XB-ART-55955Curr Biol January 1, 2018; 28 (2): 296-302.e3.
Expression of Alternative Ago2 Isoform Associated with Loss of microRNA-Driven Translational Repression in Mouse Oocytes.
Mouse oocyte maturation, fertilization, and reprogramming occur in the absence of transcription, and thus, changes in mRNA levels and translation rate are regulated through post-transcriptional mechanisms . Surprisingly, microRNA function, which is a major form of post-transcriptional regulation, is absent during this critical period of mammalian development [2, 3]. Here, we investigated the mechanisms underlying the global suppression of microRNA activity. In both mouse and frogs, microRNA function was active in growing oocytes but then absent during oocyte maturation. RNA sequencing (RNA-seq) of mouse oocytes uncovered that the microRNA effector protein AGO2 is predominantly expressed as an alternative isoform that encodes a truncated protein lacking all of the known essential domains. Full-length Ago2 as well as the related Argonautes (Ago1, Ago3, and Ago4) were lowly expressed in maturing mouse oocytes. Reintroduction of full-length AGO2 together with an exogenous microRNA in either mouse or frog oocytes restored translational repression of a target reporter. However, levels of endogenous transcripts remained unchanged. Consistent with a lack of microRNA activity, analysis of transcripts with alternative polyadenylation sites showed increased stability of transcripts with a longer 3'' UTR during oocyte maturation. Redundant mechanisms protecting endogenous transcripts and the conserved loss of microRNA activity suggest a strong selection for suppressing microRNA function in vertebrate oocytes.
PubMed ID: 29307557
PMC ID: PMC5790201
Article link: Curr Biol
Genes referenced: ago2 ago3 cdkn2c dicer1 myc rpl7 xrn1 zp3.2
GO keywords: oocyte maturation
Article Images: [+] show captions
|Figure 4. Isoforms with Longer 3′ UTRs Are More Stable in Maturing Oocytes (A) Percentage of distal alternative polyadenylation site usage in GV and MII oocytes. Significant increases in distal alternative polyadenylation usage are colored in orange; significant decreases are colored in blue. Each dot represents one gene. (B) Number of significant changes in alternative polyadenylation ratios resulting in longer 3′ UTRs or shorter 3′ UTRs during the GV to MII transition. (C) Same as (A), except in mouse ESCs and EpiCs. (D) Same as (B), except in mouse ESCs and EpiCs. (E) Boxplots showing change in 3′ UTR length for genes with a significant change in alternative polyadenylation during the GV to MII transition. Left plot shows the ratio of the longer 3′ UTR relative to the shorter 3′ UTR for genes where the distal polyadenylation site is more stable (n = 463 genes). Right plot shows the ratio of the shorter 3′ UTR relative to the longer 3′ UTR for genes where the proximal polyadenylation site is more stable (n = 129 genes). (F) RNA-seq coverage showing stabilization of distal alternative polyadenylation site in GV to MII transition at two genes (Pafah1b1 and Srpk1). Both genes are on the negative strand (transcribed from right to left). Two GV replicates, two MII replicates, Gencode annotated 3′ UTR, and miR-15a TargetScan predicted target site are shown. See also Figure S4 and Table S2.|
|Figure S1 - Schematic of reporter UTRs. Related to Figure 1. Schematic of reporter UTR sequences illustrating miR-15a binding to the perfect and bulged sequences in the 3’ UTR of Renilla luciferase.|
|Figure S3 – Correlations between arrays. Related to Figure 3. A) Log2 quantile normalized intensity values between replicates of LSL-GFP-myc-hAGO2/LSL-GFP-myc-hAGO2; Zp3-Cre+ and Zp3-Cre- mouse GV oocytes injected with miR-15a mimic.|
|Figure S4 – Evaluation of additional potential mechanisms that impact miRNA activity. Related to Figure 4. A) Luciferase assay in HEK-293T cells transfected with psiCHECK2, a vector containing Firefly luciferase and Renilla luciferase with either no let-7 sites, mutated sites, or 4X Bulge sites in the 3’ UTR for Renilla luciferase. psiCHECK2 was co-transfected with either GFP, full length hAGO2, or truncated oocyte AGO2. Luciferase data is a ratio of Renilla signal over Firefly signal. Full and truncated AGO2 is not significantly different from GFP control in all cases. All error bars represent standard deviation. N.S. = not significant. B) RNA-Seq expression of Xrn1 in mouse embryonic stem cells and mouse GV oocytes.|
References [+] :
ABE, Global gene silencing is caused by the dissociation of RNA polymerase II from DNA in mouse oocytes. 2010, Pubmed