Introduction: DAZL (deleted in azoospermia-like) is an RNA binding protein essential for germ cell entry into meiosis and later stages of germ cell maturation, and its absence is associated with infertility in vertebrates. Efforts to study DAZL and its in vivo RNA targets have been largely limited to mouse, thus human-specific investigations are required.
Methods: Human DAZL protein was immunoprecipitated from 17 week ovarian lysate bound RNAs were sequenced using the Illumina HiSeq platform. 3′UTR-luciferase assays and polysome profiling were utilised to confirm translational regulation by DAZL of novel target RNAs. RNAi knockdown of Dazl in e13.5 fetal mouse ovary cultures was used to investigate functional consequences of DAZL regulation of newly identified targets.
Results and discussion: Differential gene expression analysis using DESeq2 found 429 RNAs that were significantly enriched by DAZL immunoprecipitation (Padj<0.01). Bioinformatic analysis revealed the presence of at least one consensus mouse Dazl binding element in the 3′UTR of >97% of the potential targets, indicating similarities between mouse and human DAZL binding sites. Gene-set enrichment analysis identified the gene ontology meiosis (GO:0007126), with RNAs involved in cohesin establishment and DNA repair, which are novel findings, as well as in synaptonemal complex formation. Increased luciferase activity (P<0.004) and polysome profiling (P<0.05) demonstrated that DAZL regulates the translation of novel human target RNAs SMC1B, RAD51, TEX11 and SYCP1 via their 3′UTR. Creation of a Dazl hypomorph using RNAi knockdown in e13.5 fetal mouse ovary cultures resulted in decreased expression of target RNAs. Furthermore, Dazl hypomorph oocytes had a smaller nuclear diameter than scrambled RNAi controls after 13 days of culture (P=0.0047) ongoing studies are investigating potential impact on meiotic progression.
These data demonstrate novel RNA targets of DAZL in human fetal oocytes and identified pathways by which DAZL may contribute to lifelong oocyte quality in women.
[Supported by grants from the Medical Research Council].
11 Jul 2016 - 11 Jul 2016