Annotating from phenotypes

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Introduction

Mutants can provide useful insights into a protein's function. GO annotations based on a phenotype should represent the normal function that can be inferred from the mutant. GO does not aim to capture individual phenotypes; use phenotype annotation resources for this purpose. The following guidelines should help determine how to annotate the function of a protein that can be inferred from its observed phenotypes.

What is the normal molecular function/biological process?

  • Remember that annotations are inferences from the evidence to a normal function/process.
  • You can only annotate a gene product as being 'involved in' a biological process if the MF can be placed within the set of MFs that make up that process.
    • To help determine whether this is the case, it is useful to create a GO-CAM model in Noctua or consult a pathway or process model from a recent paper or review.
  • If there is no MF known, any phenotype can only be annotated to ‘acts upstream of or within’ OR consider not making a GO annotation - it’s OK!
    • Q: Is it possible to have a gene product with a novel or unknown MF (or just a protein binding MF) that is otherwise well characterized genetically (and maybe somewhat biochemically?) such that it could be placed within a given pathway or process?
  • Example: nuclear pore: BRR6 is involved in nuclear envelope organization, when mutated, causes nucleocytoplasmic transport defects, but is NOT involved in nuclear transport
    • Cite paper(s) here?

Being ‘required for’ a process does not mean a protein is ‘involved in’ a process

It is common for authors to state that a gene or gene product is required for a given process. However, the true meaning of this statement can vary and it is thus the responsibility of the curator to determine whether the gene or gene product's activity is, indeed, an integral part of a process. To help make this decision, it may be useful to think about the gene's role in a concentric circle of processes, starting with its MF. For a given MF, what is the most proximal process in which the gene is involved? For a transcription factor, the most proximal process would be regulation of transcription. From there, what is the next most proximal process? In a development context, for example, this might be cell fate specification. Moving outward, what phenotypes might be indicative of defects in cell fate specification? If, for example, the cell was a particular class of neurons, then one of the defects may manifest as a change in a behavior. Is the transcription factor thus 'involved in' the behavior? No, but by annotating the TF to regulation of transcription (perhaps with relevant target genes) and specification of the particular cell types, it is possible capture the most relevant aspects of that gene's function. The defects in behavior could then be captured with phenotype annotations.

apoE example? https://www.uniprot.org/uniprot/P02649

Pleiotropic effects should not usually be captured

Pleiotropy and ‘required for’ a process does not mean a protein is ‘part of’ a process

For example: splicing factors are often required for cell cycle transition, but they are not part of the cell cycle transition A good clue is viability of mutants: inviable mutants often have pleiotropic phenotypes or they have a strong terminal phenotype that can easily be misinterpreted (cell cycle transition blocks/checkpoints, chromosome mis-segregation, etc) Beware of read-outs: DNA replication, apoptotic DNA fragmentation, etc)

Cell proliferation, cell migration and apoptosis

  • Mutants showing increased/decreased cell proliferation, cell migration and apoptosis need to be analyzed carefully. If we don’t know the underlying molecular/cellular mechanism, these annotations should not be made
  • Mutually exclusive terms: cell proliferation should not be used for proteins involved generally in growth or division.
  • I would also add to this list: lethality, low brood size, slow growth, perhaps also sluggish locomotion.

Mutant phenotypes and regulation terms

  • It can be difficult to assess whether a gene or gene product regulates a process based on mutant phenotypes alone. As annotation to regulation terms in the BP ontology require an understanding of the molecular basis for that regulation, mutant phenotypes may more often be used as supporting, rather than definitive, evidence for a gene's regulatory role. As with annotating to BP terms from phenotypes more generally, consider what is known about the MFs involved in the process and use that information to guide your annotation practice.
  • Mutants annotated to ‘regulation’ with no molecular function annotation (sometimes an annotation to a protein complex of with a known function) should be examined closely) should be reviewed.

Mutant phenotypes and other BP relations

When there is new knowledge, older IMP annotation should be reviewed and removed as required - link to section on removing annotations?