Characterization of N6-methyladenosine long non-coding RNAs in sporadic congenital cataract and age-related cataract

Abstract

Aim: To characterize the N⁶-methyladenosine (m⁶A) modification patterns in long non-coding RNAs (lncRNAs) in sporadic congenital cataract (CC) and age-related cataract (ARC).

Methods: Anterior capsule of the lens were collected from patients with CC and ARC. Methylated RNA immunoprecipitation with next-generation sequencing and RNA sequencing were performed to identify m⁶A-tagged lncRNAs and lncRNAs expression. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses and Gene Ontology annotation were used to predict potential functions of the m⁶A-lncRNAs.

Results: Large amount of m⁶A peaks within lncRNA were identified for both CC and ARC, while the level was much higher in ARC (49 870 peaks) than that in CC (18 688 peaks), yet those difference between ARC in younger age group (ARC-1) and ARC in elder age group (ARC-2) was quite slight. A total of 1305 hypermethylated and 1178 hypomethylated lncRNAs, as well as 182 differential expressed lncRNAs were exhibited in ARC compared with CC. On the other hand, 5893 hypermethylated and 5213 hypomethylated lncRNAs, as well as 155 significantly altered lncRNA were identified in ARC-2 compared with ARC-1. Altered lncRNAs in ARC were mainly associated with the organization and biogenesis of intracellular organelles, as well as nucleotide excision repair.

Conclusion: Our results for the first time present an overview of the m⁶A methylomes of lncRNA in CC and ARC, providing a solid basis and uncovering a new insight to reveal the potential pathogenic mechanism of CC and ARC.

Keywords: N6-methyladenosine; RNA modification; age-related cataract; congenital cataract; epigenetics; long non-coding RNA.

Figure 1. The bioinformatics pipeline for m⁶A methylated lncRNAs

1) Paired-end reads were harvested from DNBSEQ-T7 sequencer, and were quality controlled by Q30. After 3′ adaptor-trimming and low quality reads removing by cutadapt software. The reads were aligned to the reference genome with Hisat2 software. Methylated m⁶A peaks were identified with MACS software, and differentially methylated m⁶A regions were defined with DiffReps software. 2) The coordinates of lncRNA mature transcripts were fully collected from three well-known transcriptome databases (NCBI, UCSC, Ensembl). The overlapped regions between differntially methylated m⁶A regions and lncRNA transcripts were identified with Bedtools, while these lncRNAs with overlapped regions were defined as “differentially m⁶A methylated lncRNA”. 3) The differentially m⁶A lncRNAs were annotated as 5 types (intronic, antisense, sense-overlapping, bidirectional and intergenic) according to the genomic location relative to nearby protein-coding genes with in-house scripts written in Perl&SQL. lncRNA: Long non-coding RNA; m⁶A: N⁶-methyladenosine.

Figure 2. Overview of m⁶A modification patterns within lncRNAs

A, B: Venn diagram showing the overlap of m⁶A peaks within lncRNAs in CC and ARC groups, and in ARC-1 and ARC-2 groups. C, D: The overlap of gene transcripts within lncRNAs in CC and ARC groups, and in ARC-1 and ARC-2 groups. E, F: The number of m⁶A peaks per lncRNA between CC and ARC groups, and between ARC-1 and ARC-2 groups. G: Consensus sequences in CC and ARC, ARC-1 and ARC-2 respectively. m⁶A: N⁶-methyladenosine; lncRNA: Long non-coding RNA; CC: Congenital cataract; ARC: Age-related cataract; ARC-1: ARC patients under the age of 70; ARC-2: ARC patients above the age of 80.

Figure 3. Landscape of m⁶A-modified lncRNAs in lens epithelial cells

A, B: Hierarchical clustering analysis showed that there were significant differences in the m⁶A methylation patterns within lncRNAs between CC and ARC and not so dramatical between ARC-1 and ARC-2. C, D: Distribution profiles on chromosomes of m⁶A sites within lncRNAs between CC and ARC and between ARC-1 and ARC-2. E, F: The number of m⁶A peaks within lncRNA on each chromosome between CC and ARC groups and between ARC-1 and ARC-2. G: Distributions of genomic origins of differentially distributed m⁶A lncRNAs. m⁶A: N⁶-methyladenosine; lncRNA: Long non-coding RNA; CC: Congenital cataract; ARC: Age-related cataract; ARC-1: ARC patients under the age of 70; ARC-2: ARC patients above the age of 80.

Figure 4. Overall lncRNA expression patterns between groups

Scatter plots (A, B), hierarchical clustering (C, D), and volcano (E, F) of the RNA-seq data between CC and ARC and between ARC-1 and ARC-2. lncRNA: Long non-coding RNA; CC: Congenital cataract; ARC: Age-related cataract; ARC-1: ARC patients under the age of 70; ARC-2: ARC patients above the age of 80.

Figure 5. The GO project to describe gene and gene product attributes

A-C: Major GO terms significantly enriched for hypermethylated lncRNA-associated genes in ARC compared with those in CC; D-F: For hypomethylated lncRNA-associated genes in ARC compared with those in CC; G-I: Hypermethylated lncRNA-associated genes in ARC-2 compared with those in ARC-1; J-L: Hypomethylated lncRNA-associated genes in ARC-2 compared with those in ARC-1. GO: Gene ontology; lncRNA: Long non-coding RNA; CC: Congenital cataract; ARC: Age-related cataract; ARC-1: ARC patients under the age of 70; ARC-2: ARC patients above the age of 80.

Figure 6. KEGG pathways for pathway analysis

A: Hypermethylated lncRNA-associated genes in ARC compared with those in CC; B: Hypomethylated lncRNA-associated genes in ARC compared with those in CC; C: Hypermethylated lncRNA-associated genes in ARC-2 compared with those in ARC-1; D: Hypomethylated lncRNA-associated genes in ARC-2 compared with those in ARC-1. KEGG: Kyoto Encyclopedia of Genes and Genomes pathways; lncRNA: Long non-coding RNA; CC: Congenital cataract; ARC: Age-related cataract; ARC-1: ARC patients under the age of 70; ARC-2: ARC patients above the age of 80.