A Bioinformatic Study - the Department of Computer Science

The Mode of Mutual Regulation by Host and Viral miRNAs
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 1 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 2 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bioinformatic Limitation
Target Prediction is noisy:
an excess of predicted targets for
each miRNA (some are False
Positives).
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 2 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bioinformatic Limitation
Target Prediction is noisy:
an excess of predicted targets for
each miRNA (some are False
Positives).
Bioinformatic Question
Can we combine target prediction with new regulation scenarios and
additional information sources, to narrow the target genes list to more
reliable candidates?
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 2 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Biological Background
Multiplicity: One miRNA can target more than one gene.
Cooperativity: One gene can be targeted by more than one miRNA.
A wider view of the regulation process, in which we consider miRNAs
as part of a larger regulatory network, rather than the narrow view of
single miRNA-single gene, may yield more robust results.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 2 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Our Hypothesis
Viral miRNAs might take advantage of the regulation that already exists in
the cell, to increase the translational repression to the virus benefit.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 2 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Our Hypothesis
Viral miRNAs might take advantage of the regulation that already exists in
the cell, to increase the translational repression to the virus benefit.
New Bioinformatic Goal
Explore the combined effect of host and viral miRNAs on host pathways
(Cooperating mode of regulation).
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 2 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Our Hypothesis
Viral miRNAs might take advantage of the regulation that already exists in
the cell, to increase the translational repression to the virus benefit.
New Bioinformatic Goal
Explore the combined effect of host and viral miRNAs on host pathways
(Cooperating mode of regulation).
Combine target prediction results with
Host and viral miRNA expression in cells infected with virus,
Biological pathway annotations
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 2 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
The Bi-Targeting Algorithm
Goal: To find groups of genes that are mutually regulated by the same
host and viral miRNAs - modules.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 3 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Representing the miRNA-mRNA Relations
Using target prediction and/or experimental data.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 4 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Constraints
No. of host miRNAs ≥ qh
No. of viral miRNAs ≥ qv
No. of targeted mRNAs ≥ qg
Enrichment p-value < p
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 5 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Constraints
No. of host miRNAs ≥ qh
No. of viral miRNAs ≥ qv
No. of targeted mRNAs ≥ qg
Enrichment p-value < p
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 5 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
The task of finding miRNA-mRNA modules is formulated as bi-clustering
problem.
(Yoon and Micheli, 2005; Peng et al, 2009)
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 6 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
The task of finding miRNA-mRNA modules is formulated as bi-clustering
problem.
(Yoon and Micheli, 2005; Peng et al, 2009)
⋄ The miRNA set is composed of human and viral miRNAs.
⋄ A new type of clustering is needed.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 6 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 7 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Using the modules
The genes in the modules may serve as candidates for experimental
target validation.
The modules may help understanding the role of miRNAs in host-viral
interactions.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 7 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Bi-Targeting Algorithm
The general idea:
Enumerate groups of genes that are mutually targeted by both host and
viral miRNAs. The genes are taken from a pathway/process.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 8 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Results
Cooperation between Epstein-Barr virus (EBV) and human miRNAs in
EBV infection.
Epstein Barr Virus (EBV)
A herpes virus, common in humans, that
preferentially infects B lymphocytes and epithelial
cells.
Associated with human malignancies: Burkitt’s
lymphoma, Hodgkin’s lymphoma, Nasopharyngeal
carcinoma.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 9 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Data Sources and Datasets:
miRBase and Ensembl Biomart: genomic sequences and tissue
context information.
◮ 866 human mature miRNAs.
◮ 39 viral mature miRNAs.
◮ 7208 3’UTR sequences (from 4773 genes).
miRNA Atlas (Landgraf et al, 2007):
◮ 8 tissue samples expressing both human and EBV miRNAs.
Gene Ontology (GO) annotations: participation of genes in
biological processes.
◮ 72 categories (with 10-300 genes) related to apoptosis, immune
response, signal transduction, proliferation, and cell cycle.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 10 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
The Target Prediction Algorithm was applied on the miRNAs and
mRNAs in the dataset, resulting in:
◮ 283362 human miRNA/human mRNAs pairs,
◮ 11663 EBV miRNA/human mRNAs pairs.
The Bi-Targeting Algorithm was applied on 576 pairs of (Atlas
sample,GO category), resulting in 34 modules.
Enrichment of each module was determined by hyper-geometric score
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 11 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
The Selected Module
GO Category: Induction of Apoptosis
(111 genes)
Atlas sample: hsa-Burkitt-DG-75
(18 human and 16 viral miRNAs)
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 12 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
The Selected Module
GO Category: Induction of Apoptosis
(111 genes)
Atlas sample: hsa-Burkitt-DG-75
(18 human and 16 viral miRNAs)
(Trigiante G and Lu X, 2006.)
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 12 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
The Selected Module
GO Category: Induction of Apoptosis
(111 genes)
Atlas sample: hsa-Burkitt-DG-75
(18 human and 16 viral miRNAs)
(Trigiante G and Lu X, 2006.)
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 12 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
The Selected Module
GO Category: Induction of Apoptosis
(111 genes)
Atlas sample: hsa-Burkitt-DG-75
(18 human and 16 viral miRNAs)
(Trigiante G and Lu X, 2006.)
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 12 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Conclusions
Our modules suggest a possible cooperation between human and
EBV miRNAs in the task of preventing apoptosis, promoting cell
growth, and evading the immune response.
This cooperation can lead to an increased degree of translational
repression.
The combination of several information sources in the task of module
detection minimizes the effect of noise and errors accompanying each
information source, yielding more reliable results.
Veksler-Lublinsky I, et al. Gene bi-targeting by viral and human miRNAs.
BMC Bioinformatics 11:249, 2010.
The full list of modules at: http://www.cs.bgu.ac.il/∼vaksler/BiTargeting.htm.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 13 / 43

The Mode of Mutual Regulation by Host and Viral miRNAs
Biological Evidence to Our Hypothesis
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 14 / 43

miRNA Compensating Regulation
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 15 / 43

miRNA Compensating Regulation
The Next Goal
Explore the compensating mode of regulation by host and viral miRNAs.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 16 / 43

miRNA Compensating Regulation
The Next Goal
Explore the compensating mode of regulation by host and viral miRNAs.
Biological Observations
Viral infection results in changes in the expression of some host miRNAs,
because of the:
changes induced by the virus, that may benefit the virus:
and/or
host response to the infection:
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 16 / 43

miRNA Compensating Regulation
The Next Goal
Explore the compensating mode of regulation by host and viral miRNAs.
Biological Observations
Viral infection results in changes in the expression of some host miRNAs,
because of the:
changes induced by the virus, that may benefit the virus:
and/or
host response to the infection:
Some viruses encode miRNAs that mimic host miRNAs.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 16 / 43

miRNA Compensating Regulation
The Next Goal
Explore the compensating mode of regulation by host and viral miRNAs.
Biological Observations
Viral infection results in changes in the expression of some host miRNAs,
because of the:
changes induced by the virus, that may benefit the virus:
and/or
host response to the infection:
Some viruses encode miRNAs that mimic host miRNAs.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 16 / 43

miRNA Compensating Regulation
The study of the compensating mode of regulation:
Combine target prediction results with
miRNA expression in infected vs. uninfected cells
Biological pathway annotations
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 16 / 43

miRNA Compensating Regulation
Extending the Bi-Targeting Algorithm
A new version of the algorithm which computes quasi-modules (missing
edges in the module are allowed).
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 17 / 43

miRNA Compensating Regulation
Extending the Bi-Targeting Algorithm
A new version of the algorithm which computes quasi-modules (missing
edges in the module are allowed).
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 17 / 43

miRNA Compensating Regulation
Extending the Bi-Targeting Algorithm
A new version of the algorithm which computes quasi-modules (missing
edges in the module are allowed).
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 17 / 43

miRNA Compensating Regulation
Extending the Bi-Targeting Algorithm
A new version of the algorithm which computes quasi-modules (missing
edges in the module are allowed).
Motivation for quasi modules
Most-to-most interactions
Edges could be missed by target
prediction
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 17 / 43

miRNA Compensating Regulation
Extending the Bi-Targeting Algorithm
A new version of the algorithm which computes quasi-modules (missing
edges in the module are allowed).
Motivation for quasi modules
Most-to-most interactions
Edges could be missed by target
prediction
Advantage: Bigger and more significant modules.
Disadvantage: Additional parameters are introduced into the search.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 17 / 43

Results
miRNA Compensating Regulation
Modules of viral and human miRNAs and their target human genes, where
the viral miRNAs compensate for the function of human miRNAs, in
Human Cytomegalovirus (HCMV) infection .
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 18 / 43

miRNA Compensating Regulation
Data Sources and Datasets:
miRBase, Deep-Sequencing data and Ensembl Biomart:
◮ 866 human mature miRNAs and 22 viral mature miRNAs.
◮ 10925 3’UTR sequences (from 5351 genes).
Expression data in un-infected vs. infected tissue: 28 human
miRNAs that were down-regulated upon infection (> two fold).
KEGG pathway annotations: participation of genes in biological
pathways, e.g., immune system, cell growth and death, and signal
transduction.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 19 / 43

miRNA Compensating Regulation
The Selected Module
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 20 / 43

miRNA Compensating Regulation
The Selected Module - Supporting Evidence
Natural killer (NK) cells:
Play an important role in
eliminating virus-infected cells in
early stages of the infection.
CMV encodes numerous proteins
that interfere with NK cell activity.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 21 / 43

miRNA Compensating Regulation
The Selected Module - Supporting Evidence
PIK3R3 and BRAF expressions in
HCMV infection (Challacombe et al,
2004):
increase 1 hour post infection; decrease
24 hours post infection
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 21 / 43

miRNA Compensating Regulation
The Selected Module - Supporting Evidence
ITGAL and PAK1
Were shown to have antiviral activity
(good candidates to be down regulated
by HCMV):
ITGAL is involved in NK-cell
mediated DNA-fragmentation of
CMV-infected cells (Ito et al, 1996).
PAK1 activates antiviral signaling
pathways in Hepatitis C virus(Ishida
et al, 2007).
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 21 / 43

miRNA Compensating Regulation
The Selected Module - Supporting Evidence
hsa-miR-155
KSHV and MDV-1 encode miR-155
orthologs
EBV → ↑ miR-155
miR-155/viral orthologs assist to
the replication of the virus
HCMV miRNAs compensate for the
functions of hsa-miR-155
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 21 / 43

Conclusions
miRNA Compensating Regulation
Some of our modules contain genes that have an antiviral activity.
The host is motivated to up regulate them, and the virus needs the
opposite effect.
Modules that link the viral miRNAs and the human miRNAs under the
compensating mode, may help in understating:
the role of viral miRNAs in the viral combat to survive , and
the role of host miRNAs in antiviral responses.
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 22 / 43

Acknowledgements
My Advisors
Prof. Klara Kedem
miRNA Compensating Regulation
Prof. Michal Ziv-Ukelson
Our Collaborators
Virology Department, BGU:
Dr. Yonat Shemer
Prof. Zvi Bentwich
Mr. Mesfin Meshesha
Faculty of Medicine, TAU:
Dr. Noam Shomron
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 23 / 43

miRNAs in Viral-Host Interactions
viral miRNAs ⇒ viral genes
◮ maintain replication and latency (evading the host-immune system).
◮ ebv-miR-BART2 → ebv-BALF5
viral miRNAs ⇒ host genes
◮ host immune response, apoptosis, interferon system, signal
transduction, cellular proliferation.
◮ hcmv-miR-UL112 → human-MICB
host miRNAs ⇒ viral genes
◮ inhibiting viral replication
◮ hsa-miR-32 → PFV genome
host miRNAs ⇒ host genes
◮ host miRNAs may be exploited by the virus
◮ HIV → ↓hsa-miR-17/20a → ↑ PCAF → ↑ HIV transcription
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 24 / 43

Statistical Significance
Enrichment of the genes in the
explored category (hyper-geometric
p-value):
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 25 / 43

Statistical Significance
Enrichment of the genes in the
explored category (hyper-geometric
p-value):
Isana Veksler-Lublinsky (BGU) Seminar May, 2012 25 / 43