| Abstract |
miRNAs are small regulatory RNA molecules that bind to partially complementary sequences at the 3΄UTR of mRNAs and impair their translation. Each miRNA is able to target multiple transcripts exerting in this way a pleiotropic effect on the regulation of gene expression. Nowadays, the identification and further experimental justification of miRNA:mRNA interactions attract intense scientific interest. It has recently been reported that key-factors of miRNA biogenesis are involved in the control of germ stem cell division and maintenance in Drosophila melanogaster.
Initially, this study provides in-vivo experimental evidence for the negative post-transcriptional regulation of gene CG10222 in Drosophila by miR-13 through the 3΄UTR sequence. “Sensor” transgenic lines were therefore produced and analyzed, where the reporter gene EGFP was fused to the wild type or appropriately mutated versions of CG10222-3΄UTR. EGFP expression in third instar larvae remained low in the presence of the wild type CG10222-3΄UTR, while it was restored to normal levels when the binding site for miR-13 was mutated. Correspondingly, miRNA-free cell clones exhibited high sensor expression levels. It was also observed that the post-transcriptional negative regulation imposed by miR-13 on CG10222 prevailed over the endogenous transcriptional rate. In parallel, transgene spontaneous silencing, as a by-product of the genetic transformation of Drosophila, was also studied.
Based on the response of EGFP/CG10222-3΄UTR towards miR-13, sensor lines became a valuable tool in investigating the miRNA activity pattern in the male germ line. Through the analysis of EGFP expression in testes taken from third instar larvae and adult individuals, it was observed that miR-13 was highly active only in the early pre-meiotic sperm progenitor cells. As spermatogenesis reached the spermatocyte growth stage, miR-13 activity gradually disappeared. The same activity pattern was revealed after analyzing sensor lines for other miRNA implying that all germ line expressed miRNA exhibit this general activity profile.
The detection of the expression pattern for one member of the miR-2/miR-13 subfamily revealed that it was further produced in late pro-meiotic spermatocyte populations, where it was no longer active. This observation suggested the presence of an active mechanism that intercepted miRNA activity during the spermatocyte growth period. When the sensor lines were tested under spermatocyte arrest (sa) and off-schedule (ofs) mutant backgrounds, the reporter exhibited silencing in all cell types. On the contrary, the silencing pattern was not affected in always early and boule mutants, although these genes are activated during the same spermatogenesis stage. The combined findings of this study lead to a model where miRNAs are expressed and act in the early sperm progenitor cells. As germ line differentiation proceeds through the spermatocyte growth period, sa and ofs genes gradually block miRNA activity until the point where miRNA expression ceases.
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Χαρακτηρισμός προτύπων δράσης μικρών RNA στην αρσενική γαμετική σειρά της Drosophila melanogaster
