SiRNA GO annotation manual

From GO Wiki
Jump to navigation Jump to search

siRNA-mediated regulation of gene expression

Small interfering RNAs (siRNAs) are ~20-24nt regulatory non-coding RNAs (ncRNAs), generated from dsRNAs in a Dicer-dependent manner from exogenous (viral) RNAs or endogenous transcripts. Similar to miRNAs, they are loaded onto Argonaute (AGO) proteins to form RISC-type complexes and target mRNAs via base-pairing. Unlike miRNAs, they generally 'target' the transcripts they are derived from and are therefore fully complementary. SiRNAs have been shown to act in the cytosol to inhibit the translation of mRNAs and in the nucleus to direct heterochromatin formation. Between species, there is more variation in the way they are generated and deployed. Plants have the most complex siRNA system (PMID:37772967). For example, siRNA may be derived from endogenous transcripts which form dsRNA from long inverted repeats that form stem-loops, or overlapping transcripts from opposite strands and instances of endogenous transcripts that can be converted to dsRNA by RNA-dependent polymerase. The dsRNA is processed into siRNAs by Dicer enzymes. The length and the modifications of the siRNA direct the specificity of the AGO protein loading and the mode of gene silencing. SiRNAs have been shown to direct post-transcriptional and transcriptional gene silencing, but the specific processes are highly dependent on the organism.

It must be noted that is not always possible to associate GO terms with the siRNA itself, as it may lack an annotatable identifier (e.g. RNAcentral URS or gene ID), as siRNAs may be generated somewhat 'randomly' e.g. from exogenous dsRNAs. RNAcentral v24 contains over ~44,000 siRNA sequences, the vast majority from plants which have an extensive endogenous siRNA system (for a review see The plant siRNA landscape). The proteins involved in the biological processes, however, should be annotated regardless of the availability of a distinct and annotatable siRNA sequence.

siRNA-mediated post-transcriptional gene silencing

Like miRNAs, siRNAs act as part of an RNA-induced silencing complex (RISC) to act in post-transcriptional gene silencing (PTGS). This results in the decreased translation of the mRNA either because of the destruction of the target sequence or the inhibition of translation itself.

GO terms for the annotation of siRNA-mediated PTGS

Terms for the annotation of siRNA-mediated PTGS are in the GO branch 'regulatory ncRNA-mediated post-transcriptional gene silencing' (GO:0035194)

 		|_regulatory ncRNA-mediated post-transcriptional gene silencing (GO:0035194)
 		  |_siRNA-mediated post-transcriptional gene silencing (GO:0140766)
 		    |_siRNA-mediated gene silencing by inhibition of translation (GO:0070549)
 		    |_siRNA-mediated gene silencing by mRNA destabilization (GO:0090625)

The depth of annotation is determined by the level of experimental detail. In short, where the siRNA causes the mRNA and protein product to decrease, then 'siRNA-mediated gene silencing by mRNA destabilization' (GO:0090625) should be used, where the siRNA causes the protein product to decrease but the mRNA levels remain unchanged, then this can be used to infer that the process is 'siRNA-mediated gene silencing by inhibition of translation' (GO:0070549). The parent term should be used where this distinction cannot be made i.e. just a decrease in protein levels. Protein product levels are often assayed using a reporter construct, in animal cells this is often via luciferase and in plants via green fluorescent protein (GFP). The mechanism of siRNA-mediated gene silencing by mRNA destabilization is generally mediated via the AGO endonuclease activity in the RISC, which occurs when there is perfect complementarity between the target and the guide RNA. As siRNAs are derived from the target sequences themselves, unlike miRNAs, perfect complementarity often exists between the siRNA and the target. However, instances of siRNA-mediated gene silencing by inhibition of translation have been shown in plants for virus-derived siRNA and stress-induced endogenous 22nt siRNAs (PMID:32376953,PMID:24889238).


Where the species of siRNA is annotatable (i.e. the sequence is identified and an ID is available to attach an annotation to), then it may be possible to annotate the molecular function: 'mRNA base-pairing translational repressor activity' (GO:1903231).


A decision tree is provided below to guide the choice of term used based on the level of evidence provided.


Link to SiRNA Decision Tree 2024 PDF File:SiRNA Decision Tree 2024.pdf

Link to SiRNA Decision Tree 2024 PowerPoint File:SiRNA Decision Tree 2024.pptx


'Fig 1. Decision Tree for the GO terms and annotation extensions used for capturing targets of siRNAs.

siRNA-mediated transcriptional gene silencing

SiRNAs can enter the nucleus as part of a RISC-type 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:26238358). 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 and the use of each is explained below. It should be noted that separate terms have been created for siRNA-mediated heterochromatin formation processes that are initiated by DNA methylation.

GO terms for the annotation of siRNA-mediated transcriptional repression via heterochromatin formation

 |_regulatory ncRNA-mediated heterochromatin formation (GO:0031048)
 	|_ siRNA-mediated heterochromatin formation (GO:0141194)
 		|_siRNA-mediated facultative heterochromatin formation (GO:1902795)
	 	|_siRNA-mediated pericentric heterochromatin formation (GO:0140727)
 		|_siRNA-mediated retrotransposon silencing by heterochromatin formation (GO:0141007)
 		|_ gene silencing by siRNA-directed DNA methylation (GO:0080188)
		 	|_retrotransposon silencing by siRNA-directed DNA methylation (GO:0141010)

1. facultative heterochromatin formation The expression of genes can be controlled by the switching of chromatin from euchromatin, which is permissive for transcription, and heterochromatin, that is refractory to transcription. This latter type of chromatin is referred to as facultative, as it is responds to changes i.e., genes turned on or off in response to a stimulus. This occurs via the base-pairing of siRNA in RISC-type complex, leading to the recruitment of chromatin modifiers and the enrichment of histone H3 methylated on lysine 9 (H3K9me). The term 'siRNA-mediated facultative heterochromatin formation' (GO:1902795) should be used when siRNAs are used to repress transcription from a gene and not for repression of transcription in constitutive heterochromatin. Note: that 'gene silencing by siRNA-directed DNA methylation' (GO:0080188) should be used when the mechanism of repression is initiated by DNA methylation.

2. pericentric heterochromatin formation The formation of constitutive heterochromatin at the pericentric repeats in fission yeast (Schizosaccharomyces pombe) is mediated by an siRNA-dependent process (PMID:26238358). These regions produce transcripts that are processed into siRNAs. The siRNAs are incorporated into an RNA-induced initiation of transcriptional gene silencing (RITS) complex, which contains an AGO homolog. RITS is targeted to the locus by siRNA-base pairing with nascent transcripts. A small RNA loaded onto the RITS complex targets the nascent noncoding RNA by base-pairing interactions and recruits a H3K9 methyltransferase complex, which promotes heterochromatin formation by the deposition of repressive chromatin marks, and the RNA-directed RNA polymerase complex, leading to the formation of dsRNA and hence the formation of more siRNAs in a positive feedback to keep these domains repressed. This process should be annotated with the term, 'siRNA-mediated pericentric heterochromatin formation' (GO:0140727). Note that although this process is most well characterised in fission yeast, it is not restricted to this organism. For example, it has been shown that siRNAs processed from endogenous dsRNA precursors are required for heterochromatin silencing in the adult soma and for the targeting of Su(var)39 and H3K9 methylation at the pericentromere (PMID:19948966).

3. siRNA-directed DNA methylation In plants, siRNAs can mediate heterochromatin formation via DNA methylation of cytosine at the C-5 position. This occurs via the base-pairing of siRNA in RISC-type complex, leading to the recruitment of DNA methyltransferases (DNMTs). This should be annotated using the term 'gene silencing by siRNA-directed DNA methylation' (GO:0080188). Where this results in the suppression of transposable element (TE) activity, this should be annotated with the term 'retrotransposon silencing by siRNA-directed DNA methylation' (GO:0141010).

4. retrotransposon silencing siRNAs generated from transcripts from TEs can mediate suppression of activity by heterochromatin formation. This occurs via the base-pairing of siRNA with nascent TE transcripts in RISC-type complex, leading to the recruitment of chromatin modifiers. Where this is initiated by histone modification, the term 'siRNA-mediated retrotransposon silencing by heterochromatin formation' (GO:0141007) should be used. Where this is initiated by DNA methylation, this should be annotated with the term 'retrotransposon silencing by siRNA-directed DNA methylation' (GO:0141010).