1. An example of GO annotation from a primary paper Rebecca E. Foulger (UniProt Curator) GO Annotation Camp, June 2005 PMID:12972596 http://www.ebi.ac.uk/GOA

2. 2 proteins can be annotated from this paper: HBXAP_HUMAN UniProt ID:Q96T23 (Rsf-1) SMCA5_HUMAN UniProt ID:O60264 (hSNF2H) and Loyola et al. provide experimental evidence for biological process (BP), molecular function (MF) and cellular component (CC) annotations for both Rsf-1 and hSNF2H.

3. NB: Rsf-1 and hSNF2H were previously shown to form the RSF complex, and have therefore already been annotated to the GO term: RSF complex ; GO:0031213 (in PMID:9836642)

4. Starting on Page 6762, 2 nd paragraph in left column… Rsf-1 nucleus ; GO:0005634 | IDA hSNF2H nucleus ; GO:0005634 | IDA NB: There are two sets of evidence that the subunits of RSF localize to the nucleus: 1/ the complex was purified from a nuclear fraction. 2/ antibody staining. For any given paper, if both the GO term and the evidence code are identical the annotation is only recorded once. To assign cellular component annotations….

5. Figure 2B: Subcellular localization of Rsf-1 and hSNF2H, visualised with specific antibodies. Localization of hSNF2H is restricted to a chromosome during mitosis. Both Rsf-1 and hSNF2H localize to the nucleus. NB: Using GO, it is not yet possible to state that hSNF2H is nuclear during interphase and restricted to a chromosome during metaphase. Both CC GO terms are attributed to hSNF2H, and this temporal information is captured in UniProt in free text.

6. Pg 6762, half-way down left hand column… The GO term ‘mitotic chromosome ; GO:0005708’ is obsolete. When a GO term is made obsolete, the ‘comments’ often suggest alternative GO terms that may be appropriate to use.

7. hSNF2H: condensed chromosome ; GO:0000793 | IDA ‘Open mitosis’ occurs in humans, so GO:0000793 can be used to annotate hSNF2H.

8. Can add GO molecular function ‘protein binding’ annotations based on these assays…… Pg 6762, half way down left hand column… To assign Molecular Function ‘protein binding’ annotations….

9. Figure 2C: Immunoprecipitation experiments support ‘protein binding’ GO annotations.

10. NB: For protein binding GO terms, always do reciprocal annotations. UniProt ID for hSNF2H Rsf-1 protein binding ; GO:0005515 | IPI with O60264 hSNF2H protein binding ; GO:0005515 | IPI with Q96T23 UniProt ID for Rsf-1

11. hSNF2H: ATPase activity ; GO:0016887 | IDA Rsf-1: NOT ATPase activity ; GO:0016887 | IDA See next slide about use of the ‘NOT’ qualifier in GO Pg 6762, 2 nd paragraph in the right hand column … To assign a Molecular Function ‘ATPase activity’ annotation….

12. Use of the ‘NOT’ qualifier in GO annotation: The ‘NOT’ qualifier is used when a GO term might otherwise be expected to apply to a gene product, but an experiment etc. proves otherwise. Because the recombinant RSF complex shows ATPase activity, it is informative to record for this paper (using the ‘NOT’ qualifier) that the Rsf-1 subunit of RSF does not possess this activity. For further documentation on the ‘NOT’ qualifier, refer to the GO web site at: http://www.geneontology.org/GO.annotation.shtml

13. hSNF2H: ATP binding ; GO:0005524 | IC from GO:0016887 (ATPase activity) See next slide about use of the IC (inferred by curator) evidence code in GO

14. IC: Inferred by curator. To be used for those cases 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. For this paper, the authors show that hSNF2H has ATPase activity. It can therefore be reasonably inferred that hSNF2H can bind ATP. The IC evidence code is used to support the ‘ATP binding’ annotation to show that a curator has made this inference. For evidence code documentation, see: http://www.geneontology.org/GO.evidence.shtml

15. To assign a Biological Process GO annotation…. Page 6762, half way down right hand column…

16. hSNF2H: chromatin assembly ; GO:0031497 | IDA Rsf-1: chromatin assembly ; GO:0031497 | IDA

17. Rsf-1: histone binding ; GO:0042393 | NAS hSNF2H: contributes_to histone binding ; GO:0042393 | NAS See next slide about use of the ‘contributes_to’ qualifier in GO. Page 6762, 3 rd paragraph of right hand column… To assign a Molecular Function ‘histone binding’ annotation…. NB: When data is not shown in a paper but the results are still described, the NAS evidence code is generally used to support GO annotation (subject to judgement by the annotator). If time allows, we would also annotate Ref (13) to see if we can assign histone binding annotation to Rsf-1 with an experimental evidence code.

18. Use of the ‘contributes_to’ qualifier in GO: 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. All gene products annotated using 'contributes_to' must also be annotated to a cellular component term representing the complex that possesses the activity. hSNF2H already has the cellular component GO annotation RSF complex ; GO:0031213, and therefore a ‘contributes_to’ histone binding annotation can be made. For further documentation on the ‘contributes_to’ qualifier, refer to the GO web site at: http://www.geneontology.org/GO.annotation.shtml

19. hSNF2H: DNA binding ; GO:0003677 | IDA Page 6762, last paragraph of right hand column… Page 6764, bottom of the page, left hand column… To assign a Molecular Function ‘DNA binding’ annotation….

20. The DNA binding properties of Rsf-1 are slightly more complicated…… Page 6765, left hand column…

21. Rsf-1 negative regulation of DNA binding ; GO:0043392 | IDA At the time of curation a term for ‘negative regulation of DNA binding’ did not exist. Therefore a SourceForge item was made to request that this new term be added to GO.

22. Rsf-1 CC nucleus ; GO:0005634 | IDA MF protein binding ; GO:0005515 | IPI with O60264 histone binding ; GO:0042393 | NAS NOT ATPase activity ; GO:0016887 | IDA BP chromatin assembly ; GO:0031497 | IDA negative regulation of DNA binding ; GO:0043392 | IDA hSNF2H CC nucleus ; GO:0005634 | IDA condensed chromosome ; GO:0000793 | IDA MFprotein binding ; GO:0005515 | IPI with Q96T23 ATPase activity ; GO:0016887 | IDA ATP binding ; GO:0005524 | IC from GO:0016887 DNA binding ; GO:0003677 | IDA contributes_to histone binding ; GO:0042393 | NAS * BPchromatin assembly ; GO:0031497 | IDA Summary of GO annotation for PMID:12972596 * RSF complex ; GO:0031213 annotated from PMID:9836642