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 BornThe 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 functionMore 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

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miRNA Revolution