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Introduction to microRNA
miRNA Workshop
Ravi K. Gutti
Division of Newborn Medicine
Children´s Hospital Boston and Harvard Medical School
Ravi. [email protected]
May 18, 2010
http://catalyst.harvard.edu
Agenda
– Introduction and History
– Workflow in miRNA experiments
– Finding miRNA targets by target search
databases
– miRNA validation and experimental
considerations
– Applications
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Why study miRNA?
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Non-Coding RNA: Formerly known as
“JUNK”
Non-Coding RNA: A Key to
Eukaryotic Complexity?
What is miRNA?
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Characteristics of miRNAs
•
•
•
•
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Small non-coding double stranded RNAs
Approximately 19-22 nt long
Repress activity of complementary mRNAs
Regulate 30% of mammalian gene products
1 miRNA = hundreds of mRNAs
“you can change a phenotype by modulating a single
miRNA” Thomas Wurdinger, HMS
• Have been described in invertebrates and vertebrates:
worms, fungi, plants, and mammals
• Many are conserved between vertebrates and
invertebrates
History
History
• lin-4, first miRNA to be described in C. elegans; important in
development of the worm from larva to adult.
• let-7, was also described in C. elegans (Reinhard BJ et al, 2000) as
critical to stop the stem-cell-like divisions of seam cells and induce
their fully differentiated state. Reduced let-7 expression is
associated with human cancers and cancer stem cells, thus
suggesting that let-7 in humans also promotes terminal
differentiation and is a tumor suppressor.
• 1998-Fire and Mello, experiments in C. elegans, first to show that
dsRNA is much more potent at inhibiting gene expression than
antisense RNA. Set the stage for understanding the role of miRNAs
in development and gene regulation. (Nobel Prize in Physiology and
Medicine, 2007).
Genomic Organization
Biogenesis
-Primary-miRNA is transcribed in the
nucleus, and is usually several
kilobases long; posses 5’ cap and a
poly-A tail.
-Cleaved in the nucleus by Drocha
enzyme to 70nt hairpin transcript (premiRNA).
-Transported to the cytoplasm by
Exportin 5 through nuclear pores.
-Cleaved by Dicer enzyme (RNase III
enzyme) into 19-22nt ds-transcripts.
-700 Human genes, 490 mouse genes.
1400 possible human sequences but
733 cloned or detected. Only ~120
tested.
Esquela-Kerscher, A., and Slack, F.J. (2006). Nature Reviews 6, p. 263
siRNA
miRNA
Why analyze miRNA
patterns?
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Why Analyze miRNA Expression
Patterns?
•Complete role played by each sequence not yet determined
• Functional analysis tools not yet widely available
Knockdown, Over-Expression, Reporter Assays
• Correlate miRNA expression profiles with:
Biological Phenotypes, Protein & Gene Expression Levels
• Discover miRNA sequences regulating genes involved in the
biological process of interest
Relevance of miRNA to Human Biology
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Cancer. miRNAs have been found to be downregulated in a number of tumors , and in some cases the reintroduction of these
miRNAs has been shown to impair the viability of cancer cells. The value of miRNA profiles in tumor diagnostics is well established.
For instance, strong up and down regulations of 16 miRNAs have been shown in primary breast tumors, and these markers may aid
in the development of drug-resistance and treatment-selection tests. Underlining the important role miRNA plays in oncology is the
formation of several new companies which seek to expand development of miRNA-based therapeutics.
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Age-Related Diseases. Evidence is accumulating that many age-related diseases are associated with a decreased control of cell
signaling that occurs in mid-life. The miRNA control of such systems as the cell cycle, DNA repair, oxidative stress responses and
apoptosis, has been shown to become abnormally expressed in mid-life. It is highly likely that continued research will reveal
important associations with the aging process, and may lead to therapeutics that can improve the quality of life.
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Heart Disease. Two heart-specific miRNAs were deleted in mouse models resulting in abnormal heart development in a large
proportion of the offspring. While these lethal effects were expected, other studies show a more subtle role for miRNA in the heart.
When miR-208 was eliminated, the mice appeared normal. Defects were revealed only when their hearts were stressed. These
results show that comprehensive miRNA studies may be valuable in the diagnosis of heart disease.
•
Neurological Diseases. Numerous reports have demonstrated the role of miRNAs in neural development. Evidence for a role in
Parkinsons disease comes from animal model studies published last year, showing that loss of miRNAs may be involved in the
development and progession of the disease. In cell culture experiments, transfer of small RNA fragments partially preserved miRNA
deficient nerve cells. While these results and others point to an important role for miRNA in neurodegenerative disorders, much more
work is needed to delineate the exact role of miRNAs in this important area.
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Immune Function Disorders. Recent miRNA deletion studies have revealed a central role in the regulation of the immune response.
The deletion of miRNA-155 impaired T and B cell differentiation in germinal centers, and greatly decreased antibody and cytokine
production. Two additional studies deleting miRNA-181 and 223 were found to control T cell response and granulocyte production,
respectively. As more roles for miRNAs in the immune response are found, the list of immune function disorders with a miRNA
component is certain to expand also.
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miRNA Profiling Applications
Work flow in miRNA
Experiments
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RNA Quality Assessment
Problems
• How do we find microRNA genes?
• Given a microRNA gene, how do we find its
targets?
How do we find microRNA genes?
• Biological approach
– Small-RNA-cloning to identify
new small RNAs
• Most microRNA genes are tissuespecific
miR-124a is restricted to the brain and spinal cord in
fish and mouse or to the ventral nerve cord in fly
miR-1 is restricted to the muscles and the heart in
mouse
miRNA Targets
•In plants, the identification of mRNA targets is straight forward
because most miRNAs and their target mRNAs have exact or
nearly exact complementarity.
•In animals, the tendency of miRNAs to bind their mRNA
targets with imperfect sequence homology poses considerable
challenges with target prediction.
• Several computational approaches have been developed to
facilitate experimental design and predicting miRNA targets.
•Computational target prediction identifies potential binding
sites according to base-pairing rules and across species
conservation conditions.
MicroRNA Targets Prediction and
Analysis
Target Identification
•The duplex for miRNA hsa-miR-579 and its target LRIG3 is partitioned into
two parts, the seed part and the out-seed part
•Six to eight nucleotides at the 5’ end of the mature miRNA sequence are very
important in the selection of target site
• Most of the computational tools developed to identify mRNA target
sequences depend heavily on complementarity between miRNA seed
sequence and the target sequence
•Most methods mainly use sequence complementarities, thermodynamic
stability calculations and evolutionary conservation among species to
determine the likelihood of formation of a productive miRNA-mRNA duplex
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How to make more accurate
predictions?
• Incorporating mRNA UTR structure to predict microRNA
targets (Robins et al. 2005)
– Make sure the predicted target is “accessible”.
– Not forming basing pairing itself.
Other properties of microRNA targets
• MicroRNA targets are conserved across species.
(Stark et al. 2003)
• Tend to appear in clusters.
For lins, comparison is between C. elegans and C. briggsae.
For hid, comparison is between D. melanogaster and D. pseudoobscura.
Another property of microRNA targets
• Sequence conservations of target sites
– Better complementary to the 5’ ends of the
miRNAs.
Interesting properties of microRNA targets
• Clusters of microRNA targets
– Extensive co-occurrence of the sites for
different microRNAs in target 3’ UTRs.
MicroRNA Targets Prediction
Databases
Scientific Use Case Scenarios
• What are the target genes of a given miRNA?
• What are the miRNAs targeting a given gene
and what are their binding sites?
• What is the tissue expression profile of a given
miRNA (or set of miRNAs)?
• What is the miRNA expression profile in a given
tissue (or set of tissues)?
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Nat Methods. 2006 Nov;3(11):881-6
miRNA
Tools
FEBS Journal 276 (2009) 2150–2156
miRNA
Tools
Target prediction tools covered in today’s
workshop
• Targetscan
• miRBase
• Argonaute
TargetScan
(Lewis et al., Cell 2003)
• TargetScan predicts biological targets of miRNAs by searching
for the presence of conserved 8mer and 7mer sites that match
the seed region of each miRNA. As an option, nonconserved
sites are also predicted.
• Also identified are sites with mismatches in the seed region
that are compensated by conserved 3' pairing.
• In mammals, predictions are ranked based on the predicted
efficacy of targeting as calculated using the context scores of
the sites. TargetScanHuman considers matches to annotated
human UTRs and their orthologs, as defined by UCSC wholegenome alignments.
http://www.targetscan.org/
Targetscan
(Lewis et al. Cell 2003)
Given a microRNA that is
conserved in multiple species and
a set of orthologous 3’ UTR
sequences:
1.
2.
3.
4.
5.
6.
Use 7 nt segment of the miRNA
as the ‘microRNA seed’ to find the
perfect complementary motifs in
the UTR regions.
Extend each seeds to find the
best energy
Assign a Z score.
Give a rank (Ri) according to that
species.
Repeat above process.
Keep those genes for which Zi >
Z_c and Ri < R_c.
miRBase
• Aims to provide integrated interfaces to comprehensive
miRNA sequence data, annotation and predicted gene
targets.
• miRBase takes over functionality from the miRNA
Registry and fulfils three main roles: miRBase
Sequences, miRBase Targets and miRBase Registry.
http://microrna.sanger.ac.uk/
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Sections of miRBase
miRBase contains 3 main sections:
• miRBase Sequences contains all published miRNA
sequences, genomic locations and associated
annotation.
• miRBase Targets is a newly developed database of
predicted miRNA target genes.
• miRBase Registry provides a confidential service
assigning official names for novel miRNA genes prior to
publication of their discovery
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Argonaute (miRWalk)
Nucleic Acids Res. 2006 Jan 1;34(Database
issue):D115-8
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Curated database
•
Argonaute collects latest information from both literature and other
databases. In contrast to databases on miRNAs like miRBase::Sequences,
NONCODE or RNAdb, Argonaute hosts additional information on the origin
of an miRNA, i.e. in which host gene it is encoded, its expression in different
tissues and its known or proposed function, its potential target genes
including Gene Ontology annotation, as well as miRNA families and proteins
known to be involved in miRNA processing.
•
Additionally, target genes are linked to an information retrieval system that
provides comprehensive information from sequence databases and a
simultaneous search of MEDLINE with all synonyms of a given gene. The
web interface allows the user to get information for a single or multiple
miRNAs, either selected or uploaded through a text file. Argonaute currently
has information on miRNAs from human, mouse and rat.
http://www.ma.uni-heidelberg.de/apps/zmf/argonaute/interface
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http://www.ma.uni-heidelberg.de/apps/zmf/argonaute/interface
Single microRNA Module
e.g. hsa-let-7a-1, mmu-mir-16-2, rno-mir-30c-2
HUMAN
MOUSE
RAT
Result for Single miRNA Exact Search
Origin Detail for Single miRNA
Link to PubMed
Link to PubMed for each Reference
miRNA Target Validation and
Considerations in RNAi
Experiments
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miRNA Target Validation
• Several in vitro methods useful to validate miRNA function:
• RNA interference
• protein analysis using SILAC
•Over-expression and knockdown functional studies - There are three basic
approaches to induce RNA interference:
• synthetic RNAi and siRNA duplexes
• vectors carrying RNAi cassette expressing shRNA or artificial miRNAs
• in vitro transcription and dicing of dsRNA to generate pools of siRNA.
•SILAC studies - Although miRNA profiling can show changes based on miRNA
activity, it is not always indicative of translation inhibition.
• Protein analysis by mass spectrometry combined with stable isotopic
labeling by amino acids in cell culture (SILAC) has strong correlation with
miRNA activity.
• Relies upon the metabolic incorporation of 'light' or 'heavy' forms of
the amino acid into the proteins to distinguish differences between
cell populations (i.e. normal and malignant breast tissue)
Considerations for RNAi Experiments
•Method of siRNA production. If a good target sequence is
unknown, or if you have had difficulty knocking down your gene
using an oligonucleotide approach, use effective method such as
transfecting a diced pool of siRNA and validate data using Stealth™
RNAi , siRNA or RNAi vectors.
•Delivery method(s)/cell type to use. Options for delivering
siRNA include transfection and viral delivery. It is very important that
the transfection efficiency be optimized. Even if an siRNA were100%
effective (and most are only 70-95% effective), if only 50% of the
cells are transfected you will never see better than a 50%
knockdown.
•Select appropriate controls.
Considerations for RNAi Experiments
•Measure loss of message or protein function. The best
method to verify loss of message is real time RT-PCR (much more
sensitive than RT-PCR). If protein has long half-life, assays may
need to be done at 48 or even 72 hours.
•Functionality. A visual assay can be done to identify a
phenotypic change , such as looking for apoptotic cells.
Alternatively, a functional cell based assay can be completed to
analyze the effects of gene knockdown , such as a kinase or ion
channel assay - or design your own cell based assay.
• Level of knockdown desired. If you are using a target that is
knockdown to 70% or 80%, there may still be other potential targets
that could result in more effective knockdown.
miRNA Applications
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Examples of miRNA Functions &
Relevance of miRNA to Human Biology
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Role of miRNA in Disease and Cancer
Role of miRNA in Development and
Neurodegeneration
miRNA in Cancer
Significance of miRNA
Potential miRNA Associated Products in Clinical Trials
Thank You
http://catalyst.harvard.edu
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