Linking maternal and somatic 5S rRNA types with different sequence-specific non-LTR retrotransposons

Abstract

5S rRNA is a ribosomal core component, transcribed from many gene copies organized in genomic repeats. Some eukaryotic species have two 5S rRNA types defined by their predominant expression in oogenesis or adult tissue. Our next-generation sequencing study on zebrafish egg, embryo, and adult tissue identified maternal-type 5S rRNA that is exclusively accumulated during oogenesis, replaced throughout the embryogenesis by a somatic-type, and thus virtually absent in adult somatic tissue. The maternal-type 5S rDNA contains several thousands of gene copies on chromosome 4 in tandem repeats with small intergenic regions, whereas the somatic-type is present in only 12 gene copies on chromosome 18 with large intergenic regions. The nine-nucleotide variation between the two 5S rRNA types likely affects TFIII binding and riboprotein L5 binding, probably leading to storage of maternal-type rRNA. Remarkably, these sequence differences are located exactly at the sequence-specific target site for genome integration by the 5S rRNA-specific Mutsu retrotransposon family. Thus, we could define maternal- and somatic-type MutsuDr subfamilies. Furthermore, we identified four additional maternal-type and two new somatic-type MutsuDr subfamilies, each with their own target sequence. This target-site specificity, frequently intact maternal-type retrotransposon elements, plus specific presence of Mutsu retrotransposon RNA and piRNA in egg and adult tissue, suggest an involvement of retrotransposons in achieving the differential copy number of the two types of 5S rDNA loci.

Keywords: 5S ribosomal RNA; embryogenesis; maternal rRNA; target-specific retrotransposon; zebrafish.

FIGURE 1.

5S rRNA variant types and their expression in zebrafish. (A) Chromosomal localization and organization of the maternal-type (blue) and somatic-type 5S (red) rRNA main clusters in the zebrafish genome. The colored parts on the chromosomes indicate the areas with 5S rRNA genes. In the zoomed-in parts, each colored rectangle is an individual 5S rRNA gene and each white rectangle is a MutsuDrS1 retrotransposon with two potential ORFs (black). (B) Sequence comparison of the 5S rRNA variant types. Maternal, Seq1 sequence; somatic, Seq87 sequence. Internal control regions are shown as colored boxes. A box (yellow); internal element (gray); C Box (light brown) (Schramm and Hernandez 2002; Martins and Wasko 2004). The probes used for Northern blotting are indicated with colored lines. (C) Expression of the 5S rRNA maternal and somatic types indicated by percentage of total 5S rRNA sequencing reads. (Adult FT) Adult female tail; (Adult MWB) adult male whole-body. (D) Northern blot analyses with total RNA from zebrafish eggs and adult (male) tissue and probes as indicated in B. Probe 5S-M recognizes a sequence specific for the maternal-type 5S RNA; probe 5S-S a sequence specific for somatic-type 5S RNA; and probe 5S-C a common sequence between the two 5S RNA variants. Each panel contains lanes from the same gel. In the second panel, the contrast was adjusted as the LNA 5S-S probe gave a very bright signal.

FIGURE 2.

Retrotransposon family MutsuDr linked to 5S rRNA loci. (A) 5′ half of the two rRNA types that include all nucleotide differences. Indicated are the sequence-specific MutsuDr 3′ target sites in the maternal- (blue) and somatic-type (red) 5S rDNA sequence. Small arrows show the insertion sites. (B) Tree of the different MutsuDr retrotransposon subfamilies constructed based on their genomic consensus DNA sequences. (C) An example of the maternal-type 5S rRNA genome organization on chromosome 4 (Supplemental File SF8): expressed 5S rRNA gene copies (5Sseq variant number, blue arrow), nonexpressed 5S rRNA gene copies (NE, gray arrow), and MutsuDr retrotransposon elements (brown arrow). The 5S RNA NE elements right downstream from a Mutsu element are remnants of the 5S rRNA gene that was disrupted by the insertion of the retrotransposon. (D) Relative expression of MutsuDr RNA (brown) and MutsuDr piRNAs (green) as indicated by the fraction of the sequencing reads found for each type. (Embryos) The combined samples as indicated in Figure 1C; (adult FT) adult-female tail.