Annotation Conf. Call 2016-02-08
From GO Wiki
Bluejeans Conference Line
- Recurring meeting URL: https://bluejeans.com/993661940
Review New IGI Documentation
- Following on from the 2016-01-12 call where we discussed how to annotate co-transfection experiments, below is a draft of new IGI documentation for curation.
- For this call, I'd like to review the new documentation, and solicit input for examples.
- For reference, here is the link to the existing documentation on the web site: http://geneontology.org/page/igi-inferred-genetic-interaction
- Also, please see Ruth's github ticket on the existing IGI documentation: https://github.com/geneontology/go-annotation/issues/1327
IGI: Inferred from Genetic Interaction
• Genetic interactions such as suppression, enhancement, synergistic (synthetic) interaction, etc. • Co-transfection experiments in which more than one gene is expressed in a heterologous system to assess functional interaction • Functional complementation in which a gene from one species is used to complement a mutation in a different species • Rescue experiments in which expression of one gene rescues the phenotype of a mutation in another gene • Inference about one gene drawn from the phenotype of a mutation in a different gene??
The IGI evidence code is used for annotations based on experiments reporting the effects of perturbations in the sequence or expression of one or more genes or gene products. IGI is also used for experiments that interrogate functional interactions between two or more genes or gene products when co-expressed, for example, in a cell line. Additional uses of IGI include functional complementation experiments, phenotypic rescue, and inferences made about one gene drawn from the phenotype of mutations in a different gene (?).
Key to deciding whether or not to use the IGI or IMP (Inferred from Mutant Phenotype) evidence code is consideration of the point of reference (i.e., what is being compared) to determine a possible interaction. If experiments interrogate the effects of multiple mutations or differences from the control, then use IGI. If experiments interrogate the effects of a single mutation or difference from the control, then use IMP.
The IGI evidence code requires curators enter a stable database identifier for the interacting entity in the With/From field of the Gene Association File (GAF). Independent interactors may be captured in the With/From field by separating each entry with a pipe. If the interaction experiment involves multiple perturbations simultaneously, e.g. triply mutant strains, then the respective interactors are separated with a comma.
Genetic interactions such as suppression, enhancement, synergistic (synthetic) interactions, etc. This use of the IGI evidence code refers to the more “traditional” genetic interaction experiments performed in model organisms, such as Saccharomyces cerevisiae, as well as more recent approaches adopted in a number of different systems such as RNA-mediated knockdown or genome editing techniques. Note that genetic interaction experiments may be performed with both loss- and gain-of-function mutations. Consequently, curators will need to use their expertise to determine whether interaction phenotypes resulting from gain-of-function mutations are informative about the normal, wild type role of a gene or gene product.
Example 1: Double loss-of-function mutations resulting in enhancement of a mutant phenotype Localized cell wall degradation is essential for proper cell fusion in the fission yeast, Schizosaccharomyces pombe. This process is accomplished by the localized action of degradative enzymes including several distinct glucanases that act on different polysaccharides. Deletion of multiple glucanases in S. pombe results in decreasing efficiency of cell fusion indicating that each enzyme contributes additively to this process.
exg3 fungal-type cell wall disassembly involved in conjugation with cellular fusion (GO:1904541) PMID:25825517 IGI agn2 agn2 fungal-type cell wall disassembly involved in conjugation with cellular fusion (GO:1904541) PMID:25825517 IGI exg3
Example 2: Gain-of-function mutation The response to axonal injury requires the activities of MAP kinase and cAMP signaling pathways that are required, for example, for signaling growth cone formation. In C. elegans, the activity of the upstream-most kinase in one of the MAPK signaling pathways, DLK-1, is stimulated by Ca2+ influx mediated by the EGL-19 voltage-gated calcium channel. EGL-19’s regulatory role in the MAPK-mediated axon regeneration pathway was determined, in part, through doubly mutant animals containing an egl-19 gain-of- function mutation and a dlk-1 loss-of-function mutation that showed a reduced axon regenerative response when compared to egl-19(gf) alone.
EGL-19 positive regulation of MAPK cascade involved in axon regeneration (GO:1904922) PMID:20203177 IGI DLK-1
Note that in this example, reciprocal IGI annotations are not made, as the GO term selected for EGL-19 does not make sense for DLK-1.
Example 3: Synergistic (synthetic) interactions Disruption of the MSB2 gene in S. cerevisiae has no appreciable effects on the cell's ability to activate the High-Osmolarity Glycerol (HOG) pathway upon osmotic stress, or on cellular growth on high-osmolarity media. To identify potential osmosensors in the SHO1 branch of the HOG pathway, the authors screened for a mutant that is osmosensitive only in an msb2Δ background and recovered mutations in the HKR1 gene. Like MSB2, mutations in HRK1 alone confer no osmosensitivity to the cells.
MSB2 hyperosmotic response (GO:0006972) PMID:17627274 IGI HKR1 HKR1 hyperosmotic response (GO:0006972) PMID:17627274 IGI MSB2
Co-transfection experiments Co-transfection experiments include those experiments where two or more gene products are expressed in a heterologous system, such as a cell line, for the purposes of interrogating a functional interaction between them.
In C. elegans, the response to dauer pheromone, a mixture of small molecules, is mediated by G protein-coupled receptors (GPCRs). Genetic analysis has implicated two GPCRs, SRBC-64 and SRBC-66, in a signaling pathway that responds to specific components of dauer pheromone. To assess the biochemical role of SRBC-64 and SRBC-66, the gene products were expressed singly or in combination in HEK293 cells. Only when expressed in combination were the GPCRs able to enhance forskolin-stimulated cAMP production.
SRBC-64 G-protein coupled receptor signaling pathway (GO:0007186) PMID:19797623 IGI SRBC-66 SRBC-66 G-protein coupled receptor signaling pathway (GO:0007186) PMID:19797623 IGI SRBC-64
Functional complementation Functional complementation refers to experiments in which a gene from one species complements a mutation in another. For these annotations, the With/From column should list the identifier for the endogenous gene that is complemented by the heterologously expressed gene being annotated. In annotations from cross-species functional complementation experiments, the gene referred to in the With/From column will thus be from a different species than the gene being annotated.
Example C. elegans contains two genes, lgg-1 and lgg-2, with sequence similarity to the Saccharomyces cerevisiae ubiquitin-like protein Atg8 that is required for autophagosome biogenesis. Transformation of lgg-1, but not lgg-2, into atg8 deletion mutants in nitrogen starvation medium results in increased survival compared to atg8 mutants alone, indicating that lgg-1 can functionally complement budding yeast atg8.
lgg-1 macroautophagy (GO:0016236) PMID:20523114 IGI atg8
Rescue experiments One way in which functional interactions between two or more genes is assessed is through phenotypic rescue experiments. In these experiments, the expression of one gene is used to complement, or rescue, the mutant phenotype resulting from mutations in a second gene. Rescue experiments may be used to help determine the order in which gene products act within a biological pathway or process.
Example The planar cell polarity pathway is critical for a number of biological processes including epidermal wound repair. Activity of the GRHL3 transcription factor is essential for efficient wound repair in mice and human cell lines. Wound repair requires activation of the RhoA small GTPase to effect the cellular polarization, actin polymerization and epidermal migration critical to wound closure. The gene encoding the RhoGEF RhoGEF119, a RhoA GTPase activator, is a transcriptional target of GRHL3, and RHOGEF119 activity is also required for wound repair. Expression of human RhoGEF119 in human Grhl3-kd cell lines rescues the actin polymerization defects resulting from loss of Grhl13, indicating a role for RhoGEF119 in regulation of actin cytoskeletal organization during wound repair.
ARHGEF19 positive regulation of actin cytoskeleton organization (GO:0032956) PMID:20643356 IGI GRHL3 GRHL3 positive regulation of actin cytoskeleton organization (GO:0032956) PMID:20643356 IGI ARHGEF119
When NOT to use IGI Some experiments assess a functional interaction between one or more gene products by examining the effects that mutations in one gene have on the properties of another. These types of experiments are annotated using the IMP (Inferred from Mutant Phenotype) evidence code and the target, or affected gene product, may be captured as an Annotation Extension. The key here is that the genetic perturbation is directed at only one of the gene products in the experiment. For example, treatment of cells with GSK3B antagonists results in nuclear accumulation of the GATA6 transcription factor. This experiment indicates that GSK3B negatively regulates GATA6 localization.
GSK3B negative regulation of protein localization to nucleus (GO:1900181) PMID:23624080 transports_or_maintains_localization_of GATA6