MICRO RNA

What is RNA?
• Ribonucleic acid
• Ribonucleotides (Ribose, base, & phosphate)
• Types
• Coding: messenger RNA (mRNA)
• Non-coding:
• Ribosomal RNA (rRNA)
• Transfer RNA (tRNA)
• Small nuclear RNA (snRNA)
• Small nucleolar RNA (snoRNA)
• Interference RNA (RNAi)
• Short interfering RNA (siRNA)
• Micro RNA (miRNA)
mRNA Structure
• Coding region
• Untranslated regions
• 5’ UTR
• 7methyl-G cap
• Bound by cap binding proteins
• Translation regulation
• 3’ UTR
• Stability elements
• Subcellular localization (zip codes)
• poly(A) tail
mRNA
Timeline for RNAi Dicsoveries

Nature Biotechnology 21, 1441 - 1446 (2003)

Non-Coding RNA: Formerly
known as “JUNK”
Non-Coding RNA: A Key to
Eukaryotic Complexity?
What is miRNA?
 Characteristics of miRNAs

• 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 (pre-
miRNA).

-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
miRNA Details
• Originate from capped & polyadenylated full length precursors (pri-
miRNA)
• Hairpin precursor ~70 nt (pre-miRNA)
• Mature miRNA ~22 nt (miRNA)
• First discovered in 1993 by Victor Ambros at Harvard (lin-4)
• Let-7 discovered in 2000 by Frank Slack as a postdoc at Harvard
(Ruvkun lab)
Illustration of
miRNA
processing
Another View

Microprocessor
Complex
Processing
bodies are
sites of
storage
and/or
degradation
of mRNA
Image: wiki
miRNA transcription and maturation
miRNA transcription
and maturation

1. Nuclear gene to primary-miRNA

2. Cleavage to miRNA precursor by
Drosha Rnase III
3. Transported to cytoplasm by
Ran-GTP/Exportin 5
4. Loop cut by DICE
5. *duplex is short-lived and cut by
helicase to single strand RNA
forming RNA-induced silencing
complex (RISC)/maturation

Kadri et al 2009
Enabling machinery
Summary of Players
• Drosha and Pasha are part of the “Microprocessor” protein complex
(~600-650kDa)
• Drosha and Dicer are RNase III enzymes
• Pasha is a dsRNA binding protein
• Exportin 5 is a member of the karyopherin nucleocytoplasmic
transport factors that requires Ran and GTP
• Argonautes are RNase H enzymes
Players
What are the functions of miRNA?
• Involved in the post-transcriptional regulation of gene expression
• Important in development
• Metabolic regulation (miR-375 & insulin secretion)
• Multiple genomic loci (different expression patterns?)
Differences in miRNA Mode of Action
miRNA Registry
• http://www.sanger.ac.uk/Software/Rfam/mirna/index.shtml
• Latest release contains 1620 2909 predicted and verified miRNAs
• 227 321 predicted and 131 223 experimentally verified in Homo
sapiens
• Mouse and human are highly conserved
• Human is not conserved with plants
siRNA miRNA
Why analyze miRNA patterns?
 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
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.

• 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.

• 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.

• 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.
miRNA Profiling Applications