A novel class of small RNAs in mouse spermatogenic cells

Abstract

Small noncoding RNAs, including small interfering RNAs (siRNAs) and micro RNAs (miRNAs) of approximately 21 nucleotides (nt) in length, have emerged as potent regulators of gene expression at both transcriptional and post-transcriptional levels in diverse organisms. Here we report the identification of a novel class of small RNAs in the mouse male germline termed piwi-interacting RNAs (piRNAs). piRNAs are approximately 30 nt in length. They are expressed during spermatogenesis, mostly in spermatids. piRNAs are associated with MIWI, a spermatogenesis-specific PIWI subfamily member of the Argonaute protein family, and depend on MIWI for their biogenesis and/or stability. Furthermore, a subpopulation of piRNAs are associated with polysomes, suggesting their potential role in translational regulation.

Figure 1.

PAGE analysis of a novel small RNA species. (A) A 15% PAGE gel containing equal amounts of total RNA from 24-dpp miwi^+/− (+/−) and miwi^−/− (−/−) testes and visualized with ethidium bromide. A closed arrowhead points to the novel small RNA species. (B) A 15% PAGE gel containing equal amounts of ³²P-end-labled total RNA from adult mili^+/− (+/−) and mili^−/− (−/−) testes, with or without treatment by RNase or DNase. (M) 10-nt DNA (A) or RNA (B) molecular-weight ladder; (O) 45-nt DNA oligomer only. (Closed arrowhead) The novel small RNA species; (open arrowhead) DNA oligomer.

Figure 2.

Sequence analyses of piRNAs. (A,B) Length distributions of piRNAs from this study (A) and from the Imai set (B). (C,D) Sequence logos showing conserved motifs among 40 cloned piRNAs (T is used to replace U).

Figure 3.

Distribution of piRNA-encoding sequences in the genome. The chromosomes were drawn in scale and aligned by their centromere positions. piRNAs from this study and from the Imai set are indicated in red and black, respectively. The number of piRNAs in a particular cluster is indicated by the number next to the cluster.

Figure 4.

Tissue and spermatogenic stage specificity of piRNA expression. (A) PAGE analysis of total RNA extracts from liver (Li), brain (Br), kidney (Ki), testis (Te), ovary (Ov), spleen (Sp), embryonic stem cells (E), intestine (In), caudal epididymus (Ep), lung (Lu), heart (H), and stomach (St), showing that piRNAs are only detected in the testis by ethidium-bromide staining. (B) Northern blot analysis of RNA from A, showing that piRNA-T4 is only detectable in the testis. (C) PAGE analysis with ethidium-bromide staining showing that piRNAs are only detected in the miwi^+/− testis at 24 dpp. (D) PAGE analysis with ³²P-end-labeled total RNA indicating that piRNAs are first detectable at 22 dpp in mili^+/− testes, but are absent in 22-dpp mili^−/− testes. (E) Northern blot analysis of RNA from C, showing that piRNA-T1, piRNA-T2, and piRNA-T4 are detectable in 24 dpp, but not 16-dpp, miwi^+/− testes. (F) Negative image of PAGE analysis with ethidium-bromide-stained PAGE gel showing that piRNAs are present in 24, 28, 32, and 36 dpp testes. (G) Northern blot analysis of total RNA from F, showing that piRNA-T4 is increasing in abundance from 24–32 dpp.

Figure 5.

piRNAs are associated with MIWI and polysomes. (A) A₂₆₀ profile of 24 dpp miwi^+/− (+/−) and miwi^−/− (−/−) testicular extracts separated by sucrose density gradient fractionation with RNP, 40S, 60S, monosomal, and polysomal fractions indicated. (B) PAGE analysis of RNA from pooled RNP, monosome, and polysome fractions of the adult miwi^+/− testicular extracts end-labeled with ³²P. piRNAs, indicated by the arrowhead, were quantified with the NIH Image J software. (C) PAGE analysis of RNA from pooled polysome fractions in A stained with ethidium bromide. piRNAs are indicated by the arrowhead. (D) Northern blots of total RNA and polysome-associated RNA from C probed with three individual piRNAs. miRNA miR-16 was used as a loading control. (E) Negative image of ethidium-bromide-stained PAGE gel containing RNA coimmunoprecipitated with MIWI antibody (αMW) or its preimmune sera (Pre), and 10 μg of testicular RNA input (I). (M) 10-nt DNA ladder.