More than a Messenger:
The Secret Life of RNA
Radhika Mathur
Matt Schwartz
Ilana Kelsey
Biological & Biomedical Sciences
Harvard University
1
Roadmap for the evening
1.  Introduction to RNA and its Messenger Roles
2.  Non-Messenger Roles of RNA
3.  RNA and Healthcare
2
1. Introduction to RNA and
its Messenger Roles
3
DNA: The Source of Information
•  Inherited genetic material present in every cell of the body
DNA
Nucleus
Cytoplasm
4
DNA: The Source of Information
•  DNA stores information in segments called genes.
Gene B
Gene A
•  The sequence of each gene contains the information required to form
a single protein.
Protein A
Protein B
5
Proteins: Functional Molecules of the Cell
•  Present within the cell, on the cell surface, and outside of cells
•  Highly diverse in structure and function
•  What are some of these functions?
–  Enzymes –  Membrane channels
–  Transporters
–  Hormones –  Antibodies
6
How is information contained in genes?
•  DNA consisting of 4 ‘bases’: Adenine, Cytosine, Thymine, and
Guanine
•  A and T are complementary, C and G are complementary
•  The human genome is a sequence of ~3 billion bases of DNA
7
How is the information on genes accessed?
Gene A
DNA
8
How is the information on genes accessed?
Gene A
Sequence of Gene A: TGACCG
Protein A
9
How is the information on genes accessed?
Gene A
Messenger RNA
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DNA
•  4 distinct bases
RNA
•  4 distinct bases
•  Length ~3 billion bases
•  Length ~1000 bases
•  Double stranded
•  Single stranded
•  Confined to the nucleus
•  Travels outside the nucleus
•  High stability
•  Low stability
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Transcription
•  Process by which information contained on a gene is copied to a matching
strand of Messenger RNA.
DNA
RNA
12
Transcription
A ACA G
U
AC
GA C C
13
Translation
•  Process by the instructions on Messenger RNA are used to create protein.
RNA
Protein
14
Translating the Genetic Code
G A C C
A A
C U
C A G A
Ribosome
•  Reads 3 bases at a time •  AAC
Asparagine
•  AGA
Arginine
•  CUG
Leucine
•  ACC
Threonine
15
Transcription & Translation
CU
A A C A G A
G A C C
16
The Messenger Role of RNA
DNA
Transcription
RNA
Translation
Protein
“The Central Dogma of Molecular Biology”
17
Questions?
18
The Messenger Role of RNA
DNA
RNA
Protein
19
Is Messenger RNA just an intermediate?
A A C A GA
CU
GA C C
A CU G
UG
G
A
UAG UC
A CU A
UC
G
A
CA A UU
20
Images from Shuttersotck (Nazlizart) and Meregalli et al, (2011) DOI: 10.5772/24013. Is Messenger RNA just an intermediate?
•  The function of a cell is dependent upon the proteins it contains.
MUSCLE CELLS
Myosin
PANCREATIC CELLS
Insulin
•  Every cell contains the genes required to create every protein.
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Messenger RNA allows cells to specialize
PANCREATIC CELLS
MUSCLE CELLS
Insulin
Gene
Insulin
Gene
CU
A A C A G A
G A C C
Myosin
Gene
UAG UC
A
G
GU
A CU G
Myosin
Gene
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Messenger RNAs in Development
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Messenger RNAs in Development
•  Cells differentiate by transcribing messenger RNAs from lineage-specific
genes
A G
CA A UU
A C U A
U C
CU
AACA GA
UAG UC
U
AG
GA C C
U G
GA C
•  Transcription factors regulate which messenger RNAs are transcribed
+ MyoD
A C C
UG
C
A A C A G A
24
Summary
•  RNA plays a critical messenger role by carrying information from DNA
outside of the nucleus, where it is used to create protein.
•  Genes are transcribed to messenger RNAs, which are then
translated to proteins.
•  The messenger RNAs that are transcribed in a cell determine which
proteins are created and how the cell functions.
•  Transcription of lineage-specific genes allows cells to differentiate, and
is essential for the development of multicellular organisms.
25
Roadmap for the evening
1.  Introduction to RNA and its Messenger Roles
2.  Non-Messenger Roles of RNA
3.  RNA and Healthcare
26
2. Non-Messenger Roles of
RNA
Citation goes here, do not copy/paste URLs if possible!
27
RNA: The Messenger
DNA
RNA
Protein
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Human Genome is 3 Billion Bases
•  What portion of
the genome
encodes proteins?
•  How much of the
genome is
transcribed? (National Human Genome Research Institute)
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RNA: More Than A Messenger
DNA
RNA
Protein
76%
3%
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76%
What is the
other 73%?
Noncoding RNA
3%
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Coding vs Noncoding RNA
•  Coding Transcripts:
–  messenger RNA
–  made into proteins
–  3% of the genome
•  Noncoding Transcripts:
–  Not made into proteins
–  Complex structure and folding
–  Regulate gene expression
–  73% of the genome
(William G Scott)
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0 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 Noncoding RNA Publications by Year
16000 14000 12000 10000 8000 6000 4000 2000 33
RNA Alphabet Soup
DNA
siRNA, piRNA
snRNA,
snoRNA
lncRNA
RNA
microRNA
tRNA,
rRNA
Protein
34
Questions?
35
microRNAs
•  ~22 base single-stranded RNAs
•  Function by base pairing with their target mRNAs
•  Often have many targets
36
Caenorhabditis elegans
• 
• 
• 
• 
Model system since 1963, Sydney Brenner
1 mm transparent round worms
Free living, eat bacteria, 10,000 per petri dish
3 day live cycle, 2-3 week lifespan, many offspring
(Zeynep F Altun, WormClassroom)
37
The First microRNA
•  lin-4 microRNA discovered 1993 in roundworm
–  lin-4 mutant “adults” maintain larval tissues
–  Don’t develop adult tissues
–  Continual molting
L1
(WormAtlas.org, Adapted from Reinhardt BJ et al., 2000)
L2
38
The First microRNA
lin-4 mutant
Wild Type
•  Identified a small 22 base microRNA, lin-4
22
(Adapted from Lee et al., 1993)
39
The Second microRNA
•  let-7 microRNA discovered 2000 in C. elegans
–  7 years later!
L3
L4
(WormAtlas.org, Adapted from Reinhardt BJ et al., 2000)
Adult
40
let-7 microRNA
• let-7 found across
bilateral animals, including
humans
(Adapted from Pasquinelli AE et al., 2000)
41
Human microRNAs
•  let-7 also the first discovered human microRNA (2000)
–  Humans have 18 microRNAs in the let-7 family
–  Each with 1000-8000 of their own predicted targets
•  Recent estimates suggest there are ~4500 human
microRNAs (Friedlander et al., 2014)
(microRNA.org)
42
Long noncoding RNAs (lncRNA)
DNA
RNA
lncRNA
Protein
43
Long noncoding RNAs (lncRNA)
•  Do not encode proteins
•  Size Range: 200 – 100,000’s
bases
•  May activate or repress gene
expression
•  Fold into complex structures
•  Estimates suggest as many as
>113,000 lncRNAs in the
human genome (LNCipedia.org)
(Adapted from Novikova et al., 2014)
44
lncRNA Mechanisms
•  Activate or Repress Gene Expression
–  DNA
–  RNA
–  Protein
(Adapted from Novikova et al., 2014)
45
Xist lncRNA
•  Discovered 1992
•  Required to silence expression of one of the
female X chromosomes
(Adapted from Zlir’a and YassineMrabet)
46
Xist lncRNA
(Adapted from Reinius et al., 2010)
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RNA Comparison
mRNA
microRNA
lncRNA
Coding Potential
Protein
Noncoding
Noncoding
Regulatory Potential
Mainly No
Yes
Yes
Folding
Maybe?
No
Yes
Interactions
Sequence
Sequence
Sequence or
Structure
Size
Long
Short
Long
48
Summary
•  Although only 3% of the genome encodes proteins,
~76% of the genome is transcribed
•  Much of that transcription represents noncoding
RNAs
•  microRNAs are small (~22 base) noncoding RNAs
which silence gene expression
•  Long noncoding RNAs (>200 – 100’s kb) likely
represent multiple distinct classes of ncRNA and
activate or repress gene expression by sequence or
structural interactions
49
Questions?
50
Roadmap for the evening
1.  Introduction to RNA and its Messenger Roles
2.  Non-Messenger Roles of RNA
3.  RNA and Healthcare
51
3. RNA and Healthcare
Outline
•  What can cause disease?
•  How do traditional drugs work?
•  Depleting too much protein in order to treat
disease
•  Adding protein back in diseases caused by loss
of a gene
53
What Can Cause Disease?
•  Too much of a
protein
–  Cancer
–  Viral infections
–  Huntington’s Disease
54
What Can Cause Disease?
•  Not enough of a
protein
–  Cystic fibrosis
–  Tumor syndromes
caused by gene loss
–  Macular degeneration,
vision loss
55
What are drugs, and when do we use
them?
•  Drugs are small molecules that bind to proteins in
the cell
•  Usually used to turn off a protein
•  Sometimes turn on a protein
56
How do drugs work?
Traditional method
of drugging with
small molecules
Traditionally
“undruggable”
targets
57
58
USING RNA IN DISEASES
CAUSED BY TOO MUCH
PROTEIN
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What is siRNA?
siRNA = small interfering RNA
Gene of Interest
60
Advantages of siRNA as a drug
•  siRNA prevents proteins from being made
•  Can target any protein
–  Don’t need to “get lucky” with a synthesized drug
molecule
•  Can target mutated protein
–  If there is an unmutated copy, it will be unaffected
•  BUT: do need to carefully test for off-target
effects
61
What types of diseases could be
targeted by siRNA?
•  Examples:
–  Viruses
•  Ebola
•  Hepatitis virus (B and C)
–  Cancer
–  Diseases that rely on protein-protein interactions
–  Huntington’s Disease
62
What is Huntington’s Disease?
•  Genetic neurodegenerative disorder
–  One parent with disease has 50% chance of passing
on to child
•  Usually begins between 35-44
–  But 6% begin before 21, more aggressive disease
•  Symptoms:
–  Slow loss of muscle control
–  Brain degeneration into eventual dementia
•  No cure; average life expectancy from
diagnosis is 20 years
63
Huntington’s Disease
Htt
Mutant Htt
64
Huntington’s Disease
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Huntington’s Disease
siRNA targets mutated protein
66
Huntington’s Disease
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Summary
•  siRNA treatments target unwanted proteins
before they are made
•  What it could be used for
–  Diseases caused by over-production of proteins
–  Proteins that have been considered “un-druggable”
68
QUESTIONS?
USING RNA IN DISEASES
CAUSED BY LACK OF A
PROTEIN
72
Recap: mRNA
Protein coding
transcript
73
Advantages of mRNA as a drug
•  May not need to be as extensively tested as
drugs
–  Off-target effects
–  Efficacy
–  Clearance from body
•  mRNA are inherently natural products
74
What types of diseases could be
treated with mRNA?
•  Good for diseases characterized by a loss of a
certain protein
–  Cancers driven by loss of a tumor suppressor
•  BRCA loss in breast cancer, for example –  Cystic fibrosis
–  A variety of other rare diseases
75
What is cystic fibrosis?
• 
• 
• 
• 
Genetic disorder
Life expectancy 37-40
Approximately 30,000 people in U.S.A.
Symptoms
–  Difficulty breathing
–  Lung infections
–  Poor growth/poor weight gain
•  No cure; often requires lung transplant
76
Cystic Fibrosis
Outside
Inside
77
Summary
•  mRNA treatments add back needed proteins
to the body
•  Useful for diseases caused by loss of a protein
78
QUESTIONS?
THESE SOUND GREAT –
WHY AREN’T THEY
ALREADY IN USE?
Challenges to RNA Delivery
•  Hard for RNA to cross membrane
–  Charge
–  Size
Outside
Inside
81
Challenges to RNA Delivery
•  Unstable as a molecule, so needs to be
protected from its environment
82
One Method for Delivering RNA
Outside
Inside
83
One Method for Delivering RNA
Outside
Inside
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One Method for Delivering RNA
Outside
Inside
One Method for Delivering RNA
Outside
Inside
Grand Summary
•  RNA is the messenger between DNA and protein •  RNA expression varies by cell-­‐type in order to determine cell funcGon •  Most of the genome (76%) is transcribed but only a small porGon (3%) encodes proteins •  Noncoding RNAs are not translated into protein and play a variety of roles in the regulaGon of gene expression at the level of DNA, RNA, and proteins •  RNA treatments may one day be used in a variety of diseases 87
QUESTIONS?
Thank you!
SITN would like to acknowledge the following
organizations for their generous support.
Harvard Medical School
Office of Communications and External Relations
Division of Medical Sciences
The Harvard Graduate School of Arts and Sciences (GSAS)
The Harvard Graduate Student Council (GSC)
The Harvard Biomedical Graduate Students Organization (BGSO)
The Harvard/MIT COOP
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