Non Coding RNAs and Disease Flashcards by sita clark

2006

In 2006, Andrew Fie and Craig Mello won the Nobel prize for medicine for their work on “RNA interference- gene silencing by double stranded RNA”
RNA interference pathway is an Epigenetic method where gene silenced by small RNA’s binding to them, blocking their transcription or translation
-RNA now has function

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ncRNAs are involved in disease

Pathogenesis: development and progression of disease
Therapeutics: treatment of disease
Biomarkers: diagnosis and prognosis of disease
-drug and protein serum tests

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Central Dogma of molecular Biology

1958 Francis Crick, one of the discoverers of the DNA double helix structure, described that genetic info flows from DNA –> RNA –> protein in a simple linear manner
Specialised Mechanisms:
1. RNA –> DNA. Reverse Transcription. seen in virus
2. RNA –> RNA
mRNA encode proteins
Translation RNA’s= Non-coding RNA’s = rRNAs and tRNAs = ribosomal and transfer RNAs

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RNA importance in the future

Total RNA

1) Coding RNA 1.5%–> mRNA
2) Noncoding RNA 98.5% (has regulatory roles)—>
a) Long ncRNAs (>200nt)
- Signal, Decoy, Guide, Scaffold IncRNA
b) Small ncRNAs (

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Organism Complexity

Can judge the increasing organism complexity, by the amount of transcribing RNA that it makes

same number of protein coding genes as other species
huge amount of Non-coding RNA, that allows for complexity
e. g Bacteria vs Yeast vs Worms vs Humans

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Non-coding RNA publications

Rapid increase in the number of publications due to increase in mRNA interest

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What are Non-coding RNAs?

Classified into many subtypes within two major groups, defined by size (not function)
-200nt “long”ncRNAs
All forms have varied regulatory roles within the cell, some well characterised (tRNA), some not

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Examples of Small ncRNAs

70-100 nt trasnfer RNAs (tRNA)
-carry a/acids for building growing peptide chain during translation
70-120 nt small nucleolar RNAs (snoRNA)
-involved in translation. modify ribosome. involved in direct splicing of mRNAs (can make different protein isoforms). associated with many different disease types
18-26 nt microRNAs (niRNA)
-distinctive hairpin structure
-work v similarily with siRNA (the F and M won nobel prize for)

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tRNAs general

Identified in 1958
-well characterised non-coding RNA
-clover leaf structure
-bind to read mRNA, identify in triplets which a/acids should come to the growing peptide
Vital for correct translation of proteins
-error in tRNA = problems with protein production

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tRNA in human disase

MELAS syndrome:
Mitochondrial myopathy, Encephalomyopathy, Lactic Acidosis and Stroke-like episodes
-Maternal Mitochondrial inheritance
-tRNA normally encoded on mitochondrial DNA that is 12 kilobases, inherited through mother
-this extra piece of DNA/mitcohondrial genome encodes quite alot of these tRNAs
-A3243G mutation tRNALeu ^UUR causes a/acid misincorporation
-A–>G switch
-effects mutiple organs
-errors in mitochondria genome tRNAs results in a single nucleotide mutation

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How are tRNA’s mutated

tRNA are a gene

can be mutated in the same way as any other protein coding mRNA

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snoRNA general

snoRNAs act as a guide for RNAs for modification of ribosomal RNA and tRNA’s
are crucial in ribosome biogenesis = translation of RNA into protein
-names in 1981
-discovered in late 60s
2x forms:
-C/D box, methylation (modify ribosomal RNA so they function properly) (if they dont function properly there will be translation problems)
-H/ACA box, pseudouridylation

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snoRNA transcription

snoRNA sit within protein coding genes

snoRNA host gene
1. Primary RNA transcription (including all exons and introns with their snoRNAs)
2. RNA processing (splicing of exons and removal of introns) –>
highlights that the “junk” discarded proteins actually contain regulatory RNAs
ensures youre not wasting anything
a) Cytoplasm (mRNA used for protein)
b) Further processing–> snoRNAs
-> nucleolus

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snoRNA and human disease

Prader-Willi and Angelman syndromes
-Loss of imprinted(methyl groups) region C15q11-13 (long arm), paternally expressed, included six snoRNAs (chromosome 15 long arm has methyl groups expressed only from father’s copy)
-SNORD116 mice show a defect in growth and feeding regulation (have different roles in different syndromes. losing more than one gene, and more than one function, changing the effect on more than one gene ==> get an effect on more than one organ)
-SNORD115 also causes alternative splicing of seratonin receptor 2C so loss of the sno causes different isoforms of 5-HC2cR –> growth retardation, increased energy expenditure
Illustrates how snoRNAs are directly associated with the development of disease

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microRNAs

• Initially identified in C. elegans in 1993 and found conserved in animals in 2000
Mutations (knocked out) in the C. elegans let-7 (lethal) miRNA gene result in developmental abnormalities that often lead to rupturing of the worms (leak out of insides/burst of vulvo) and lethality
• First associated with human disease in 2002, miR-15/-16 deleted in chronic lymphocytic leukemia (Calin GA et al PNAS 2002, 99(24):15524-9) (pair of microRNA in region, and their loss was directly associated wiht the development of the leukemia)
• So well studied now – online database (inks different microRNAs with different disease states)
• In miRBase V21 (June 2014) contains 2588 miRNAs

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Pathway of miRNA (microRNA)

RNA pathway miRNA binds to a Target mRNA (messenger RNA)
a) blocks translation of that protein
b) causes RNA to be degraded
-overall decreasing the expression of the target RNA
Is an Epigenetic mechanism
-expression of miRNA is regulated like most other genes
-can be inherited with disease states

microRNAs and Cancer

Cancer: miRNAs repress the translation of oncogenes and tumour suppressors
(miRNA itself is working as an oncogene or a tumour surpressor gene)
-many of the hall marks of cancer are regulated directly by micro RNAs (Cell adhesion and invasion. Apoptosis. Epigenome. Cell Proliferation cycle. Genomic Stability. Differentiation)

microRNAs and Down syndrome

Down syndrome: Gain of C21 DNA
– C21 carries 5 miRNAs, (all tumour supressor genes)
–two of which repress the production/translation of protein MeCP2.
– (Lower levels of MeCP2 may lead to) abnormal brain development (also lost in neurodevelopmental disorder Rei syndrome)
– Decreased risk of solid tumours in DS, all the miRNAs are known tumour suppressor genes.
Increased expression of C21 miRNAs in hippocampus from DS pa)ent. Kuhn DE et al, BBRC 2008, 370(3).

microRNAs and human disease (Neurodegeneration)

NEUROdegeration:
-Huntington's Disease
-Parkinson's Disease
-Alzhiemers Disease
-Amyotrophin Lateral Sclerosis
MiRNA regulate mRNAs (transcription factors) which directly regulate cell functions --> phenotypes

microRNA and Sporadic Alzheimer’s disase

Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer’s disease correlates with increased BACE1/ b-secretase expression
BACE1 is the protein which nibbles off Alzheimer’s protein –> Senile Plaque development
-lose the regulatory microRNA, lose ability to control/repress this gene –> increased Senile Plaque development

Long non-coding RNAs

Harder to classify into groups. know less about them.
• >200nt in size
• Can look like mRNAs (protein-coding genes) with introns and exons and a poly-adenylated tail
-main different is that they DONT encode protein (as is Non-coding RNA)
-really varied in their functions
• Functions are highly varied
-lncRNAs cannot easily be grouped into functional families like ‘miRNAs’ and ‘snoRNAs’

lncRNA mapping

The Human Genome Project has identified transcribed regions in our DNA that do not encode for proteins.(non doing regions)
-can identify which genes sit within this region
UCSC (University of California, Santa Cruz) genome browser with ENCODE (Encyclopedia of DNA elements) data
-try to identify new non-coding RNA and new mCRA
-identify genes that sit within the region
-XIST gene

How the Landscape has changed having Long non-coding RNA

Increased Techniques + Increased knowledge
= allowed increased information. more able to answer questions
1999-2002 Identification of Tumour Suppressor genes on C11p15 in Ovarian Cancer
-UCSC Genome Browser on Human July 2003 Assembly
-gives so much more information that before (more known genes)
—->2009
-more genes identified
-more tracks on UCSC browser identifying and annotating mRNA
—-> 2010
miR-210 links hypoxia with cell cycle regulation and is deleted in human epithelial ovarian cancer
-functions like a Tumour surpressor gene

lncRNAs and disease

– XIST and X-inacOvaOon
– HOTAIR and Cancer
– H19 on chromosome 11, many diseases
– lncRNA and Prader-Willi syndrome

lncRNAs and XIST

XIST (LINC0001 found in 1991) LINC1
• X-inactive specific transcript 17kb (very long)
(encoded from X chromosome) (involved in inactivation of one of the X chromosomes. XIST randomly wraps around on chromsome = inactivation )
-dosage compensation
• Familial skewed X inactivation, in females where one copy of the X chromosome should be randomly repressed, the loss of XIST causes a ‘skew’ to where one chromosome is
-preferentially repressed >80% of the time.
Tortoise Shell cats = Mosaicism
• Skewed inactivation = X-linked diseases such as
-Rets syndrome,
-Haemophilia and even
-spontaneous abortion due to CX genetic imbalance.
80% repression/ Inacitvation/ Mosaicism= Increased Incidence of X-linked diseases

IncRNAs and XIST and Tortoise Shell cats

Maternal X chromosomes O allele Orange
+ Paternal X chromosome o allele Black
–> Random X inactivation (changes patterning of Mum Orange and Dad Black –> Mosaicism)
–> Portion of embryo showing clones of cells
–> Mosaic adult
-some cells express O
-some cell express o

lncRNAs and HOTAIR

HOTAIR (NCRNA00072- found in 2007)
• HOX transcript antisense RNA in the mRNA(protein coding cluster) HOXC gene cluster on C12, 2.2kb (chromosome 12. Pretty long)
• Increased expression of HOTAIR in breast cancer and metastases and induces cell invasion.
-Epigenetic mechanism.
-allows cells to trigger formation of metastes and invade around the body
- this lcRNA works to to directly regulating machinery inolved in transcription
• Oncogenic ncRNA involved in many forms of cancer

lncRNAs and H19

H19 (LINC00008- first published in 1984)
Imprinted (expressed by either paternal or maternal copy of gene)
Maternally(Mother) expressed C11p15.5 imprinted RNA associated with diseases of either UNDER or OVERgrowth
-Beckwith- Wiedemann syndrome,
-Silver-Russell syndrome (dwarfism),
-Wilms tumour (kidney) and cancers (overgrowth).
• If both H19 switched off by methylation, excess IGF2 is produced = overgrowth = cancer
• If both H19 switched on (demethylated) = too little IGF2 = dwarfism
-Within lncRNA H19 is a miRNA
-miR-675 regulates normal placental development (can be spliced out to make another function RNA)
Overexpression of miR-675 and
H19 are seen in colorectal cancer (Tsang WP 2010 Carcinogenesis)

H19 methylation mechanism

H19 directly regulates transcription of IGF2 gene
Insulin Growth Factor - v important in growth regulation
- if repressed by methylation
1) Paternal: IGF2 On –> H19 OFF
-H19 is switched off by methylation= IGF2 is made in abundance
2)Maternal: IGF2 OFF –> H19 On
-if H19 doesnt have sufficient methylation it is Active = Supresses/Regulates IGF2 = insufficient growth (dwarfism)

lncRNA and miRNA

H19 lncRNA contains miRNA

alot of mRNA or lncRNA that have smaller miRNA which have a role
dont waste anything in out body

SnoRNA-lncRNA overlap

Prader-Willi syndrome
• SNORD116 also encodes a lncRNA which ‘mops up’ Fox2 protein normally. (LongncRNA is made up of repeat blocks of snoRNA) (is then chopped up into snoRNA)
-the sno-IncRNAs mop up/collect the Splicing Factor Fox2
-regualtes RNA splicing
Prader Willi = deleted SNORD116 = no lncRNA or snoRNA made = No FOX2 mopped up = change in mRNA Splicing = Abnormal protein production= Abnormal Development/ Neuronal Functions
• When SNORD116 is lost in PWS, the extra free Fox2 causes abnormal splicing of genes associated with
neuronal function. (Yin et al 2012 Mol. Cell 48:2)

Importance of ncRNAs?

We have 30,000 mRNAs which account for only 1.5% of the transcriptome!
– ncRNAs have utility as biomarkers of disease
– Single Nucleotide Polymorphisms (SNP) in ncRNA
genes associate with disease
– ncRNAs could be novel therapeutic targets for treatments

SNPs in ncRNAs

SNPs= small nucleotide polymorphisms
-ANRIL (antisense noncoding RNA in the INK4 locus)
ANRIL gene contains lots of SNPS throughout
-Study which SNPs are associated with/found in certain diseases =
-slight changes/different SNPs in the ANRIL gene can be associated with a huge range of diseases
-ANRIL SNPs found in:
glioma (red),
basal cell carcinoma (purple),
nasopharangeal cancer (black), breast cancer (blue),
coronary disease (orange),
intercranial aneurysm (grey),
type 2 diabetes (green)

ncRNA biomarkers

miRNA levels associate with cancer and clinical-pathological characteristics
-biomarkers used to predict and progositcate
-look for miRNA levels in cancer tissues.
-where it has come from (pick pest treatment)
-most effective drug
-aggresiveness of tumour
Tissue sampling of tumour tissues is standard and assays are now available to assess their miRNA levels for predictors of treatment responses, tissue origin and outcome.
miRNAs are also very stable (so small. resist degradation by enzymes) so they can be detected in archival samples, which also simplifies sample storage and postage.
-formal in blocks
-blood spots
miRNAs in mesenteric lymph and
plasma associate with acute
pancreatitis severity in a rat model (Blenkiron et al, 2014 Annals of Surgery).

ncRNA and non-invasive blood tests

Non-Invasive
Dont want to take biopsy/take tissue
-very popular
-take ncRNA that are passaged around the body
(perform LIQUID BIOPSIES)
-miRNAs re Very STABLE as packaged in vesicles
-identify miRNAs that are associated with disease
(released by tumour, released by body due to tumour being present, released by necrotic organ etc)
-increased particular blood miRNA = increased pancreatitis severity
miRNAs in mesenteric lymph and
plasma associate with acute
pancreatitis severity in a rat model
2. miRNAs are also very stable (so small. resist degradation by enzymes) so they can be detected in archival samples, which also simplifies sample storage and postage.
-formal in blocks
-blood spots

Types of Biomarkers

Not just miRNA
-now usually in blood plasma
lncRNA

ncRNA therapeutics

Identification of disease associated microRNAs allows new pathways for therapies
- targets miRNA
- switching on and off miRNA in a disease
- Hepatitis C (HCV) = miRNA-122 (Neurovesin drug)
- regulatory
lncRNA - upregulate gene expression
- try to regulate how genes are used
- epigenetics in a cell

ncRNA summary

• 98.5% of all RNA is ‘non-coding’ (small RNAs are associated with disease)
• Many different types and flavours with regulatory functions in the cell
• New techniques such as RNA-sequencing are identifying and revealing ncRNAs as important in disease:
a) onset and
b) progression
• Study of these new ‘genes’ has successfully led to therapeutics and biomarkers for use in the clinic (success straight away!)

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Non Coding RNAs and Disease Flashcards

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