Abstract
BackgroundMicroRNA (miRNA) play a significant role in the pathogenesis of complex neurodegenerative diseases including age-related macular degeneration (AMD), acting as post-transcriptional gene suppressors through their association with argonaute 2 (AGO2) - a key member of the RNA Induced Silencing Complex (RISC). Identifying the retinal miRNA/mRNA interactions in health and disease will provide important insight into the key pathways miRNA regulate in disease pathogenesis and may lead to potential therapeutic targets to mediate retinal degeneration.MethodsTo identify the active miRnome targetome interactions in the healthy and degenerating retina, AGO2 HITS-CLIP was performed using a rodent model of photoreceptor degeneration. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data was performed to identify the cellular location of AGO2 and key members of the microRNA targetome in the retina. AGO2 findings were verified by in situ hybridization (RNA) and immunohistochemistry (protein).ResultsAnalysis revealed a similar miRnome between healthy and damaged retinas, however, a shift in the active targetome was observed with an enrichment of miRNA involvement in inflammatory pathways. This shift was further demonstrated by a change in the seed binding regions of miR-124-3p, the most abundant retinal AGO2-bound miRNA, and has known roles in regulating retinal inflammation. Additionally, photoreceptor cluster miR-183/96/182 were all among the most highly abundant miRNA bound to AGO2. Following damage, AGO2 expression was localized to the inner retinal layers and more in the OLM than in healthy retinas, indicating a locational miRNA response to retinal damage.ConclusionsThis study provides important insight into the alteration of miRNA regulatory activity that occurs as a response to retinal degeneration and explores the miRNA-mRNA targetome as a consequence of retinal degenerations. Further characterisation of these miRNA/mRNA interactions in the context of the degenerating retina may provide an important insight into the active role these miRNA may play in diseases such as AMD.
Highlights
MicroRNA play a significant role in the pathogenesis of complex neurodegenerative diseases including age-related macular degeneration (AMD), acting as post-transcriptional gene suppressors through their association with argonaute 2 (AGO2) - a key member of the ribonucleic acid (RNA) Induced Silencing Complex (RISC)
In the study presented here, we revealed that the majority of the retinal AGO2-bound miRNA transcriptome (miRnome) was comprised of only a small miRNA subset, with miR-124-3p being highly represented
A twodimensional principal component analysis (PCA) was employed to visualize differences in RNA profiles between the groups, which showed clear inter-group clustering with clear segregation between Photo-oxidative damage (PD) and dim retinas (Fig. 1B)
Summary
MicroRNA (miRNA) play a significant role in the pathogenesis of complex neurodegenerative diseases including age-related macular degeneration (AMD), acting as post-transcriptional gene suppressors through their association with argonaute 2 (AGO2) - a key member of the RNA Induced Silencing Complex (RISC). Once incorporated into RISC, miRNAs guide binding of AGO to the 3′ untranslated (3′ UTR) of the mRNA target(s) through recognition of the miRNA seed region (typically 6–8 nucleotides long, commonly referred to as 6-mer, 7-mer and 8-mer seed region) [8] This process results in post-transcriptional gene silencing through translational repression or mRNA degradation [9, 10]. Due to their selective targeting ability, miRNAs have emerged as key orchestrators of the mammalian transcriptome As such, their dysregulation has been implicated in the pathogenesis of multiple inflammatory diseases, cancers, neurological disorders as well as retinal degenerative diseases including age-related macular degeneration (AMD) [12,13,14,15,16], a disorder that affects the central vision due to progressive damage to the light-sensing photoreceptor cells in the macular region. Retinal degenerations such as AMD have complex and multi-faceted etiology, with causal links to the pathogenesis of how retinal degeneration develops in AMD remaining elusive [17,18,19,20,21,22]
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