Abundant and dynamically expressed miRNAs, piRNAs, and other small RNAs in the vertebrate Xenopus tropicalis

Abstract

Small regulatory RNAs have recently emerged as key regulators of eukaryotic gene expression. Here we used high-throughput sequencing to determine small RNA populations in the germline and soma of the African clawed frog Xenopus tropicalis. We identified a number of miRNAs that were expressed in the female germline. miRNA expression profiling revealed that miR-202-5p is an oocyte-enriched miRNA. We identified two novel miRNAs that were expressed in the soma. In addition, we sequenced large numbers of Piwi-associated RNAs (piRNAs) and other endogenous small RNAs, likely representing endogenous siRNAs (endo-siRNAs). Of these, only piRNAs were restricted to the germline, suggesting that endo-siRNAs are an abundant class of small RNAs in the vertebrate soma. In the germline, both endogenous small RNAs and piRNAs mapped to many high copy number loci. Furthermore, endogenous small RNAs mapped to the same specific subsets of repetitive elements in both the soma and the germline, suggesting that these RNAs might act to silence repetitive elements in both compartments. Data presented here suggest a conserved role for miRNAs in the vertebrate germline. Furthermore, this study provides a basis for the functional analysis of small regulatory RNAs in an important vertebrate model system.

Figure 1.

Expression of small RNAs and core protein components in X. tropicalis. (A) Total RNA was isolated from X. tropicalis adult liver and stage I and stage II oocytes. RNA was size-selected using the miRVana kit. Ten micrograms of this RNA was subjected to β-elimination or not as indicated and analyzed on a denaturing gel after 5′ end-labeling. Arrows indicate RNA that likely represent miRNAs, siRNAs, and piRNAs. (B,C) The expression of core components of small RNA pathways in Xenopus oocytes, eggs, and somatic tissues was assayed using RT-PCR and qRT-PCR. For each experiment equivalent oocyte total RNA was reverse transcribed. E, egg; L, liver; I, intestine; N.D., not done. (B) RT-PCR experiment. Control experiment without the addition of reverse transcriptase. RT-PCR primers are listed in Supplemental material, Armisen_SupData4.xls. (C) qRT-PCR for the four Piwi-related genes described in X. tropicalis.

Figure 2.

(A,B) Length distributions of filtered short RNA sequences in oocyte and somatic cell libraries. (A) Counting total reads per library at each length. (B) Counting unique tags per library at each length.

Figure 3.

(A–D) Distribution of small RNA (sRNA) types in oocytes (A,B) and somatic cells (C,D), by unique tag (A,C) and by read (B,D) count. Filtered tags were identified either by BLAST similarity to known RNA types (miRNA, tRNA, rRNA, other noncoding RNA [oncRNA]), or otherwise by occurrence in “blocks” of neighboring tags, or not. Blocks were defined by groups of tags of a given type with no gaps between neighbors greater than a fixed value (200 bases). Tag types used were single locus tags, tags with 10+ loci, and all tags, defining low copy number, high copy number, and mixed blocks, respectively. Tags not in blocks were termed isolated. (E, top) Schematic view of general method for defining short RNA blocks. (E, bottom) Different families of blocks are made using only unique mapping tags (low copy number), tags with ten or more loci (high copy number), and all tags.

Figure 4.

(A–C) Expression of three miRNAs (miR-101, miR-202-5p, and miR-148a) was assessed during oogenesis and in eggs. Stages I and II, III and IV, and V and VI were pooled. One hundred and fifty oocytes were used for each experiment. (D–F) The expression of the same three miRNAs was assessed in early stage oocytes (stages I and II) and a number of adult somatic tissues using Northern blotting. Ten micrograms of total RNA were used for each lane. EDC was used to crosslink small RNAs to the membrane prior to hybridization. 5S rRNA is shown as a loading control.

Figure 5.

(A) piRNA piR-1 (5′-TGAAGACGGACAGAAGATGGGTTAATTATTT-3′) expression was validated using Northern blotting of early stage oocytes (stages I and II). One hundred and fifty oocyte equivalents were used to analyze small RNA expression. A miR-101 probe was used as a control. β-Elimination was performed to assay for 2′O-methyl-modified 3′ nucleotides. EDC was used to crosslink small RNAs to the membrane prior to hybridization. (B) Experiments were performed as in A, but with probes for piR-2 (5′-TGAATTGTAGAACAATGTACAGGTACACCAT-3′) and miR-202-5p. (C) Expression of piR-1 and miR-148a was assessed in a number of adult somatic tissues and early stage oocytes (stages I and II) using Northern blotting. Ten micrograms of total RNA were used for each lane. 5S rRNA is shown as a loading control. (D) Northern blot analysis of immunoprecipitated piRNAs using piwil1.1/piwil1.2 specific antibody (PI: pre-immune).

Figure 6.

(A) Base composition at positions 1 and 10 was analyzed separately for piRNAs and 20–24 nt RNAs (siRNAs). (B) Analysis of 5′ and 3′ base overlap for unique tags. (C) Overlap of piRNA populations cloned from different stages of oogenesis. We considered only unique tags with ≥10 reads. (D) Overlap between piRNA clusters and annotated genes. (E) Identification of endo-siRNA in Xenopus tropicalis. The expression of endo-siRNA1 (5′-ACGGCCGGGGGCATTCGTATT-3′) was validated using Northern blotting after β-elimination, to assay for 2′O-methyl-modified 3′ nucleotides. miR-148a was used as a control for β-elimination as well as a loading control.