Expression of maternal and somatic rRNA variants in zebrafish

A) Organization of maternal (yellow) and somatic (blue) rDNA genes in the zebrafish genome. The 5S rRNA genes are encoded separately, while 18S, 5.8S, and 28S are derived from a single 47S pre-rRNA transcript containing additional spacer sequences (ITS1 and ITS2 (internal transcribed spacers)) that are removed in several processing steps. Expansion segments (ES3S and ES31L) are indicated by lighter-colored boxes and PCR primer icons (see Supplementary Fig. S1A). B) Relative expression levels of the four rRNAs (28S, 5.8S, 18S, and 5S), comparing maternal (yellow) and somatic (blue) variants. C) Fraction of maternal versus somatic rRNAs, obtained by quantifying reads with variant-specific SNPs (single nucleotide polymorphisms) during the first five days of development, in adult somatic organs, and in testes of 5 different males. D) Expression of maternal and somatic rRNA variants in whole embryo/larva lysate and FACS-sorted PGCs. ES, expansion segment. FACS, fluorescence activated cell sorting.

Expression of alternative ribosomal core proteins in zebrafish development

A) Heatmap of protein expression (normalized IBAQ values) for variant ribosomal protein pairs (paralogs, top; alternative isoforms, bottom) associated with zebrafish ribosomes at different developmental stages. Below, relative levels of maternal (yellow) and somatic (blue) rRNA variants at these developmental stages are indicated separately. B) Heatmap of mRNA expression of paralogs and alternative isoforms for ribosomal protein variants during embryogenesis. See also Supplementary Fig. S5. The relative levels of maternal (yellow) and somatic (blue) rRNA variants at these developmental stages are indicated in the scheme below the heat map. C) Volcano plot based on mass spectrometry data showing fold enrichment of proteins in the ribosome fraction of 5 dpf larvae versus 6 hpf embryos (n = 3 for each time point). Pairs of ribosomal protein variants with significantly different associations are shown in different shades. Non-significant but detected variants are shown in green. Permutation-based false discovery rates (FDRs) are shown as dotted (FDR < 0.01) and dashed (FDR < 0.05) lines. All significantly enriched or depleted proteins are listed in Supplementary Table S1. TPM, transcripts per million. iBAQ, intensity Based Absolute Quantification.

Location of sequence and structural differences in rRNA variants

A) Map of the maternal zebrafish ribosome isolated from 6 hpf embryos (PDB-7OYB; Leesch and Lorenzo-Orts et al., 2023). Maternal rRNA sequences are shown in surface representation (left) and cartoon representation (right) in shades of yellow. Locations of nucleotide differences in zebrafish somatic rRNAs are highlighted at the bottom (see legend for color code). A cross-section through the model is shown in the right, revealing that most of the sequence differences are located on the surface and not in the inside of the ribosome’s core. B) Sequence differences in maternal and somatic rRNA variants mapped onto rRNA secondary structure predictions derived from R2DT (Sweeney et al., 2021). 18S rRNA, 5.8S and 5S: structural predictions for Danio rerio somatic rRNAs; 28S rRNA: structural prediction for the Homo sapiens 28S rRNA, with the ES regions adjusted based on modelling of the zebrafish sequence in the Vienna RNAfold package (Lorenz et al., 2011). Circles indicate positions of nucleotide differences between the maternal and somatic rRNAs. Regions not modeled in the cryo-EM structure obtained from 6 hpf zebrafish embryos due to poor density are shown in gray.

Structural differences of intersubunit bridge regions in maternal and somatic ribosomes

Intersubunit bridges (listed in the first column) are connections between 40S SSU (composed of the 18S rRNA and 33 RPs) and 60S LSU (composed of the 5S, 5.8S, 28S rRNAs, and 46 RPs). Bridges composed of rRNA sequences with differences between maternal and somatic types are marked. H, helix. ES, expansion segment.

Maternal and somatic ribosomal subunits are translationally active in 24 hpf embryos

A) Representative polysome profile with continuous UV absorbance (A260) reading from sucrose density gradients containing lysate from 24 hpf zebrafish embryos for which the relative levels of each rRNA variant is shown in the inset. Position in the gradient and collected fractions are indicated. B) Gel electrophoretic analysis of PCR-based detection of maternal and somatic small (18S) and large (28S) rRNA variants in the individual factions indicated in (A). See Supplementary Fig. S1B for the specificity of the primers and Supplementary Table S3 for lengths of PCR products from maternal and somatic rRNAs. bp (base pair), H (helix), ES (expansion segment).

In vivo evidence for hybrid ribosome formation in 1 day post-fertilization embryos

A) Scheme summarizing the generation of embryos with tagged ribosomes by crossing Tg(Mat-RiboFLAG) females to wildtype males and Tg(Som-RiboFLAG) males to wildtype females (see Methods). B) RT-qPCR analysis of the relative amounts of maternal and somatic 28S rRNAs detected in input or eluate (IP) fractions of a FLAG-IP experiment, which used EDTA-treated lysates containing either tagged maternal (Tg(Mat-RiboFLAG)) or somatic (Tg(Som-RiboFLAG)) 60S subunits as input. C) RT-qPCR analysis of the relative amounts of maternal and somatic 28S and 18S rRNAs detected in input or eluate (IP) fractions of a FLAG-IP experiment. Cycloheximide (CHX) was added during lysis of embryos containing either tagged maternal (Tg(Mat-RiboFLAG)) or somatic (Tg(Som-RiboFLAG)) 60S subunits. Monosome fractions from RNAse-treated lysates were used as input. In (B) and (C), three biological replicates from independent natural crosses were used, and significance was calculated by t-test (if not indicated, p-value > 0.05).

Enrichment of PGC-localized mRNAs with maternal ribosomes at 1 day post-fertilization

A) Schematic of the experimental strategy. eGFP-nanos3’UTR containing mRNA was injected into 1-cell stage zebrafish embryos from which two different experimental approaches were used to investigate the ratio of maternal and somatic ribosomes in PGCs and to test which ribosomes are bound to PGC-localized mRNAs. B) Brightfield and eGFP images of 1 day post-fertilization embryos, previously injected with only water, eGFP-nanos3’UTR mRNA, or biotinylated eGFP-nanos3-3’UTR mRNA. Scale bars indicate 500 μm. C) Ratio of maternal (yellow) versus somatic (blue) 18S and 28S rRNA in FACS-isolated eGFP-positive (PGCs) and eGFP-negative (somatic) cells analyzed by RT-qPCR. D) Ratio of maternal (yellow) versus somatic (blue) 18S and 28S variants associated with biotinylated eGFP-nanos3-3’UTR mRNAs isolated by RIP (RNA immunoprecipitation). Non-biotinylated eGFP-nanos3-3’UTR mRNA served as control. Non-specific (background) amplification was determined using IPs from wild-type (uninjected) lysate and was set to zero. In (C) and (D), three biological replicates from independent natural crosses were used, and significance was calculated by t-test (if not indicated, p-value > 0.05).

Detection of maternal and somatic rRNAs in embryonic development

A) Schematic representation of the rRNA expansion segments (ES) depicted in Fig. 1A which are targeted for a PCR-based fragment length polymorphism (FLP) assay. Either ES3S (helical regions H9a and H9b) in the 18S rRNA or ES31L (helical regions H79a and H79b) in the 28S rRNA are PCR amplified using a single primer pair (indicated by primer icons) detecting both maternal and somatic rRNA variants in the same reaction. Resulting PCR products differ in size due to the different lengths of ES regions in each rRNA. Supplementary Table S3 contains the relevant PCR conditions and primer sequences. B) Gel electrophoretic analysis of PCR-based detection of maternal and somatic rRNA variants at the indicated developmental times. For each developmental time assayed, three individual embryos, each from an independent natural cross comprising one wildtype female and one wildtype male, were used for RNA extraction and cDNA synthesis. C) Averaged (n=3) line profiles of densitometry scans (ImageJ) from each lane of the gel shown in (B). The regions representing signal from maternal and somatic 18S rRNA PCR FLP are indicated and used for relative quantification in Fig. 1B. bp, base pair. NRT, no RT control. NTC, no template control. M, 100 bp marker.

Analysis of rRNA variant expression in different tissues

Gel electrophoretic analysis of PCR-based detection of maternal and somatic A) 18S rRNA and B) 28S rRNA variants in different adult zebrafish tissues (n=3, each). Positive control reactions using 2 dpf and egg cDNAs are included for reference. See Supplementary Fig. S1 and Supplementary Table S3 for indicated lengths of PCR products from maternal and somatic rRNAs. H, helix. bp, base pair. M, 100 bp marker.

Transcription of rRNA variants in zebrafish and primordial germ cells (PGCs) from 1 through 10 days post fertilization

Expression of maternal (yellow) and somatic (blue) ITS1 sequences, indicative of de novo rRNA transcription, in A) whole embryos/larvae and in B) FACS-sorted PGCs. Public dataset PRJNA597223 (Redl et al. 2021) was obtained from the Sequence Read Archive at NCBI for analysis.

Detecting rRNA variants in cell lines, tumors, and regenerating fins

A) Gel electrophoretic analysis of PCR-based fragment length polymorphism (FLP) 18S rRNA variant detection from the indicated cell lines. Diagnostic PCR lengths for maternal and somatic 18S rRNAs are indicated. Positive control reactions using 3 hpf, 24 hpf, and adult fin clip cDNAs are included to compare maternal-only, maternal and somatic, or somatic-only band patterns, respectively. B) Gel electrophoretic analysis of 18S rRNA variant detection from the indicated excised tumors. See Materials and Methods for a complete description of each cell line and tumor. C) Gel electrophoretic analysis of the 18S rRNA FLP assay using cDNA made from regenerating fin blastema excised at the indicated times post initial amputation. NTC, no template control. AB9 (fibroblast cells), ZMEL (melanoma cells), ZF4 (fibroblast cells), BRAF (melanoma tumor), GNAQ (melanoma tumor), MPNST (malignant peripheral nerve sheath tumor).

Expression of alternative ribosomal core protein mRNAs in zebrafish oogenesis and adult tissues

Heatmap of mRNA expression of paralogs and alternative isoforms encoding ribosomal protein variants assayed A) over progressive stages of oogenesis and B) across adult tissues and organs. The relative levels of maternal (yellow) or somatic (blue) rRNA variants at these developmental stages is indicated in the scheme below each heat map.

Sites that differ between maternal and somatic rRNAs in zebrafish are enriched in evolutionarily less conserved regions

A) Map of the maternal zebrafish ribosome isolated from 6 hpf embryos (PDB: 7OYB; Leesch and Lorenzo-Orts et al., 2023). Maternal rRNA sequences are shown in surface representation in shades of yellow; ribosomal proteins are shown in cartoon representation in various colors. B) Positions of sequence differences in zebrafish maternal and somatic rRNA variants mapped onto rRNA secondary structure predictions of human 28S, 18S, 5.8S and 5S rRNAs, color-coded for site-specific Shannon Entropy values, which were downloaded from Ribovision2 (https://ribovision2.chemistry.gatech.edu/; Bernier et al., 2014). A higher magnification view of the 5.8S rRNA is shown in C), highlighting the co-occurrence of evolutionarily variable nucleotides (high Shannon Entropy values) with sites that differ between zebrafish maternal and somatic rRNAs.

Generation of Tg(Mat-RiboFLAG) and Tg(Som-RiboFLAG) lines

A) Cartoon depicting the generation of two transgenic lines containing either FLAG-tagged maternal or FLAG-tagged somatic ribosomes (see Materials and Methods for details). Mosaic F0 were raised to adulthood and transmission of either transgene via the germline was screened for in B) 48 hpf embryos using a green heart marker (cmlc2 promoter driving eGFP) also encoded on the plasmid (Kwan et al. 2007) as indicated by the white arrow. As expected, only half of the progeny derived from a Tg(Som-RiboFLAG) male and a wildtype female express the transgene. Scale bars indicate 500 μm. eGFP, enhanced green fluorescent protein. Tg, transgene.

Transgenic expression 3xFLAG-eGFP-Rpl10a

Scheme summarizing the generation of embryos with FLAG-tagged ribosomes. Brightfield and fluorescent images of developing zebrafish embryos from either A) maternally-provided Tg(Mat-RiboFLAG), B) paternally-provided Tg(Som-RiboFLAG), or C) paternally-provided Tg(Mat-RiboFLAG) parents at the indicated hours post-fertilization. Embryos depicted in (A) contain maternally deposited 3xFLAG-eGFP-Rpl10a at 0 hpf while embryos depicted in (B) begin expression of 3xFLAG-eGFP-Rpl10a after 6 hpf. See Supplementary Fig. S9 for expression at 48 hpf. As expected, progeny derived from paternally-provided Tg(Mat-RiboFLAG) show no transgene expression. Scale bars indicate 500 μm.

Incorporation of Tg(3xFLAG-Rpl10a) into translating ribosomes

A) Representative polysome profiles with continuous A260 reading from 24 hpf embryos. Position in the gradient and collected fractions are indicated. B) Western blots of polysome gradient fractions containing lysate from 24 hpf embryos with either maternally-provided Tg(Mat-RiboFLAG) or paternally-inherited Tg(Som-RiboFLAG) detecting the expression of transgenic FLAG-Rpl10a, and a negative control Western blot of polysome gradient fractions containing lysate from 24 hpf embryos with paternally-provided Tg(Mat-RiboFLAG) lacking the expression of transgenic FLAG-Rpl10a. kDa, kilodalton.

Proteins differentially associated with maternal and somatic zebrafish ribosomes.

Proteins significantly enriched or depleted in ribosomes isolated from 6 hpf embryos versus 5 dpf larvae (permutation-based FDR <0.05).

Primer sequences and PCR conditions used to detect maternal and somatic rRNA variants by PCR-based fragment length polymorphism (FLP) and RT-qPCR.