1. Introduction to GO Annotation Eurie Hong (SGD), Michelle Gwinn (TIGR), Tanya Berardini (TAIR), Karen Pilcher (DictyBase), Russell Collins (FlyBase), Carol Bastiani (Wormbase), Doug Howe (ZFIN), Stacia Engel (SGD)

2. What is a GO annotation? References Gene (protein coding gene, functional RNA) GO TermEvidence code IMP, IGI, IPI, ISS, IDA, IEP, TAS, NAS, ND, RCA, IC Qualifiers NOT contributes_to colocalizes_with With/From Supporting evidence for certain evidence codes

3. What is an annotation? Strategies for identifying literature to annotate Identifying the correct annotation Molecular Function Biological Process Cellular Component Extent of annotation for a single gene product Strategies for annotating a genome

4. Which type of literature is appropriate for annotation? Papers with experimental evidence for GO process, function or component annotation Mutant phenotype descriptions Enzymatic activity assays Localization studies Papers describing phylogenetic studies for GO function annotation (ISS) Reviews (Textbooks) (Meeting abstracts)

5. Strategies for reading a paper for annotation Abstract Results/Figures Materials and Methods Discussion

6. Which granularity of GO term is appropriate for annotation? Molecular Function Souza et al. (1998) YakA, a protein kinase required for the transition from growth to development in Dictyostelium. PMID: 9584128

7. Background YakA was identified as a developmental mutant YakA is an ortholog of the yeast Yak1p The protein kinase domain of YakA is similar to both serine/threonine kinases and tyrosine kinases PMID: 9584128

8. YakA belongs to the DYRK family YakA is a member of the DYRK family of protein kinases (dual-specificity tyrosine-regulated kinase)

9. The Experiment Assay for YakA protein kinase activity YakA + γ 32 P-ATP + MBP (substrate) Look for presence of 32 P in substrate in the presence of YakA PMID: 9584128

10. The Result PMID: 9584128

11. GO Term for Annotation protein kinase activity ; GO:0004672 MBP (myelin basic protein) is a generic substrate Kinase specificity not determined; no phospho- tyrosine antibodies used, for example Definition: Catalysis of the transfer of a phosphate group, usually from ATP, to a protein substrate.

12. Searching for Terms in DAG- Edit Search term name that contains: kinase 359 results protein kinase 60 results protein kinase activity 20 results

13. Search Output in DAG-Edit

14. Sibling Terms in DAG-Edit

15. Child Terms in DAG-Edit

16. Parent Terms in AmiGO

17. Evidence Code The evidence code for the protein kinase activity term is IDA (Inferred from Direct Assay) Although endogenous substrates were not tested, the authors clearly showed kinase activity with a direct assay

18. Granular Terms Using ISS protein serine/threonine kinase activity ; GO:0004674 protein tyrosine kinase activity ; GO:0004713 (Inferred from Sequence or structural Similarity)

19. How is Biological Process different form Molecular Function? Molecular Function… “Elemental activities, such as catalysis or binding, describing the actions of a gene product at the molecular level. A given gene product may exhibit one or more molecular functions.” Biological Process… “A phenomenon marked by changes that lead to a particular result, mediated by one or more gene products.” is about the protein. is about the organism. are the activities that a protein specifically and directly does. are the organism uses those activities for. Rho1 has GTPase activity…and the organism uses that activity for gastrulation, axon guidance, germ cell migration, etc … A hammer hammers nails…and builds houses. for example

20. Important points: Process is a migration of germ (pole) cells. It is the movement of cells from one side of the epithelium to the other. It is one step in a three step process.

28. Is a new term needed?

29. New term might be appropriate because it would describe a discrete, separable process, thus providing additional useful information to the user. Also, a new term(s) permit linking two similar processes that are currently separate in GO, but are connected in the literature. cell migration (is a) transepithelial cell migration (is a) pole cell transepithelial migration (is a) cellular extravasation cell migration (is a) germ cell migration (is a) pole cell migration (part of) pole cell transepithelial migration

30. Annotating to the Cellular Component Ontology Carol Bastiani, Caltech

32. Experiment: Immunolocalization of LIN-10 with a LIN-10 antibody.

33. Vulval epithelial cells can be distinguished from ventral cord neurons by their larger size and the presence of stained cell junctions (red) Localization of LIN-10 by Immunoflourescence:

34. Figure 7. LIN-10 is expressed in neurons. (A-C) Wild-type, late L3 hermaphrodite stained with anti-LIN-10 antibodies (green). LIN-10 is present in ventral cord processes (A, *), lateral neural cell bodies and processes (A and B, arrowheads), and dorsal cord processes

38. Search MGI GO Browser for neuron:

39. Choosing the evidence code:

40. In neural cell bodies, a small amount of LIN-10 appears diffusely throughout the cytoplasm, whereas the majority of LIN-10 is concentrated in discrete perinuclear structures (Figure 7, D and E), similar to perinuclear structures observed in vulval epithelial cells. To determine whether these perinuclear structures correspond to Golgi, we used ST-GFP as a marker for the trans-cisterna of the Golgi (Jamora et al., 1997). We expressed ST-GFP in transgenic worms using a heat shock promoter and examined the subcellular localization of LIN-10 and ST-GFP using anti-LIN-10 and anti-GFP antibodies. In single neurons expressing both endogenous LIN-10 and transgenic ST-GFP, the subcellular pattern of LIN-10 staining is similar to that of ST-GFP staining. Deconvolution of images obtained in double-staining experiments revealed that LIN-10 staining is closely associated with ST-GFP staining (Figure 7, F-I), but LIN-10 staining is consistently offset (by 0.2-0.5 µm) from ST-GFP staining. These results indicate that LIN-10 is localized in the trans-cisterna of the Golgi or is localized in a compartment closely associated with the trans-cisterna, such as the trans-Golgi network. Further subcellular localization of LIN-10:

41. LIN-10 is localized to: 1) Cytoplasm 2) Within or in association with a part of the Golgi apparatus/ in close association with the trans-cisterna or trans-Golgi network

42. 1) Annotate to cytoplasm:

43. LIN-10 is localized to: 1) Cytoplasm 2) Within or in association with a part of the Golgi apparatus/ in close association with the trans-cisterna or trans-Golgi network

44. 2)Annotate to Golgi apparatus, evidence code IDA:

45. Qualifier to use “when the resolution of the assay is not accurate enough to say that the gene product is a bona fida component member:”

46. Strategies for annotation of a genome 1. How to get a complete set of GO annotations 2. Updating GO annotations 3. Representative approaches

47. Strategies for annotation of a genome Complete a first pass –For all 3 aspects (MF, BP, CC) –For all genes that get GO annotations Proteins, RNAs, pseudogenes NOT centromeres, telomeres, LTRs, retrotransposons, ARSs –Unknowns are allowed How to get a complete set of GO annotations

48. Strategies for annotation of a genome Second pass –Replace unknowns –Update where IEA was used Info with “better” evidence code, if available –Update where other db’s are referenced Primary literature is preferred Updating the complete set of GO annotations

49. Strategies for annotation of a genome GO annotations will never be “done” Part of normal curation process –More specific information –Better evidence code Replace obsolete terms “Last reviewed” date Updating GO annotations - ongoing

50. Strategies for annotation of a genome Updating GO annotations - ongoing

51. Strategies for annotation of a genome Representative approaches