miRNA Revolution

Scientific American.com, April 2012 Pharmacogenomics. March 2009
RNA has emerged as a path to
a new world of medical treatment
By Christine Gorman and Dina Fine Maron

Biologists have identified molecules that direct and shape the organized chaos within the
body’s cells, and they have exploited those findings with thousands of drugs and
treatments.
For decades the stars of the drama came from two: DNA, or deoxyribonucleic acid and
Proteins.
Protein discoveries and gene therapy have led to such medical advances such as synthetic
insulin, next-generation anticancer drugs, hemophilia, hereditary blindness and other
previously intractable diseases.
Overlooked in this march of medical progress was a third type of biomolecule: RNA, or
ribonucleic acid.
Introduction

RNA Shines in New Roles
RNA’s basic duties in the cell was known for decades. Research over the past few years, however, has
uncovered new forms of RNA with surprising functions.
New forms of RNA can direct specialized proteins to block certain cellular processes from happening
or even silence them entirely.
Researchers are adapting these pathways to develop new, more precise medical treatments.
Over the past 8 years, the ongoing RNA revolution has resulted in more than 4300 publications
documented in PubMed alone.
To date, more than 200 experimental studies of either miRNAs or siRNAs have been registered
through the U.S. government’s database.

Financial interest
The pace of discovery has accelerated, dozens of startups have formed to capitalize on new findings
and now some promising treatments are in the offing.
Among recent ventures, Editas Medicine received $43 million in venture capital for its launch at the
end of 2013.
A slightly older company, Alnylam Pharmaceuticals, founded in 2002, received $700 million this
past January.
The funding has come “in waves,” says Robert MacLeod, vice president of oncology and exploratory
discovery at Isis Pharmaceuticals, which has raised nearly $3.8 billion since it was founded in 1989.

Supporting Role
The vast majority of today’s medicines—from aspirin to Zoloft—work by manipulating proteins, either by
blocking their function or by altering the amount that is produced.
Drawback with protein is that investigators were not able to develop drugs that act on all the proteins they
would like to target.
Why is it? Because certain proteins bury their active sites too far inside narrow channels (like, part of the
cell’s internal skeleton), or they do not even contain an active site.
This roadblock is what the new RNA medicines are designed to overcome.

A Star is Born
The groundwork for RNA’s breakout performance was laid in 1993, with the identification of the first
microRNAs.
Cells apparently use microRNAs to coordinate the production schedule of many proteins—particularly
early in an organism’s development. These short stretches of RNA attach themselves to strands of mRNA,
preventing ribosomes from making any progress in assembling a protein.
Five years later researchers made another breakthrough when they demonstrated that different short RNA
molecules effectively silenced the translation of a gene into protein by cutting up mRNA.
That landmark discovery later netted a Nobel Prize, in 2006.

About miRNA
MicroRNAs (miRNAs) are small, single-stranded, 21–23 nucleotide-long, independent functional
units of noncoding RNA. Often referred to as the 'micromanagers of gene expression'.
To date, 678 human miRNAs have been characterized; however, computational predictions
suggest that the total number of different miRNA sequences in humans may exceed 1000.
MiRNAs regulate specific genes, which are broadly involved in multiple pathways such as cell
death, cell proliferation, stress resistance and fat metabolism.
Accumulating evidence now suggests that miRNAs not only inhibit translation of, but also
destabilize its target mRNA.

Basic Plot
Cells start the process of manufacturing
proteins by copying, or transcribing, the
genetic code found in DNA into long
complementary sequences of messenger
RNA, or mRNA (shown on left of diagram).

Researchers hope to manipulate microRNA, which gives cells
the ability to change the production of specific proteins, to
treat a range of diseases.
Because the RNA of the microRNA does not have to be a
perfect match for the mRNA whose translation is being
affected, a small number of microRNAs can temporarily alter
production of many different kinds of proteins.
miRNA’s breakout performance

MiRNA biogenesis and function
(A) A miRNA gene is transcribed by RNA polymerase II,
resulting in a hairpin-shaped pri-miRNA. (B) The pri-
miRNA is further processed by Drosha/Pasha to form a
pre-miRNA, (C) which is transported from the nucleus
to the cytoplasm with the help of Exportin-5/Ran GTP.
(D) The pre-miRNA is further cleaved in the cytoplasm
by an RNase III endonuclease, Dicer (E), to release two
complementary short RNA molecules (F). (G) The
argonaut protein complex selectively binds to the guide
strand and facilitates the formation of the miRNA–RISC
assembly. (H) Upon miRNA binding the RISC complex
is activated and, by a mechanism that is still unclear,
locates its binding site in the 3′-UTR of the target mRNA
contributes to regulation of gene expression by
translational inhibition and/or mRNA degradation.

MiRNA in Action

miRNA function
More and more evidence suggests that a gain or loss of miRNA function is associated with disease progression and
prognosis.
Several studies have now established that miRNAs are differentially expressed in human cancers as compared with the
normal tissue.
Examples:
downregulation of two miRNAs, miR-143 and miR-145, in colorectal cancer
increased expression of the miR-155 precursor in pediatric Burkitt lymphoma
down-regulation of Let-7 in lung cancers.
Some miRNAs have the potential to act as an oncogene or a tumor suppressor by affecting the expression of a tumor
suppressor or an oncogene, respectively.

MiR-polymorphisms and mRNA function
Generally, miRNAs regulate gene expression of a target gene by binding to its 3′-UTR.
MiRNAs can potentially regulate expression of multiple genes and pathways.
A single miR-polymorphism can potentially affect the expression of multiple genes involved in
pathways regulating drug absorption, metabolism, disposition, stem cell function and the cell cycle.

miRNA Polymorphisms
Polymorphisms present in the target mRNA, pri-miRNA,
pre-miRNA, processed miRNA, Drosha, Dicer, exportin5-
ranGTP and in the RISC complex may affect miRNA-
mediated regulation in the cell.
The miR-polymorphisms can be present in the form of
insertions, deletions, amplifications, chromosomal
translocations and so on, leading to loss or gain of a
miRNA site/function.

MiR-polymorphisms/miR-mutations
A miRNA mutation can be defined as a mutation that interferes with miRNA function (hereafter miR-
mutations).
A miR-mutations can be present either in heterozygous or homozygous form.
A somatic cell in the human body can be profoundly influenced by a miR-mutation.
A somatic miR-mutation can potentially alter cell morphology, induce cell death or contribute to
carcinogenesis.
A miR-mutation in a germ-line cell can be transmitted to the next generation resulting in an altered
phenotype.

Examples
An example of a miRNA gain-of-function polymorphism is a G > A mutation in the GDF8 allele of
the myostatin gene in Texel sheep. The mutation creates a potential illegitimate miRNA target site
for miR-1 and miR-206 and is associated with sheep muscular hypertrophy.
An example of a loss of miRNA function polymorphism is a C>T SNP present in the 3′-UTR of
DHFR, preventing miR-24 binding.

Gain or Loss of miRNA function
If a miR-polymorphism results in a gain of miRNA
function, it will cause downregulation of both
drug-target and the drug-effector proteins
resulting in drug sensitivity and drug resistance,
respectively.

Classifications
MiRNA polymorphisms/mutations can be classified in the following three major categories:
Polymorphisms or mutations affecting miRNA biogenesis.
MiR-polymorphism/mutations in miRNA target sites.
MiR-polymorphisms/mutations altering epigenetic regulation of miRNA genes.

Polymorphisms or mutations affecting miRNA biogenesis
Polymorphisms present not only in miRNA precursors but also in the proteins involved in its
biogenesis may potentially affect miRNA mediated regulation of the cell.
MiR polymorphisms/mutations affecting microRNA biogenesis can be further subclassified
in following three categories:
In pri and pre miRNA transcripts
In mature miRNA sequences
Affecting expression of the proteins involved in various steps of miRNA biogenesis

MiR polymorphism
in miRNA target sites
MiR polymorphisms in miRNA target sites will impact only its encoded target mRNA and its
downstream effectors, hence, are more specific.
A recent genome wide association (GWA) study suggests that a gene that has more than two miRNA
target sites will have increased expression variability as compared with a gene that is not regulated
by a miRNA.
MiRNA polymorphisms/mutations in miRNA target mRNA sites can be further subclassified in
following three categories:
At a miRNA binding site
Near a miRNA binding site

MiR-polymorphisms/mutations altering epigenetic regulation of miRNA genes
Various miRNA genes are affected by epigenetic silencing due to aberrant hypermethylation.
MiR polymorphism mediated epigenetic alteration of miRNA regulation is a new, unexplored area of
research.
miR polymorphisms or miR mutations that can alter epigenetic regulation of a miRNA (methylation
or acetylation) can be a mechanism of disease progression.
Gain or loss of epigenetic regulation of an oncogene or a tumor suppressor, respectively, due to a
miR polymorphism or mutation, may have devastating effects in a cell.

Role of miR-polymorphisms in disease progression, diagnosis & prognosis
Advancements in the miRNA field indicate a clear involvement of deregulated miRNA gene
signatures in cancers, such as papillary thyroid carcinoma, chronic lymphocytic leukemia and breast
cancer.
Recent GWA studies suggest that variations present in regulatory sites are more likely to be
associated with disease and not the variations within coding region.
Following are some of the common disorders found to be associated with miR polymorphisms:
Neurological disorders
Muscular hypertrophy
Cancer
Type II diabetes

miRNA pharmacogenomics
Pharmacogenomics of miRNA is a novel and promising field of research that holds new possibilities
for tailor made medical therapy.
MiRNA pharmacogenomics can be defined as the study of miRNAs and polymorphisms affecting
miRNA function in order to predict drug behavior and to improve drug efficiency.
miR polymorphisms have potential as predictors of drug response in the clinic and will result in
development of more accurate methods of determining appropriate drug dosages based on a
patient’s genetic makeup, thus decreasing the likelihood of drug overdose.

What’s next?
Whereas medications containing microRNA are furthest along in the race to the clinic, another
generation of aspiring starlets is now waiting in the wings.
These potential medications would work even further upstream, on the DNA molecule itself.
One of the approaches is based on CRISPR sequences found in the DNA of many single- celled
organisms and was enthusiastically described in Science as the “CRISPR Craze.”

Thank you

miRNA Revolution

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