1. microRNA GO annotation

2. Why annotate miRNAs?
Direct curation of the roles of miRNAs is expected to improve miRNA functional analysis that has so far relied on indirect analysis of the gene targets’ functions
“We show that the most commonly used functional enrichment test is inappropriate for the analysis of sets of genes targeted by miRNAs.” Bleazard et al. 2015

3. miRNAs can affect gene regulation in many ways
Scope of annotation guidelines
miRNAs can affect gene regulation in many ways
The focus of our guidelines is on gene silencing by miRNA via the 3’UTR of mRNAs, including;
annotation of the protein components of the canonical mammalian miRNA processing pathway
annotation of proteins that affect the levels of miRNAs
annotation of the miRNA’s role in gene silencing
annotation of the miRNA targets
contextual annotation of miRNA regulation

4. Guideline examples
annotation of the protein components of the canonical mammalian miRNA processing pathway
annotation of proteins that affect the levels of miRNAs
annotation of the miRNA’s role in gene silencing
annotation of the miRNA targets
contextual annotation of miRNA regulation

5. Annotation of proteins involved in miR processing
Animals
All human proteins
involved in miR
processing have
been annotated
with these roles
GO terms
to apply
Over 250 annotations

6. miR processing proteins
Plants
miPEPs – newly discovered
peptide regulators of miRNA
transcription encoded by
miRNA genes

7. Guideline examples
annotation of the protein components of the canonical mammalian miRNA processing pathway
annotation of proteins that affect the levels of miRNAs
annotation of the miRNA’s role in gene silencing
annotation of the miRNA targets
contextual annotation of miRNA regulation

8. New biology = ontology developments
New GO terms added for miRNA processing
deadenylation involved in gene silencing by miRNA GO:
mRNA cleavage involved in gene silencing GO:
endoribonuclease activity, cleaving miRNA-paired mRNA GO:
pre-mRNA transporter activity GO:
regulation of production of miRNAs involved in gene silencing by miRNA GO:
During the process of annotating the proteins involved in miRNA processing, I identified some GO terms that were missing from the ontology. These have now been added and there are probably more additions and rearrangements that need to be done as we go along.
- negative regulation of production of miRNAs involved in gene silencing by miRNA GO:
- positive regulation of production of miRNAs involved in gene silencing by miRNA GO:

9. miRNA role in gene silencing
Translational repression
mRNA deadenylation
mRNA cleavage
miRNA can act via several different mechanisms. In our guidelines we are only covering the gene silencing aspect, i.e. translational repression, mRNA deadenylation, mRNA cleavage

10. All silencing roles of miRNAs now come under gene silencing by miRNA
Ontology updates – May 2015
Gene silencing
mRNA
deadenylation
mRNA
cleavage
Translation
regulation
GO: positive regulation of nuclear-transcribed mRNA poly(A) tail shortening
All silencing roles of miRNAs now come under gene silencing by miRNA

11. GO terms for miRNA roles
Translational repression
GO:
gene silencing
by miRNA
GO:
negative regulation
of translation
involved in gene
silencing by miRNA
GO:
mRNA binding
involved in
posttranscriptional
gene silencing
GO:
mRNA cleavage
involved in gene
silencing by
miRNA
GO:
deadenylation
involved in
gene silencing
by miRNA
mRNA deadenylation
mRNA cleavage

12. Increasing levels of miRNAs e.g. pri-miRNA, pre-miRNA, miRNA mimic
Evidence codes
Increasing levels of miRNAs
e.g. pri-miRNA, pre-miRNA, miRNA mimic
Evidence code = Inferred from Direct Assay (IDA)
2. Decreasing levels of miRNAs
e.g. antagomir, antisense-miRNA, blockmir, miRNA eraser or sponge, miRNA knockout
Evidence code = Inferred from Mutant Phenotype (IMP)
Different types of experiment specify which evidence code is used for an annotation

13. Guideline examples
annotation of the protein components of the canonical mammalian miRNA processing pathway
annotation of proteins that affect the levels of miRNAs
annotation of the miRNA’s role in gene silencing
annotation of the miRNA targets
contextual annotation of miRNA regulation

14. miRNA targets Predicted Sequence alignment of miRNA with mRNA
Validated binding
miRNA shown to act directly on a
mRNA by e.g. reporter assay or
Co-IP plus change in mRNA levels
Definitions created for annotation guidelines. These designations are used to help a curator decide what GO terms and annotation extensions can be used
Validated other
no evidence of direct action, but
levels of mRNA are affected in
response to miRNA, e.g. Western, qPCR

15. Guidelines for miRNA target annotation
Excerpt of the guidance table for miRNA target annotation
Annotation extension is capturing the contextual information, i.e. target, cell type

16. Guideline examples
annotation of the protein components of the canonical mammalian miRNA processing pathway
annotation of proteins that affect the levels of miRNAs
annotation of the miRNA’s role in gene silencing
annotation of the miRNA targets
contextual annotation of miRNA regulation

17. Important for pathway analysis
Capturing cell types
Important for pathway analysis
miR-195 and miR-29b regulate extracellular matrix expression in smooth muscle cells (SMCs).
Elastin expression in SMCs following overexpression of miR-195 or miR-29b as assessed by qPCR. (PMID: )

18. Important for functional analysis of miRNAs
Capturing physiological effects of silencing
Important for functional analysis of miRNAs
microRNA-133 (miR-133) modulates cAMP signaling in vitro. A, Effect of miR-133 on adenylyl cyclase (AC) activity in cardiomyocytes. Bars indicate SEM. (PMID: )

19. Summary
When annotating a miRNA we should minimally aim to capture the following:
The miRNA’s main role in gene silencing and its target(s);
gene silencing by miRNA
negative regulation of gene expression
mRNA binding involved in post-transcriptional gene silencing by miRNA target mRNA indicated in the annotation extension
2. The effect of silencing the target mRNA;
e.g. if an adenylate cyclase transcript was silenced:
negative regulation of adenylate cyclase activity
negative regulation of cAMP biosynthetic process
3. The relevance of that process it regulates;
e.g. negative regulation of adenylate cyclase activity involved in heart contraction

20. Practical use of Guidelines
The BHF-UCL cardiovascular miRNA annotation project
The aims of the microRNA GO annotation project are to:
✔ draw up guidelines for consistent literature-based annotation of miRNAs in consultation with the GO Consortium and miRNA experts
functionally annotate miRNAs that are involved with cardiovascular development and related processes
capture the targets of the cardiovascular-related miRNAs 77
174
121
372 annotations

21. Thank You miRNA experts Lars Maegdefessel Manuel Mayr Anna Zampetaki
Anastasia Kalea
Curators
Dmitry Sitnikov
Leonore Reiser
Reactome curators

22. From: MicroRNA gets down to business
George S Mack
Nature Biotechnology 25, (2007)