Annotation Conf. Call 2016-01-12

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Agenda

New Year's Welcome

  • Plans for upcoming year
    • Annotation Consistency Exercises
      • Will continue monthly exercises for 2016
      • This year, we will add discussion of LEGO representation to the calls, and compare conventional GO annotations to LEGO representations.
      • AI: Confirm rota schedule.

Ontology Updates

Deprecated Annotation Extensions

  • Rachael and Ruth have put together documentation on the deprecated extensions along with recommendations for revision.

Please document here examples of where you have questions about re-annotation of annotations with deprecated extensions.

dependent_on

Example 1

  • Co-transfection experiment that used dependent_on relation to capture requirement of both proteins to effect a GPCR signaling pathway
  • Recommendation: make direct annotations to each gene product to avoid 'reverse' annotations and use contributes_to qualifier
    • Two chemoreceptors mediate developmental effects of dauer pheromone in C. elegans.
    • To investigate whether expression of SRBC-64/66 was sufficient to confer pheromone responses in a heterologous context, we expressed one or both receptors in HEK293 cells and monitored intracellular second messenger levels upon the addition of ascarosides. Co-expression of SRBC-64 and -66, but not each receptor alone, enhanced forskolin-mediated increases in cAMP levels (Fig. 4A, B), and this increase was significantly inhibited upon addition of C6 and C9 at different concentrations (Fig. 4A-C).
    • Old annotation (one of four annotations made):
 Gene product: SRBC-64 (UniProtKB:G5ECV4)
 GO ID:	G-protein coupled receptor activity (GO:0004930) 
 GO evidence:  IDA
 Extension: dependent_on SRBC-66 (UniProtKB:O61938) 
  • Possible new annotation:
 Gene product: SRBC-64 (UniProtKB:G5ECV4)
 GO qualifier: contributes_to
 GO ID: G-protein coupled receptor activity (GO:0004930)
 GO evidence:  IDA

In this case, I will lose the information that the activity is only seen in the presence of SRBC-66.

  • Possible new annotation:
 Gene product: SRBC-64 (UniProtKB:G5ECV4) 
 GO qualifier: contributes_to
 GO ID: G-protein coupled receptor activity (GO:0004930)
 GO evidence:  IGI
 With: SRBC-66 (UniProtKB:O61938)

Should this instead be an IGI annotation?

Example 2

  • Co-transfection experiment demonstrating need for a tethering molecule to facilitate enzymatic activity
  • Recommendation: make direct annotations to avoid 'reverse' annotations and use contributes_to qualifier
    • LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans.
    • PUP-2 was previously shown to polyuridylate an artificially tethered RNA in Xenopus oocytes but was inactive without tethering. Therefore, we tested whether LIN-28 might be able to recruit PUP-2 to mediate pre–let-7 uridylation (Fig. 3c). We incubated anti-Flag immunoprecipitates from cell extracts expressing HA-Flag–PUP-2 and/or SBP–LIN-28 with radiolabeled pre–let-7 and radiolabeled UTP. HA-Flag–PUP-2 uridylated pre–let-7 only in the presence of SBP–LIN-28 (Fig. 3c). We also confirmed LIN-28–dependent uridylation of pre–let-7 by PUP-2 in vitro (Supplementary Fig. 6b).
  • Old annotation:
 Gene product: PUP-2 (UniProtKB:Q09408)
 GO ID: RNA uridylyltransferase activity (GO:0050265)
 GO evidence: IDA
 Extension: dependent_on LIN-28 (UniProtKB:P92186)
  • Possible new annotations:
 Gene product: PUP-2 (UniProtKB:Q09408)
 GO ID: RNA uridylyltransferase activity (GO:0050265)
 GO evidence: IDA
 Gene product: LIN-28 (UniProtKB:P92186)
 GO qualifier: contributes_to
 GO ID: RNA uridylyltransferase activity (GO:0050265)
 GO evidence: IDA
 Gene product: PUP-2 (UniProtKB:Q09408)
 GO ID: protein complex (GO:0043234)
 GO evidence: IDA
 Gene product: LIN-28 (UniProtKB:P92186)
 GO ID: protein complex (GO:0043234)
 GO evidence: IDA
  • OR
 Gene product: PUP-2 (UniProtKB:Q09408)
 GO ID: RNA uridylyltransferase activity (GO:0050265)
 GO evidence: IGI
 With: LIN-28 (UniProtKB:P92186) 
 Gene product: LIN-28 (UniProtKB:P92186)
 GO qualifier: contributes_to
 GO ID: RNA uridylyltransferase activity (GO:0050265)
 GO evidence: IGI
 With: PUP-2 (UniProtKB:Q09408)

Example 3

  • Enzymatic activity requires presence of another protein that acts as an upstream activator
  • Recommendation: make direct annotations to avoid 'reverse' annotations and use contributes_to qualifier
    • Since SGK-1, AKT-1, and AKT-2 all contain a highly similar kinase domain, we tested if they are able to phosphorylate their candidate substrate DAF-16 in vitro. For this purpose, we constructed transgenic wild-type and pdk-1(sa680) mutant strains expressing gfp-tagged akt-1, akt-2, or sgk-1. The fusion proteins were immunopurified from worm extracts and used for kinase assays with purified DAF-16. Both AKT-1, AKT-2, and SGK-1 purified from a wild-type background phosphorylated DAF-16 ( Figure 4A). In contrast, all three kinases purified from a pdk-1 mutant background were not able to phosphorylate DAF-16 (Figure 4A). This demonstrates that functional PDK-1 is required to activate AKT-1, AKT-2, and SGK-1 in vivo.
    • In pull-down experiments, we found that both in vitro-translated PDK-1 and DAF-16 interact with bacterially expressed, GST-tagged SGK-1, AKT-1, and AKT-2.
  • Old annotation (one example):
 Gene product: AKT-1 (UniProtKB:Q17941)
 GO ID: protein kinase activity (GO:0004672)
 GO evidence: IDA
 Extension: has_direct_input DAF-16 (UniProtKB:O16850),dependent_on PDK-1 (UniProtKB:Q9Y1J3)
  • New annotations:
 Gene product: AKT-1 (UniProtKB:Q17941)
 GO ID: protein kinase activity (GO:0004672)
 GO evidence: IDA
 Extension: has_direct_input DAF-16 (UniProtKB:O16850)
 Gene product: PDK-1 (UniProtKB:Q9Y1J3)
 GO ID: protein kinase activator activity (GO:0030295) 
 GO evidence: IMP
 Extension: has_direct_input AKT-1 (UniProtKB:Q17941)

Example 4

  • Protein interaction that only occurs in the presence of a specific DNA sequence
  • Recommendation: use occurs_at
    • The Caenorhabditis elegans JNK signaling pathway activates expression of stress response genes by derepressing the Fos/HDAC repressor complex.
    • Next we asked whether FOS-1 could interact with HDA-1. T7-FOS-1 and FLAG-HDA-1 were co-expressed in HEK293 cells. We immunoprecipitated FLAG-HDA-1 with anti-FLAG antibodies, and probed for the T7-FOS-1 on a Western blot with anti-T7 antibodies. We failed to detect an association between FOS1- and HDA-1 (Figure 7C, lane 1). However, if we transfected in the Pkreg-1::venus reporter along with T7-FOS-1 and FLAG-HDA-1, we could detect an association between FOS-1 and HDA-1 (Figure 7C, lane 3). Furthermore, removal of the TRE2 site from the Pkreg-1::venus reporter reduced this interaction (Figure 7C, lane 4). These results suggest that HDA-1 and FOS-1 can associate on the kreg-1 promoter.
  • Old annotation (one of two reciprocal annotations made):
 Gene product: FOS-1 (UniProtKB:G5ECG2)
 GO ID:	histone deacetylase binding (GO:0042826)
 GO evidence:  IPI
 With/from:	HDA-1 (UniProtKB:O17695)
 Extension:	dependent_on RNApol_II_promoter (SO:0000170),dependent_on kreg-1 gene (WB:WBGene00018725)
  • New annotation:
 Gene product: FOS-1 (UniProtKB:G5ECG2)
 GO ID:	histone deacetylase binding (GO:0042826)
 GO evidence:  IPI
 With/from:	HDA-1 (UniProtKB:O17695)
 Extension:	occurs_at RNApol_II_promoter (SO:0000170)
 Gene IDs are not currently valid for occurs_at relation, so cannot add the specific gene.  Only GO and SO IDs are currently valid.

Minutes