1. Annotating Gene Products to the GO
Harold J Drabkin
Senior Scientific Curator
The Jackson Laboratory
Mouse Genome Informatics
Bar Harbor, ME

2. What is an annotation? GO Term
An annotation is a statement that a gene product …
…has a particular molecular function
…is involved in a particular biological process
…is located within a certain cellular component
…as determined by a particular method
…as described in a particular reference.
Evidence
Code
Reference
Smith et al. determined by a direct assay that Abc2 has protein kinase activity, is involved in the process of protein phosphorylation, and is located in the cytoplasm.

3. Anatomy of an annotation
Object (previously mentioned)
GO Term from most recent GO
GO Term Qualifier (optional)
NOT, Co_localizes with, or Contributes_to
Evidence Code : IDA, IPI, IMP, IEP, IGI, ISS, IEA, TAS, NAS, or IC
Evidence Code Qualifier (required for some codes)
Used in combination with IPI, IMP, IGI, and ISS
Seq_ID or DB_ID required.
Reference: literature or database specific reference
DB_ID or PMID

4. Getting the GO http://www.godatabase.org http://www.ebi.ac.uk/ego

5. GO Evidence Codes Code Definition IEA
Inferred from Electronic Annotation
NAS
Non-traceable Author Statement
TAS
Traceable Author Statement ND
No Data
Use with annotation to unknown
IDA
Inferred from Direct Assay
*IPI
Inferred from Physical Interaction
*IGI
Inferred from Genetic Interaction
IMP
Inferred from Mutant Phenotype
IEP
Inferred from Expression Pattern
*IC
Inferred from Curator
*ISS
Inferred from Sequence Similarity
Manually
annotated

6. Annotation Strategies
Electronic (IEA)
Good for first pass
Usually based on some sort of sequence comparisons (but use ISS if paper based)
IP2GO (InterPro to GO
SPTR2GO (SwissProt to GO)
Manual (literature)

7. Literature selection
A paper is selected for GO curation of a mouse gene product if:
A paper gives direct experimental evidence for the normal function, process, or cellular location of a mouse* gene product (IDA, IMP, IGG, IPI).
A paper gives direct experimental evidence for the normal function, process, or cellular location of a non-mouse gene product AND the paper presents homology data to a mouse gene product (ISS)
The two main keywords: DIRECT and NORMAL
Or organism of your choice!!!

8. Annotation process READ the full papers!
Abstracts alone can be very misleading
Quite often, the species are not specified. Sometimes a paper uses human, mouse and rat interchangeably , or uses human for one gene and mouse for a different one.
I highly caution against using abstracts as the sole source of GO.

9. Example Annotations

10. Abstract suggests that this paper demonstrates that Ibtk
Note also that the actually species used for any assays is NOT specifically mentioned!
Abstract suggests that this paper demonstrates that Ibtk
Binds to a protein kinase
Inhibits kinase activity
Inhibits calcium mobolization
Inhibits transcription

11. Evidence used for process and function
Use most specific term
possible
Both IDA
Same piece of data (IDA) demonstrates that this gene product inhibit protein kinase activity and thus is involved in the negative regulation of protein amino acid phosphorylation

12. Both Btk and iBtk have protein binding activity to each other, IPI evidence code

13. IDA evidence code
This paper demonstrates that expression of the protein down-regulates calcium mobilization. Using the term GO: is misleading, because it is not known if this protein DIRECTLY effects this process. It would be safer to use a term for negative regulation of …., which doesn’t exist. The curator should request that the term be created and re-visit the annotation.

14. Abstract totally misses the sub-cellular localization!!!

15. The Gene Association File
Sharing Annotations
The Gene Association File

16. Annotation Sharing Amigo Browser: http://www.godatabase.org
A GO browser that tracks contributed GO annotations across species.
Uses annotation sets supplied in a specific format.

17. The Gene Association files
15 column tab delimited text file

18. Anatomy of a gene association file
Column
Content
Example 1 DB
SGD, MGI 2
DB_Object ID
MGI: 3
DB_Object_Symbol
Gras3 4
GO_ID Qualifier
NOT, co_localizes_with, contributes_to 5
GO_ID
GO: 6
DB_Ref
PMID:234567 7
Evidence_Code
IDA, etc. 8
With/From 9
GO_aspect
P (process), C (component) F (function) 10
DB_Object_Name
Grasshopper 3 homlog 11
DB_Object_Synonym
Locust III, E12Rik 12
DB_Object_Type
Gene, transcript, or protein 13
Taxon
taxon:4932 14
Date 15
Assigned_by
DB (usually same as column 1)

19. Some Special Cases

20. Annotate to finest granularity
Annotating to GO: automatically annotates to all of its parents; thus a product is annotated to both protein modification AND cytoskeleton organization

21. GO Does not annotate substrates
A gene product that has protein kinase activity is also involved in the process of protein phosphorylation
The protein that gets phosphorylated is NOT involved in the process of protein phosphorylation.

22. Qualifiers GO Term Qualifiers Evidence Code Qualifiers “NOT”
Can be used with any term
“contributes_to”
Used for molecular function
“co_localizes with”
Used with cellular component
Evidence Code Qualifiers
Sequence ID (for ISS)
Protein ID (for IPI and protein binding)
Mutant ID (for IMP)
Gene (for IGI)
GO ID (for IC)

23. The “not” GO Term Qualifier
'NOT' is used to make an explicit note that the gene product is not associated with the GO term. This is particularly important in cases where associating a GO term with a gene product should be avoided (but might otherwise be made, especially by an automated method).
e.g. This protein does not have ‘kinase activity’ because the
Author states that this protein has a disrupted/missing an ‘ATP binding’ domain.
Also used to document conflicting claims in the literature.
NOT can be used with ALL three GO Ontologies.

24. The ‘contributes_to’ qualifier
Contributes_to: An individual gene product that is part of a complex can be annotated to terms that describe the action (function or process) of the complex.
This practice is colloquially known as annotating 'to the potential of the complex‘.
This qualifer allows us to distinguish the individual subunit from complex functions e.g. contributes_to ribosome binding when part of a complex but does not perform this function on its own.
All gene products annotated using 'contributes_to' must also be annotated to a cellular component term representing the complex that possesses the activity.
Only used with GO Function Ontology
contributes_to: An individual gene product that is part of a complex can be annotated to terms that describe the action (function or process) of the complex. This practice is colloquially known as annotating 'to the potential of the complex', and is a way to capture information about what a complex does in the absence of database objects and identifiers representing complexes. Annotating individual gene products according to attributes of a complex is especially useful for molecular function annotations in cases where a complex has an activity, but not all of the individual subunits do. (For example, there may be a known catalytic subunit and one or more additional subunits, or the activity may only be present when the complex is assembled.) Molecular function annotations of complex subunits that are not known to possess the activity of the complex must include the entry 'contributes_to' in the 'Qualifier' column (see below). All gene products annotated using 'contributes_to' must also be annotated to a cellular component term representing the complex that possesses the activity.
Note that 'contributes_to' is not needed to annotate a catalytic subunit. Furthermore, 'contributes_to' may be used for any non-catalytic subunit, whether the subunit is essential for the activity of the complex or not.
Examples:
Subunits of nuclear RNA polymerases: none of the individual subunits have RNA polymerase activity, yet all of these subunits are annotated to 'DNA-dependent RNA polymerase activity' (with the 'contributes_to' note), to capture the activity of the complex. Another example is
ATP citrate lyase (ACL) in Arabidopsis: it is a heterooctamer, composed of two types of subunits, ACLA and ACLB in a A(4)B(4) stoichiometry. Neither of the subunits expressed alone give ACL activity, but co-expression results in ACL activity. Both subunits can be annotated to ATP citrate lyase activity.
eIF2: has three subunits (alpha, beta, gamma); one binds GTP; one binds RNA; the whole complex binds the ribosome (all three subunits are required for ribosome binding). So one subunit is annotated to 'GTP binding' and one to 'RNA binding' without qualifiers, and all three are annotated to 'ribosome binding', with the contributes_to qualifier. And all three are annotated to the component term for eIF2 complex.
The Qualifier documentation:

25. GO: Protein Binding
Used to annotate a gene product as being able to bind another protein
If the target protein is known, then use the IPI evidence code and the UniProt identifier in the “with” field.
If the target is not known, then use the IDA evidence code.
The gene product being annotated does not have to be a protein itself: eg: Rpph1, ribonuclease P RNA component H1, has protein binding activity (GO: )

26. ISS:Inferred from sequence similarity
Author
states
Orthology
Used by MGI curators
A direct experiment must have been performed in the non-mouse organism
If the sequence comparison and the experiment are in one paper,then the reference is the paper
If the orthology is MGI curated, then the reference is J: The experimental paper reference goes in a note.
MGI
curates
Orthology

27. IMP:Inferred from mutant phenotype
Mostly used in inferring function from knock-out mice
Uses the WITH field.
Abnormal branching
of submandibular
gland

28. Inferred from Curator (IC)
Used where an annotation is not supported by any evidence, but can be reasonably inferred by a curator from other GO annotations, for which evidence is available.
The ‘with’ field is required, and is populated by a GO id using the same reference
Example: Ref. 1 shows that a gene product has chloride channel activity (GO: :) by direct assay (IDA). A curator can then add the component annotation ‘integral to membrane’ (GO: ) using the IC evidence code and put GO: in the “with” field.
Some databases are doing IC based on ISS annotation??????
Should GOA allow this?
Often we find it hard to find evidence to fill in the Component Ontology
but we may know that ‘DNA binding’ occurs in the ‘nucleus’ or that ‘cytokines’
are secreted and therefore act ‘extracellular’.
The IC code has been included in the GO evidence codes to allow us to
use our curator judgement to annotate to GO ontologies base on a
GO term we do have evidence for.
Other examples:
If known protein possesses kinase activity, then could annotate the term ATP binding with IC code.
If known to bind DNA, then could annotate ‘nucleus’
With IC code.
Caution: The IC evidence code should not be used for something obvious. For example, if a gene product is being annotated to the function “protein kinase activity” (GO: ) by IDA, then it is also involved in the process “protein amino acid phosphorylation” (GO: ) by the same experiment (IDA).

29. Unknown v.s. Unannotated
GO has three terms to be used when the curator has determined that there is no existing literature to support an annotation.
Biological_process unknown GO:
Molecular_function unknown GO:
Cellular_component unknown GO:
These are NOT the same as having no annotation at all.
No annotation means that no one has looked yet.