Annotation Conf. Call September 24th, 2013: Difference between revisions

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[[Category:Annotation Working Group]]
==Annotation extension exercises==
==Annotation extension exercises==


==== Paper 1: PMID 17620096 (Title: Human S100A15 splice variants are differentially expressed in inflammatory skin diseases and regulated through Th1 cytokines and calcium.)====
N.B. have replaced example from the one discussed on the call since it was an invalid annotation.
http://www.ncbi.nlm.nih.gov/pubmed/17620096


Annotation Extension to be discussed for the cytoplasm annotation for S100A14.
==== Paper 1: PMID:12479811 (Title: VASA localization requires the SPRY-domain and SOCS-box containing protein, GUSTAVUS.)====
http://www.ncbi.nlm.nih.gov/pubmed/12479811
 
Annotation Extension to be discussed for the cytoplasm annotation for VAS.


{| class="wikitable" border="1"
{| class="wikitable" border="1"
Line 13: Line 16:
!Extension (Col 16)
!Extension (Col 16)
|-
|-
|S100A14, Q9HCY8
|vas, P09052
|GO:0005737 (cytoplasm)
|GO:0005737 (cytoplasm)
|IDA
|IDA
|PMID:17620096
|PMID:12479811
|part_of: CL:0000362 (epidermal cell)
|part_of: CL:0000026 (invertebrate nurse cell)
|-
|-
|}
|}




'''Evidence is from Figure 3 and text on page 687:''' "hS100A15 is predominantly expressed in the epidermis of psoriatic and atopic skin
'''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)."
Because psoriasis and atopic eczema are characterized by morphological changes in both dermis and epidermis, we investigated the distribution of the hS100A15 transcripts in normal and diseased skin by in situ hybridization. Both S100A15 isoforms were detected mainly in the epidermis of normal and inflamed skin and showed a similar distribu- tion pattern. In line with RT-PCR analysis, the expression of S100A15-S was less pronounced than S100A15-L. Within the faint staining of healthy skin, hS100A15-L transcripts were present in the cytoplasm of the basal epidermal layer (Fig. 3, arrow), whereas the differentiated epidermal layers showed weak probe reactivity to hS100A15-L. "


==== Paper 2: PMID 9799565 (Title: Role of human CYP4F2 in hepatic catabolism of the proinflammatory agent leukotriene B4.)====
==== Paper 2: PMID 9799565 (Title: Role of human CYP4F2 in hepatic catabolism of the proinflammatory agent leukotriene B4.)====
http://www.ncbi.nlm.nih.gov/pubmed/9799565
http://www.ncbi.nlm.nih.gov/pubmed/9799565
[[File:1-s2.0-S0003986198908803-main(1).pdf]]


Annotation Extension to be discussed for the leukotriene-B4 20-monooxygenase activity annotation for CYP4F2.
Annotation Extension to be discussed for the leukotriene-B4 20-monooxygenase activity annotation for CYP4F2.
Line 48: Line 51:




====Paper 3: PMID 23437011 (Title: The Caenorhabditis elegans JNK signaling pathway activates expression of stress response genes by derepressing the Fos/HDAC repressor complex.)====
====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.)====
http://www.ncbi.nlm.nih.gov/pubmed/23437011
http://www.ncbi.nlm.nih.gov/pubmed/23437011


Annotation Extension to be discussed for
 
 
Annotation Extension to be discussed for KGB-1 phosphorylation of FOS-1
 
{| class="wikitable" border="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
 
{| class="wikitable" border="1"
!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
 
{| class="wikitable" border="1"
!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).
 
 
==Discussion==
 
* 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

Latest revision as of 16:51, 9 April 2014

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.)

http://www.ncbi.nlm.nih.gov/pubmed/12479811

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.)

http://www.ncbi.nlm.nih.gov/pubmed/9799565 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.)

http://www.ncbi.nlm.nih.gov/pubmed/23437011


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).


Discussion

  • 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