Scope of the guidelines
These guidelines are intended to cover firstly, annotation of the protein components of the canonical mammalian miRNA processing pathway (Figure 1 and Winter et al. 2009) and secondly, the annotation of the role of miRNAs in gene silencing together with the targets of miRNA regulation. The focus is on negative regulation of gene expression, or gene silencing, by miRNA regulation via the 3’UTR of mRNAs, although other mechanisms of gene regulation are mentioned. Additionally, there is a section on positive regulation of transcription by miRNA, since there are an increasing number of papers now showing this mechanism.
These guidelines do not cover the annotation of proteins involved in gene silencing after the miRNA processing pathway, although these proteins are listed in Table 1 for information. The guidelines do, however, cover annotation of proteins that regulate the cellular levels of specific miRNAs (e.g. TGF-beta). Only experimental data demonstrating the role of a miRNA in a process/function through addition of an exogenous miRNA (IDA) or reduction of a miRNA (IMP) should be annotated. Co-incident expression data associated with a specific condition (IEP) should not be annotated, this information is available through other resources, such as ArrayExpress; expression alone does not confirm the involvement of a miRNA in a biological process.
Since there are some major differences between mammalian and plant miRNA biogenesis and action, there is also a section specifically describing the current status of plant miRNA biogenesis and action.
The canonical miRNA processing pathway starts with the production of the primary miRNA transcript (pri-miRNA) by RNA polymerase II with subsequent cleavage of the pri-miRNA by the microprocessor complex DROSHA–DGCR8 in the nucleus. This cleavage results in a precursor hairpin, the pre-miRNA, which is exported from the nucleus by RAN:GTP:XPO5. Once in the cytoplasm, DICER1 ribonuclease, in complex with one of the double-stranded RNA-binding proteins, TARBP2 or PRKRA (PACT), cleaves the pre-miRNA hairpin to its mature double-stranded length of around 22 nucleotides. The RNA-induced silencing complex (RISC)-loading complex, comprising of DICER1, TARBP2 (or PRKRA) and one of the argonaute proteins (AGO1-4), loads the functional strand of the mature miRNA into the RISC and the passenger strand is degraded. DICER1 and TARBP2/PRKRA then dissociate leaving the mature RISC (miRISC) consisting of AGO1-4 and miRNA. The miRNA then guides the RISC to silence target mRNAs through mRNA cleavage, translational repression or deadenylation (Figure 1).
Annotation of the miRNA processing pathway
The recommended annotations for those proteins involved in miRNA processing are shown in the left panel of Figure 1. Additional notes on some of the components of this pathway are given below.
Figure 1. The canonical mammalian miRNA processing pathway. The proteins involved in the pathway of miRNA formation are shown together with the GO terms that they are expected to be associated with. The relevant protein complex IDs could also be annotated with these terms. See also (Winter et al. 2009). Protein names: DROSHA: Ribonuclease 3; DGCR8: Microprocessor complex subunit DGCR8; XPO-5: Exportin-5; RAN-GTP: GTP-binding nuclear protein Ran; DICER1: Endoribonuclease Dicer; TARBP2: RISC-loading complex subunit TARBP2; AGO: Argonaute.
Note: A miRNA identifier (or a mRNA identifier for Argonaute) should not be included in the annotation extension field for any of the annotations to the core miRNA processing machinery. This is because this pathway is applicable to thousands of miRNAs (or mRNAs) that are involved in (or affected by) the gene silencing pathway, and so is not informative information.
DROSHA and DICER1
DROSHA and DICER1 are both ribonuclease enzymes that have the same activity but differ in the site of their cleavage. As such, both of these proteins can be annotated with “ribonuclease III activity” (GO:0004525). The definition of this term is “Catalysis of the endonucleolytic cleavage of RNA with 5'-phosphomonoesters and 3'-OH termini; makes two staggered cuts in both strands of dsRNA, leaving a 3' overhang of 2 nt.” Figure 2 depicts the cleavage site within the miRNA for both enzymes.
Figure 2. DICER and DROSHA cleavage sites.
RISC complex and the RISC-loading complex
It should be noted that, in some older papers, authors did not distinguish between the RISC complex and the RISC-loading complex. The mature RISC complex now is known to be composed of an Argonaute protein (AGO1-4) and a miRNA, whereas the RISC-loading complex is composed of an Argonaute protein (AGO1-4), DICER1, and TARBP2 or PRKRA together with the miRNA. Figure 3 shows the distinction between the RISC-loading components and those of the RISC complex.
Figure 3. RISC assembly in human cells. a. The first step is RISC loading, in this step the miRNA/miRNA* duplex is transferred from DICER1 to AGO in the RISC loading complex (RLC) followed by dissociation of DICER1 and TARBP2. b. Next, the N domain of AGO actively wedges between the miRNA strands and c. the PAZ domain of AGO unwinds the miRNA duplex. d. The passenger strand (miRNA*) dissociates from the RISC and undergoes rapid degradation, by a mechanism that is unclear. e. miRNA within the mature RISC binds to its target mRNA sites. Adapted from Stroynowska-Czerwinska, Fiszer, and Krzyzosiak 2014.
The RISC-complex may contain any of the four mammalian argonaute proteins, however only AGO2 has endoribonuclease activity and so is the only one that can cleave mRNA. The other argonautes are involved in silencing the mRNA target by translational repression, mRNA deadenylation or one of the other mechanisms briefly described in the “Modes of miRNA action” section.
These guidelines do not intend to cover the annotation of proteins involved in the miRNA-mediated mechanisms of gene expression regulation after formation of the mature RISC, only the miRNAs, however these proteins are listed in Table 1 as it may be useful to recognise them in context.
Table 1. The proteins involved in miRNA-mediated mechanisms of gene expression regulation. Adapted from Table 1 in Stroynowska-Czerwinska, Fiszer, and Krzyzosiak 2014, which contains the full table including references. No attempt has been made to suggest annotations for these proteins in these guidelines.
Modes of miRNA action
In order to be able to correctly identify GO terms that can be used to describe the functional roles of miRNAs, we must first understand the mechanisms by which miRNAs can regulate gene expression (Figure 4).
These guidelines are primarily for annotating proteins and miRNAs involved in gene silencing via the interaction between a miRNA-containing RISC complex and the 3’UTR of its target mRNA, causing either mRNA cleavage, translational repression (initiation or elongation block) or mRNA deadenylation followed by decapping and degradation of the mRNA. Other mechanisms are briefly described later for information. It should be noted that many of these mechanisms and their dependencies are still not fully understood (Wilczynska and Bushell 2014), therefore a definitive description cannot be given herein. When annotating, however, the curator should always capture the available experimental evidence and consider the intentions and interpretations of the author.
Figure 4. Post-transcriptional modes of miRNA action. miRNAs may promote degradation of mRNA via deadenylation and decapping or mRNA cleavage as well as repress translation of mRNAs by initiation or elongation block.
An mRNA is cleaved when there is near-perfect sequence complementarity between the miRNA and its target mRNA, this is most common in plants. The miRNA guides the RISC complex to the target mRNA, which is cleaved by AGO2. mRNA cleavage can only be mediated by AGO2 as AGO1, 3 and 4 do not have endoribonuclease activity.
Translational repression occurs when there is imperfect sequence complementarity between the miRNA and mRNA target. The RISC complex binds to the 3’UTR of the target mRNA and recruits additional factors, e.g. DDX6 (see Table 1), that interact with translation factors to interfere with translation initiation and/or elongation (Figure 4). Translational repression may lead to deadenylation and degradation.
This mechanism is one of the most common modes of gene silencing by miRNA (Guo et al. 2010) and occurs when there is imperfect complementarity between the miRNA and mRNA target. Deadenylation is the shortening of the poly(A) tail of the mRNA, and once the tail reaches a minimum length the mRNA is decapped and subsequently degraded. Deadenylation can also lead to translational repression of the mRNA.
Figure 5 depicts those proteins involved in mRNA deadenylation and decapping that follows binding of the miRNA to its target mRNA.
Figure 5. miRNA involvement in mRNA deadenylation and decapping.
Other miRNA-mediated mechanisms of gene regulation
In addition to targeting of the 3’UTR of mRNAs, miRNAs have been shown to regulate gene expression in other ways, as follows.
Alternative translational repression
miRNAs have been shown to repress translation by targeting either the 5’UTR or coding regions of their target mRNAs (Hausser et al. 2013 and references in Introduction of Zhang et al. 2014) as opposed to the usual 3’UTR regulation.
miRNAs have also been shown to be capable of activating translation by targeting 5’UTR or 3’UTR of mRNAs (see references in Introduction of Zhang et al. 2014), although this has only been shown in a few studies.
Regulation of transcription
miRNAs are also implicated in both the positive and negative regulation of transcription of mRNAs (see references in Introduction of Zhang et al. 2014). See the “Annotating miRNAs that activate gene expression” section for an example of positive regulation of transcription by miRNAs.
There is evidence that miRNAs may target other miRNAs to regulate their processing (Cipolla 2014).
As most of these additional mechanisms have not been as well studied as the miRNA role in post-transcriptional gene silencing via the 3’UTR, we will not provide detailed guidelines here on how to annotate each of these mechanisms.