 genes are also down-regulated while the rest remain unchanged; Among the 172 genes with up-regulated methylation, the expression of 83 genes are also up-regulated while expression of 89 genes remain unchanged.

Fig 2. Pattern of m⁶A methylation in HIV and host transcripts of HIV Tg rats.

A) Outline of HIV genome organization and gag/pol deletion in HIV Tg rats. B) m⁶A methylation in HIV RNA from HIV Tg rats (HIV-1 MeRNA-IP) is enriched in transcripts corresponding to the 5’ and 3’ of the virus with a similar overall distribution as observed in HIV transcripts in vitro [–44]; signal obtained from RNA-Seq of input RNA from the hippocampus of HIV Tg rats (HIV-1 Input). C) Distribution of m⁶A methylation in representative host genes with significant differential m⁶A methylation (*significantly differentially methylated peaks). D) PCR validation of significantly differentially methylated intervals in the genes in D (*p<0.05, *p<0.01 by t test).

Fig 3. Pathway analysis of differential m⁶A RNA methylation in the hippocampus of HIV Tg rats.

Top 40 differential m⁶A methylation pathways in the hippocampus of HIV Tg rats by Gene Set Enrichment Analysis (GSEA) (Complete list in S2 Table).

Fig 4. Representative host pathways involving differentially m⁶A methylated genes in HIV Tg rats.

A-D) Several pathways containing genes differentially m⁶A modified are involved in neural function and are indicative of synaptodendritic injury [–60], consistent with differential expression of genes in these ontology classes [52, 53]; E-G) pathways involved in inflammation and immune response were also differentially m⁶A methylated, consistent with previous observations both in humans and HIV Tg rats [52, 53]; H,I) differentially m⁶A methylated gene transcripts also included pathways related to HIV infection; J-L) and RNA metabolism and processing, e.g. splicing, which are processes in which m⁶A RNA methylation has been previously implicated [–10]. Significance is indicated in each plot (Complete list in S2 Table).

Fig 5. Correlation of m⁶A methylation and gene expression.

A) The normalized enrichment scores (NES) of pathways showing both significant differential m⁶A RNA methylation (NES of pathway methylation) and gene expression (NES of pathway expression). RNA m⁶A methylation was either directly or inversely correlated with gene expression. Pathways with increased m⁶A RNA methylation and expression are shown in red; pathways with decreased m⁶A RNA methylation and expression are shown in blue; pathways with increased m⁶A RNA methylation and decreased expression are shown in orange. B) Pathways showing increased differential m⁶A RNA methylation and expression included some pathways involved in apoptosis and tissue responses (red); pathways showing decreased differential m⁶A RNA methylation and expression included some pathways involved in neuronal trophisms and function (blue); pathways showing increased differential m⁶A RNA methylation and decreased expression included pathways involved in RNA processing and metabolism. C) Example of pathway showing increased differential m⁶A RNA methylation and expression, “KEGG Apopotosis”; D) Example of pathway showing decreased differential m⁶A RNA methylation and expression, “Reactome Developmental Biology”; E) Example of pathway showing increased differential m⁶A RNA methylation and decreased expression, “Reactome mRNA Splicing”.