PiRNA GO annotation manual

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PIWI-associated/interacting RNAs (piRNAs) are ~24-34nt regulatory non-coding RNAs (ncRNAs) that are bound by the PIWI subclade of the Agonaute family of proteins. Their association with PIWI is the main method of identifying these ncRNAs as piRNAs, as their sequence diversity makes them difficult to predict. In general, mature piRNAs are terminally 2’ O-methylated with a bias to a 5’ uridine. PIWIs, and therefore piRNAs, are restricted to animals. In most species, piRNAs are transcribed from piRNA clusters and processed from long single-stranded precursors in a Dicer-independent manner. The most common role associated to piRNAs is the maintenance of genome integrity by the suppression of transposable elements (TEs) in the germline. However, there are examples of them acting outside of this role. Like miRNAs and siRNAs, piRNAs act by base-pairing with target transcripts to mediate gene silencing. The piRNA-mediated gene silencing can occur via PIWI endonucleolytic cleavage of transcripts in the cytosol or in the promotion of heterochromatin formation at target loci. The silencing mechanism is dependent on the organism and on cell type/stage, and may be linked to the biogenesis pathway of the piRNA. For a recent review see PMID:36217279.

piRNA biogenesis

There are two very distinct mechanisms of piRNA processing: primary and secondary. In primary processing, piRNAs are generally derived from long, single-stranded precursors transcribed from piRNA clusters. The precursor piRNAs are cleaved via the endonucleolytic activity of cytosolic PIWI and mitochondrially-tethered Zucchini (orthologous to vertebrate phospholipase D family member 6, Pld6). In secondary processing, the targeting of a piRNA to a mRNA and the resulting cleavage of the target result in the creation of a secondary piRNA. These secondary piRNAs can then target piRNA precursors to produce more secondary piRNAs. This self-perpetuating loop is often referred to as a “ping-pong cycle”. In secondary piRNA processing, destruction of the target mRNA is an integral part of piRNA production and can be viewed as a type of adaptive immunity. Transcription from transposable elements results in the production of piRNAs that are independent of the initiator sequence in the mRNA and thus silence the activity of the transposable element.

Fig. 1. The two mechanisms of piRNA processing in Drosophila. A simplified scheme showing the link between piRNA processing in Drosophila and transposon silencing. A) Primary processing. The primary transcripts of piRNA clusters are shortened into piRNA intermediates, which are loaded onto Piwi proteins. They are then trimmed from the 3' end to the size of mature piRNAs, and 2'-O-methylated. In mice, Piwil4 (MIWI2) and Pld6 (MitoPLD) are the functional equivalents to Drosophila Piwi and Zucchini, respectively. B) Secondary processing. In Drosophila, secondary “ping-pong” processing is carried out by the cycling of target RNAs and piRNAs between the PIWI-class endonucleases Aubergine (Aub) and Argonaute 3 (AGO3) (PMID:31932527). In mice, the functional equivalents of these proteins are Piwil2 (MILI) and Piwil1 (MIWI). Secondary processing occurs in a molecular condensate called the P-granule or nuage. Note that this is different to the proposed model of secondary processing in the mouse germline, where Piwil2 acts in both the generation of sense and anti-sense piRNAs and then loads secondary piRNAs onto Piwil4. PiRNA-loaded Piwil4 translocates into the nucleus to mediate heterochromatin silencing via DNA methylation (PMID:29437694).

GO terms for the annotation of piRNA biogenesis

  |_regulatory ncRNA processing (GO:0070918)
     |_piRNA processing (GO:0034587)
        |_primary piRNA processing (GO:0140990)
        |_secondary piRNA processing (GO:0140965)

Annotation tips

The mechanism of biogenesis is context-dependent, and this can be used to pick the correct term. As the most extensive studies have been conducted in mice and Drosophila, these are the main examples.

piRNA processing class clues
Species Feature Primary Secondary
Any sense+anti-sense piRNA pairs No Yes
Any anti-sense piRNAs only Yes No
Mouse PIWI:Piwil1 Yes No
Mouse PIWI:Piwil2 & Piwil4 No Yes
Mouse germ cells Yes Yes
Drosophila PIWI:piwi Yes No
Drosophila PIWI:Aub & Ago3 No Yes
Drosophila follicle cells Yes No
Drosophila germ cells Yes Yes
Any Occurs in Nuage/P granule No Yes
Drosophila Occurs in Yb body Yes No
Mouse Occurs in Pi-body Yes No

Note:There may be other ways of generating piRNAs, for example phasing/inchworming piRNA species are generated in an end-on-end pattern, but as it is not clear whether these are separate pathways with their own distinct components, no term has been provided as yet. Where the mechanism is not clear, use 'piRNA processing' (GO:0034587).

piRNA-mediated post-transcriptional gene silencing

The base-pairing of piRNAs in complex with PIWIs in the cytoplasm leads the endonucleolytic cleavage of target sequences by PIWI leading to post-transcriptional gene silencing (PTGS). In mice, PTGS is mediated by Piwil1/Piwil2 and in Drosophila, Aub and AGO3. As Fig. 1. shows, in Drosophila, PTGS is intimately associated with secondary piRNA processing and so co-annotation of factors involved in these processes may be required.

GO terms for the annotation of mediated post-transcriptional gene silencing

   |_post-transcriptional gene silencing (GO:0016441)
       |_regulatory ncRNA-mediated post-transcriptional gene silencing (GO:0035194)
           |_piRNA-mediated gene silencing by mRNA destabilization (GO:0140991)
               |_piRNA-mediated retrotransposon silencing by mRNA destabilization (GO:0141009)

In most instances, the target will be a TE mRNA (PMID:36217279)and the term 'piRNA-mediated retrotransposon silencing by mRNA destabilization' (GO:0141009) should be used. Where the target of PTGS is a non-TE gene, then the term 'piRNA-mediated gene silencing by mRNA destabilization' (GO:0140991) should be used. Although this is less frequent, it has been shown to occur in the degradation of maternal mRNAs in the Drosophila syncytial embryo (PMID:20953170), the spermatid mRNAs in mice (PMID:24787618) and in female sex determination in the silkworm (PMID:33672402). In these instances, the term 'piRNA-mediated gene silencing by mRNA destabilization' (GO:0140991) should be used.

PTGS by piRNAs is often mediated by the slicer activity of the PIWI protein, as in secondary piRNA processing in Drosophila. However, other mechanisms can be used to destabilize mRNAs. During mouse spermatogenesis, Piwil1 acts by recruiting Cnot7 to promote mRNA deadenylation, which acts in concert with by Piwil1-mediated cleavage to degrade mRNAs (PMID:24787618).

These biological process terms should be used to annotate the ncRNA and protein components of the pathway. Due to their variable nature and the complexity of encoding loci, a suitable piRNA identifier may not be available to attach annotation to. However, RNAcentral v24 contains ~250k piRNA sequences across a number of species.

Where an piRNA identifier is available, the molecular function term 'mRNA base-pairing translational repressor activity' GO:1903231 should be used.

Example annotation
PMID:24828047:A single female-specific piRNA is the primary determiner of sex in the silkworm
 Annotations for Bombyx mori Fem piRNA (URS000073423D_7091)
 Molecular Function:mRNA base-pairing translational repressor activity silencing (GO:1903231)
         has_input UniProtKB:A0A024FSH6 (Masc)
 Biological Process:piRNA-mediated gene silencing by mRNA destabilization (GO:0140991)
         part_of female sex determination (GO:0030237)

piRNA-mediated transcriptional gene silencing

PiRNAs can enter the nucleus as part of a PIWI complex to base-pair with nascent mRNAs leading to the recruitment of chromatin modifiers, the deposition of repressive marks and the formation of heterochromatin (PMID:36217279). The pathways utilised may be limited to certain organisms, so care should be taken when choosing a term for annotation. The terms provided in the GO are consistent with those processes that have been experimentally shown to exist. It should be noted that separate terms have been created for piRNA-mediated heterochromatin formation processes that are initiated by DNA methylation or by histone modification.

GO terms for the annotation of piRNA-mediated transcriptional gene silencing

   |_heterochromatin formation (GO:0031507)
       |_regulatory ncRNA-mediated heterochromatin formation (GO:0031048)
           |_ piRNA-mediated heterochromatin formation (GO:0140966)
               |_piRNA-mediated retrotransposon silencing by heterochromatin formation (GO:0141006)
               |_ gene silencing by piRNA-directed DNA methylation (GO:0141176)
                   |_retrotransposon silencing by piRNA-directed DNA methylation  (GO:0141196)

In mammalian systems, piRNAs can mediate heterochromatin formation via DNA methylation of cytosine at the C-5 position (PMID:32674113). This occurs via the base-pairing of piRNA in a PIWI complex (Piwil4 in mice), leading to the recruitment of DNA methyltransferases (DNMTs). This should be annotated using the term 'gene silencing by piRNA-directed DNA methylation' (GO:0141176). Where this results in the suppression of transposable element (TE) activity, this should be annotated with the term 'retrotransposon silencing by piRNA-directed DNA methylation' (GO:0141196). In Drosophila, which lacks a DNMT3 ortholog, retrotransposon silencing occurs via the recruitment of histone modifying complexes, and in this case the term 'piRNA-mediated retrotransposon silencing by heterochromatin formation' (GO:0141006) should be used. To date, this has only been shown for TEs and not for the silencing of non-TEs genes and so, in line with GOC policies, an intermediate term has not been created for 'piRNA-mediated gene silencing by heterochromatin formation' by this means.