Annotation Conf. Call September 24th, 2013

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Annotation extension exercises

N.B. have replaced example from the one discussed on the call since it was an invalid annotation.

Paper 1: PMID:12479811 (Title: VASA localization requires the SPRY-domain and SOCS-box containing protein, GUSTAVUS.)

Annotation Extension to be discussed for the cytoplasm annotation for VAS.

DB (Col 2) GO ID (Col 5) ev.code Reference (Col 6) Extension (Col 16)
vas, P09052 GO:0005737 (cytoplasm) IDA PMID:12479811 part_of: CL:0000026 (invertebrate nurse cell)

Evidence is from Figure 2 and text on page 868: "To confirm that VAS, GUS, and EXU are at times associated within the same cytoplasmic particles, we examined the localization of these three proteins at an ultrastructural level. By simul- taneously staining the same sections for VAS, GUS, and EXU, we found them to colocalize in electron-dense structures within the nurse cell cytoplasm (Figure 2I)."

Paper 2: PMID 9799565 (Title: Role of human CYP4F2 in hepatic catabolism of the proinflammatory agent leukotriene B4.) File:1-s2.0-S0003986198908803-main(1).pdf

Annotation Extension to be discussed for the leukotriene-B4 20-monooxygenase activity annotation for CYP4F2.

DB (Col 2) GO ID (Col 5) ev.code Reference (Col 6) Extension (Col 16)
CYP4F2, P78329 GO:0050051 (leukotriene-B4 20-monooxygenase activity) IDA PMID:9799565 occurs_in: UBERON:0002107 (liver)

Evidence is from Table III

Paper 3: PMID 23437011 - This is a long paper, so only a few annotations will be discussed. (Title: The Caenorhabditis elegans JNK signaling pathway activates expression of stress response genes by derepressing the Fos/HDAC repressor complex.)

Annotation Extension to be discussed for KGB-1 phosphorylation of FOS-1

DB (Col 2) GO ID (Col 5) ev.code Reference (Col 6) Extension (Col 16)
WB:WP:CE29466 or UniProtKB:O44408 GO:0004674 (protein serine/threonine kinase activity) IDA PMID:23437011 has_direct_input: WB:WP:CE27375 or UniProtKB:G5EDK8 (FOS-1B in paper, the 331 amino acid isoform in WB and UniProtKB)

Relevant text (not inclusive, i.e., there are other statements that also support the annotation):

As FOS-1A has previously been characterized as a regulator of anchor-cell invasion during nematode development [17], we focused our investigations on the FOS-1B form (hereafter referred to as FOS-1).

Figure 1. FOS-1 is phosphorylated by KGB-1.

We further generated three FOS-1 mutants that individually changed Thr-304, Thr-316, or Thr-318 to Ala and found that the FOS-1(T304A) mutation exhibited decreased phosphorylation by KGB-1 (Figure 1D, line 3 and Figure S2). These results suggest that T304 is a major site of phosphorylation.

To confirm that KGB-1 phosphorylates FOS-1 at the Thr-304 residue, we generated anti-phospho-FOS-1 antibodies that specifically recognize FOS-1 phosphorylated at Thr-304. Transfection with active KGB-1, but not with the kinase-negative mutant KGB-1 KN, resulted in strong reactivity of FOS-1 with this antibody (Figure 1D, lanes 1, 2). In contrast, we found that the FOS-1 (T304A) mutated form could not be detected by this antibody (Figure 1D, lane 3), confirming that it was specific for FOS-1 phosphorylated at Thr-304.

Note: The phosphorylation experiments reportedly rely on an activated form of KGB-1 (activated by another protein kinase, MEK-1) but there doesn't seem to be a control in the paper that definitively shows this. Therefore, I haven't added any additional information about the role of MEK-1. We could discuss this on the call.

Annotation Extension to be discussed for KGB-1 regulation of FOS-1 oligomerization

DB (Col 2) GO ID (Col 5) ev.code Reference (Col 6) Extension (Col 16)
WB:WP:CE29466 or UniProtKB:O44408 GO:0032463 (negative regulation of protein homooligomerization) IDA PMID:23437011 has_regulation_target: WB:WP:CE27375 or UniProtKB:G5EDK8 (FOS-1B in paper, the 331 amino acid isoform in WB and UniProtKB)

Relevant text:

Indeed, GFP-FLAG-FOS-1 readily co-immunoprecipitated with T7-FOS-1 (Figure 1E, lanes 1, 2), indicating that the two proteins oligomerized, presumably as dimers.

Co-expression of active but not inactive KGB-1 resulted in reduced co-immunoprecipitation of T7-FOS-1 with GFP-FLAG-FOS-1 (Figure 1E, lanes 3, 4).

Annotation Extension to be discussed for FOS-1 regulation of kreg-1 transcription

DB (Col 2) GO ID (Col 5) ev.code Reference (Col 6) Extension (Col 16)
WB:WBGene00001345 or UniProtKB:G5EDK8, UniProtKB:G5ECG2 GO:0000122 (negative regulation of transcription from RNA polymerase II promoter) IMP PMID:23437011 has_regulation_target: WB:WBGene00018725 or UniProtKB:Q20689 (kreg-1)

Relevant text:

Treatment with fos-1 RNAi markedly increased intestinal Pkreg-1::venus expression even in the absence of Cu2+ (Figure 4).

The effect of fos-1 RNAi on expression of kreg-1 and kreg-2 was further confirmed by qRT-PCR (Figure S6).


  • Pipe vs comma in col-16: Pipe is like making a separate annotation. Pipe can be interpreted as meaning "or" and comma meaning "and"
  • with_product_of relationship: when you mention a gene name as a substrate for a enzyme, would you use with_product_of to indicate that the product is the substrate? Not necessary. We are not being specific for With column.
  • question about Mek1 phosphorylating KGB-1: Worm paper doesn't have evidence that Mek1 phosphorylates KGB-1, although they mention it. So can't capture that detail from this paper. But the yeast paper (PMID 8384702) discussed last month had evidence for two proteins (Fus3 and Far1) being kinased, all in the same paper. We discussed that situation again to refresh everybody
    • FUS3 phosphorylates FAR1. But to do this FUS3 has to be first phosphorylated by STE7.
    • So, the kinase annotation for Fus3 would have two items in col-16: has_direct_input:FAR1, requires_regulator: Ste7.
    • We will also make a kinase annotation for Ste7 with has_direct_input:Fus3.
    • in addition you could request an ID in Pro for the phosphorylated form of FUS3 and insert it into col-17 for the fus3 annotation.
  • regulation_target: Do we have to say has input gene, has output RNA? For now we will just put the gene_id in col-16 for has_regulaiton_target
  • User_label in the relationship ontology file. We can use that to make sure the users see meaningful stuff (e.g. has_direct_input vs has_substrate) We will talk about it at Barharbor. Pombase has a mapping between relationships and meaningful labels. Midori will look into adding these user labels to the relationship ontology file