Annotation Conf. Call 2016-01-12

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Agenda

Deprecated Annotation Extensions

  • Beginning with this call, we will work our way through revising annotations using 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 each gene product 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

  • 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