The most abundant maternal lncRNA Sirena1 acts post-transcriptionally and impacts mitochondrial distribution

Abstract

Tens of thousands of rapidly evolving long non-coding RNA (lncRNA) genes have been identified, but functions were assigned to relatively few of them. The lncRNA contribution to the mouse oocyte physiology remains unknown. We report the evolutionary history and functional analysis of Sirena1, the most expressed lncRNA and the 10th most abundant poly(A) transcript in mouse oocytes. Sirena1 appeared in the common ancestor of mouse and rat and became engaged in two different post-transcriptional regulations. First, antisense oriented Elob pseudogene insertion into Sirena1 exon 1 is a source of small RNAs targeting Elob mRNA via RNA interference. Second, Sirena1 evolved functional cytoplasmic polyadenylation elements, an unexpected feature borrowed from translation control of specific maternal mRNAs. Sirena1 knock-out does not affect fertility, but causes minor dysregulation of the maternal transcriptome. This includes increased levels of Elob and mitochondrial mRNAs. Mitochondria in Sirena1-/- oocytes disperse from the perinuclear compartment, but do not change in number or ultrastructure. Taken together, Sirena1 contributes to RNA interference and mitochondrial aggregation in mouse oocytes. Sirena1 exemplifies how lncRNAs stochastically engage or even repurpose molecular mechanisms during evolution. Simultaneously, Sirena1 expression levels and unique functional features contrast with the lack of functional importance assessed under laboratory conditions.

Figure 1.

Genomic organization of the Sirena1 locus in the mouse genome. Shown is composition of UCSC genome browser snapshots depicting the Sirena1 locus and its expression in a fully grown GV oocyte from total RNA-seq (37). The Sirena1 gene is localized on chr. 7 between Spty2d and Uevld genes; all three genes have the same transcriptional orientation. Expression maximum is indicated by a grey dashed line and the counts per million (CPM) value at its beginning. Dashed angular lines depict alternative splicing events observed in the RNA-seq data. The black angular dashed line depicts the most common splice variant identified during lncRNA annotation in oocytes (9). The blue rectangle represents the Elob antisense pseudogene fragment in the first exon, the black arrow indicates sense orientation of the pseudogene.

Figure 2.

Sirena1 expression (A) Most expressed genes in fully-grown GV mouse oocytes. Gene expression in fragments per million per kilobase (FPKM) was estimated from total RNA-seq data (37). Sirena1 transcript abundance was estimated from reads mapping to the first and the last exon. (B) Sirena1 expression in mouse tissues. Expression was estimated as FPKM from the ENCODE polyA RNA NGS mouse tissue raw data (GSE49417 (36)). Error bars = standard deviation (SD). (C) Localization of Sirena1 in GV and MII oocytes by smFISH. As a positive control, we used Polr2a, as a negative control, bacterial DapB RNA (control). Shown is a 5 μm slice made of a maximum projection of 0.5 μm confocal sections. RNA staining signal is red, DNA labeled with DAPI is blue. Size bar = 10 μm. (D) Temporal pattern of expression of Sirena1 (C86187 splice variant) during oocyte development and in early embryos based on RNA-seq data (8,37).

Figure 3.

Evolutionary origin of Sirena1. Positions of Spty2d and Uevld genes in mammalian genomes are syntenic. The Sirena1 gene (depicted in green) can be identified in the mouse and rat genomes, but not in gerbils and hamsters. The phylogenetic tree depicting the phylogenetic relationship of the analyzed species was generated with Timetree (82).

Figure 4.

Genomic organization of the Sirena1 locus in the rat genome. Shown is the composition of UCSC genome browser snapshots depicting the Sirena1 locus and its expression in fully grown GV oocyte RNA-seq as in Figure 1A for the mouse ortholog. Dashed angular lines depict alternative splicing events observed in the RNA-seq data. The black angular dashed line depicts the most common splice variant. Dashed yellow rectangles depict upstream regions that evolved in the rat lineage differently because of retrotransposon insertions. The blue rectangle depicts the Elob antisense pseudogene fragment upstream of the first exon. Red rectangles depict rat-specific retrotransposon insertions in the Sirena1 locus.

Figure 5.

Cytoplasmic polyadenylation of Sirena1. (A) Sirena1 RNA abundance in RNA-seq data indicates cytoplasmic polyadenylation during meiotic maturation. The plot depicts the ratios of poly(A)-selected RNA-seq data (44) relative to total RNA-seq data (37), positive numbers and negative log₂ values indicate enrichment and depletion of transcripts in poly(A)-selected RNA-seq data, respectively. In blue are labeled dormant mRNAs with a short poly(A) tail in GV oocytes, which undergo polyadenylation during meiotic maturation. Gray labels depict histone and mitochondrial genes underrepresented in poly(A)-selected RNA-seq data. (B) Sequence organization and conservation of the 3′end of Sirena1. Depicted are putative CPE elements ∼100 nucleotides from the poly(A) signal in ten rodent genomes. (C) Schematic design of reporters for analysis of cytoplasmic polyadenylation. Renilla luciferase reporter (control) was polyadenylated, other reporters were not polyadenylated. (D) Relative luciferase activity of microinjected reporters. Reporters were microinjected into GV oocytes, which were either matured for 20 h (MII) or cultured in the presence of milrinone, which prevents resumption of meiosis (GV). Activity was calculated as luciferase activity of each Renilla reporter divided by a co-injected polyadenylated firefly reporter standard. Error bars = SD. (E) Western blot analysis of MOS and CCNB1 proteins in fully-grown GV oocytes and MII eggs. (F) Relative abundance of Mos, Ccnb1, and Sirena1 transcripts in the polysomal fraction in GV oocytes and MII eggs. Three independent experiments were performed, error bars = SD. *P < 0.05 and **P < 0.01, n.s. = not significant (paired t-test). In a control experiment in the right half of the graph, polysomes were disrupted during fractionation by adding EDTA. The control experiment was performed once as a technical duplicate, shown is the average value.

Figure 6.

Elob regulation by RNAi through Sirena1. (A) Shown is expression and 21–23 nt small RNAs in Elob, Elobl and Sirena1 loci. Each locus scheme was built from UCSC browser snapshots containing four tracks. Three of them display RNA-seq data of small RNA from GV oocytes (52) or MII eggs (71,72), the fourth displays total RNA (37). Dashed lines represent maximum CPM values written at their beginning. At the bottom, conservation tracks for four mammalian genomes are shown. The Elob locus contains convergent transcription of Elob (blue) and Srrm2, which does not produce small RNAs. Elob can basepair with the Sirena1 transcript produced in trans. The Sirena1 transcript carries antisense oriented Elob pseudogene. The Elobl locus contains the Elob pseudogene, which has been incorporated into the last exon of a transcript driven by a RMER6BA retrotransposon-derived promoter. (B) Basepairing of the Sirena1 transcript with Elob and Elobl mRNAs. (C) MA plot of gene expression in Dicer knock-out oocytes. (D) MA plot of gene expression in oocytes expressing catalytically inactive AGO2. RNA-seq data come from (59).

Figure 7.

Sirena1 knock-out. (A) Schematic depiction of CRISPR cleavage sites in the Sirena1 locus. (B) qPCR analysis of Sirena1 expression in knock-out oocytes. Error bars = SD. (C) smFISH of Sirena1 in wild-type and knock-out GV oocytes. Shown is a 0.5 μm confocal section. Red color marks RNA staining, blue color marks DNA labeled with DAPI. The DapB probe set served as a negative control. Size bar = 10 μm. (D) Quantification of nuclear envelope breakdown (NEBD) in Sirena1^−/− and wild-type oocytes resuming meiosis. The number of analyzed oocytes is indicated in brackets. (E) Quantification of entry of Sirena1^−/− and wild-type oocytes into anaphase I of meiosis. The number of analyzed oocytes is indicated in brackets. (F) Long-term breeding experiment with four breeding pairs where both parents had the same genotype. The Y axis depicts the cumulative litter size of each breeding pair. A symbol crossed with a line indicates the last litter of each breeding pair. (G) Breeding performance of all females with indicated genotypes mated before 16 weeks, 16–32 weeks and 32–48 weeks of age. Error bars = SD.

Figure 8.

Gene expression analysis of Sirena1^−/− oocytes. (A) MA plots depict gene expression in fully-grown GV oocytes (left) and MII eggs (right). Significantly upregulated transcripts are depicted in red, the downregulated ones in blue. Triangles represent genes encoded by mtDNA. (B) Relative expression change of the neighboring genes. (C) qPCR validation of the increased abundance of Elob transcript in Sirena1^−/− oocytes. (D) GO annotation analysis of the cellular component of transcriptome changes. (E) qPCR analysis of upregulation of mitochondrial genes; shown is expression of genes in Sirena1^−/− GV oocytes relative to wild-type controls. * indicates P < 0.05. (F) Analysis of mitochondrial DNA amount. All error bars = SD.

Figure 9.

Changes in mitochondrial distribution in Sirena1^−/− knock-out oocytes. (A) Confocal images of living GV and MII oocytes stained with MitoTracker. A single optical section is shown. Size bar = 10 μm. (B) Mean fluorescence signal per GV oocyte. Error bars = SD. (C) Quantitative analysis of the MitoTracker signal. Each data point represents the median size of mitochondrial clusters in an oocyte estimated as median mitochondrial signal volume (μm³) per oocyte from confocal microscopy data. (D) Mitotracker signal distribution analyzed as mean radial intensity from the nucleus center. (E) Transmission electron microscopy of wild-type and Sirena1^−/− oocytes. The dashed line indicates position of the nuclear envelope. The white rectangle indicates the region magnified in the neighboring panel. Filled arrowheads point to contacts between mitochondria; empty arrowheads point to absenting contacts between mitochondria. (F) Quantification of ATP in Sirena1^−/− and wild-type oocytes using the Adenosine 5′-triphosphate (ATP) bioluminescent somatic cell assay kit (FLASC). GV oocytes were denuded and analyzed immediately (t_0 h), or were cultured without cumulus cells for an additional 20 h in the presence of IBMX (t_20 h). Each data point represents one estimation of ATP level per oocyte, the horizontal line represents the mean.