CircNAPEPLD is expressed in human and murine spermatozoa and physically interacts with oocyte miRNAs

Abstract

Circular RNAs (circRNAs) have a critical role in the control of gene expression. Their function in spermatozoa (SPZ) is unknown to date. Twenty-eight genes, involved in SPZ/testicular and epididymal physiology, were given in circBase database to find which of them may generate circular transcripts. We focused on circNAPEPLDiso1, one of the circular RNA isoforms of NAPEPLD transcript, because expressed in human and murine SPZ. In order to functionally characterize circNAPEPLDiso1 as potential microRNA (miRNA) sponge, we performed circNAPEPLDiso1-miR-CATCH and then profiled the expression of 754 miRNAs, by using TaqMan® Low Density Arrays. Among them, miRNAs 146a-5p, 203a-3p, 302c-3p, 766-3p and 1260a (some of them previously shown to be expressed in the oocyte), resulted enriched in circNAPEPLDiso1-miR-CATCHed cell lysate: the network of interactions generated from their validated targets was centred on a core of genes involved in the control of cell cycle. Moreover, computational analysis of circNAPEPLDiso1 sequence also showed its potential translation in a short form of NAPEPLD protein. Interestingly, the expression analysis in murine-unfertilized oocytes revealed low and high levels of circNAPEPLDiso1 and circNAPEPLDiso2, respectively. After fertilization, circNAPEPLDiso1 expression significantly increased, instead circNAPEPLDiso2 expression appeared constant. Based on these data, we suggest that SPZ-derived circNAPEPLDiso1 physically interacts with miRNAs primarily involved in the control of cell cycle; we hypothesize that it may represent a paternal cytoplasmic contribution to the zygote and function as a miRNA decoy inside the fertilized oocytes to regulate the first stages of embryo development. This role is proposed here for the first time.

Histone Post-Translational Modifications and CircRNAs in Mouse and Human Spermatozoa: Potential Epigenetic Marks to Assess Human Sperm Quality

“…Our data strongly suggest that SPZ-derived circNAPEPLD may function as a decoy to inhibit the anti-proliferative activity of some oocyte-derived miRNAs, thus allowing cell proliferation, a massive event during the first stages of embryo development [283]. As strongly suggested for other circRNAs, circNAPEPLD also appears to be able to bind RBPs.…”

“…Interestingly, human and murine SPZ retain two circular isoforms of NAPEPLD (that we named circNAPEPLDiso1 and circNAPEPLDiso2). Both isoforms are expressed at low levels in testis and heterogeneously fluctuate in sperm samples analyzed suggesting that, as for genetic traits, an individually variable transmission of epigenetic traits may exist as well [283]. Otherwise than previously reported, circNAPEPLDiso1 and circNAPEPLDiso2 expression appears to be independent from the linear counterparts, although there is evidence demonstrating a competition between circRNAs and linear transcripts [230,233].…”

“…Such an analysis has suggested that circNAPEPLDiso1 is potentially translatable, with a predicted ORF of 352 aa, in both humans and mice. This protein has a shorter C-terminal compared with the protein encoded by the linear transcript (393 and 396 aa in humans and mice, respectively), but concerning the biological function of circRNA-encoded proteins it is still an intriguing open question [283].…”

“…Until today, no experimental evidence exists in support to this hypothesis. The only indication regarding the delivery of a circRNA from SPZ to the oocyte comes from Ragusa et al, 2019 [283]. What a paternal circRNA in the zygote may do is still a fascinating mystery.…”

The influence of l ‐carnitine on the expression of miRNAs in asthenospermia spermatozoa and the network regulation of the associated molecules

“…Furthermore, Chioccarelli et al () detected 10,726 transcripts in human spermatozoa by circRNA microarray. Ragusa et al () found that circNAPEPLDiso1 may represent a paternal cytoplasmic contribution to the zygote and may function as a miRNA decoy inside the fertilised oocytes to regulate the first stages of embryo development. In this study, five circRNAs (hsa_circ_0003812, hsa_circ_0001337, hsa_circ_0000816, hsa_circ_0000817 and hsa_circ_0000917) were predicted to be sponges of hsa‐miR‐206, and the circRNA‐hsa‐miR‐206‐mRNA network was generated as shown in Figure d.…”