IMPACT OF SILENCING A MEIOTIC lncRNA ON SPERMATOGENESIS: DEVELOPMENT OF AN ANTISENSE OLIGONUCLEOTIDE MICROINJECTION-BASED APPROACH

Abstract

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with little or no protein-coding potential. Approximately 40% of lncRNAs are intergenic, while the remaining 60% overlap with, or are located adjacent to protein-coding genes. The biological functions of most lncRNAs, particularly of the antisense ones, remain poorly understood due to technical limitations. Current CRISPR-based approaches for lncRNA depletion are effective for those located far from coding genes, but not suitable for most of them, as they may interfere with their adjacent or overlapping genes. Conversely, RNA interference (RNAi)-mediated knockingdown acts through the microRNA pathway and is therefore more efficient at silencing cytoplasmic transcripts, with limited effectiveness in nuclear contexts. Given these limitations, our aim was to establish an in vivo knock-down strategy for lncRNAs through testicular microinjection of chemically modified antisense oligonucleotides (ASOs). This required the optimization of a delivery system via rete testis microinjection. In this study, we focus on the functional characterization of an antisense lncRNA showing meiotic differential expression. RNA-FISH revealed its nuclear localization in prophase I meiotic cells, closely associated with paired homologous chromosomes, and suggesting a potential role in homology search during meiosis. Using this approach, we achieved ~50% reduction of the target lncRNA. Although this silencing efficiency is lower than that typically expected for RNAi (>70%), it was enough to significantly reduce testis size and seminiferous tubules diameter. TUNEL assays showed increased testicular cell death upon decreased levels of the lncRNA. While the precise function of this lncRNA remains to be determined, our findings highlight its importance in meiosis. Furthermore, the development of this methodology represents an important advance, as it enables the in vivo functional characterization of lncRNAs in a complex system such as the testis, and may be extended to the study of antisense lncRNAs in other organs and tissues.