Discovery of a new cellular process has been ! exploited to knock down ! expression of genes in eukaryotes!!
Tissue culture cells! C. elegans!
Regulation of mRNA expression by small RNAs! 1. RNA interference (RNAi)! [Link] (miRNA)!
C. elegans is an excellent organism for ! genetic analysis of development!
C. Elegans is a great organism! for studying development because:! The cell lineage is known *! Striking phenotypes can be! observed if there are develop-! mental defects!
*Genes responsible for regulating! developmental timing are! heterochronic!
Weakened cuticle=bursting through vulva!
Discovery of a critical mechanism in gene expression started with an unexpected outcome of a simple experiment !
Caenorhabditis elegans 3
m7G
Translation of! mRNA into Protein!
Control: Provide the sense RNA ! and nothing should happen!
m7G
3 5 Antisense RNA inhibition! of Translation (modest)!
BUT, sense RNA also inhibited???!
m7G
3 5 Antisense RNA inhibition! of Translation! control experiment ! not working!!!! 3
m7G
Sense RNA inhibition?!
The Solution: double-stranded RNA ! directs mRNA Degradation!!
In vitro txn was producing a! sense AND a contaminating! antisense --> dsRNA! (Andy Fire & Craig Mello, 1998)! Using puried sense + AS causes! potent response!! How does dsRNA direct degradation ! of a mRNA target?!
Mechanism of RNA interference!
double-stranded RNAs
RNaseIII
RNaseIII
Dicer
siRNAs
~22nt
mRNA
RISC complex
RNase is ! Argonaut !
Experimental use of RNAi!
Specic RNAs can be targeted using siRNAs ! "(small interfering RNAs)! "--model organisms and mammalian cells!!!!! ! " "Very POWERFUL tool.!
Ex. RNAi against Hst-2 leads to a delay in egg laying!
Wang et al. 2005
Ex. RNAi against survivin decreases tumors in mice!
siRNAs can be used to knockdown gene expression! These are introduced by the experimentalist!
What is the biological function of RNAi!
1. Protection against double-stranded RNA viruses!
2. Transposon silencing--prevent chromosomal ! translocations. !
Nobel Prize Medicine 2006!
Andrew Fire! Stanford U. ! USA !
Craig Mello! U Mass Worcester! USA!
Taken from the Nobel web site!
Regulation of mRNA expression by small RNAs! 1. RNA interference (RNAi)! [Link] (miRNA)!
The discovery of microRNA genes! Genetic Analysis of Development!
Functional complementation of a developmental! timing defect identied the gene lin-4! Cloning and analysis of WT lin-4! showed that no protein product was! generated, only a 21-ntd. RNA! Subsequent analysis showed that! that lin-4 hybridized to the 3UTR! of another RNA lin-14.!
lin-4 is the RNA! lin-14 is the target!
The 21nt lin-4 RNA regulates the lin-14 mRNA ! via complementary sites in its 3UTR!
3UTR
lin-4
lin-4
lin-4
lin-4
lin-4
lin-4
lin-4
lin-14
A
A A
5C
AU
3
CUCAUGCUCUCAGGAAC
GAGUGUGAGAGUCCUUG
3U
5
AA
C
C C
UC
UUCUACCUCAGGGAAC
GAGGUGGAGUCCCUUG
3U
5
U
AA
A C
C A
C
Gene expression regulated by a tiny RNA!
transcription
GENE
The RNA! and! its Target!
tiny RNA
Cell, 1993
miRNA REGULATION
Pri- miRNA
RNaseIII
RNaseIII
RNA INTERFERENCE
double-stranded RNAs
Drosha
Pre- miRNA
RNaseIII
RNaseIII
miRNA
~22nt
Dicer
RISC complex
RNaseIII
RNaseIII
Dicer
siRNAs
~22nt
OR
RISC complex
mRNA
RISC complex
Difference between miRNA and siRNA ! target recognition!
Conservation of the let-7 miRNA!
5ccggUGAGGUAGUAGGUUGUAUAGUuugga3
5aaauUGAGGUAGUAGGUUGUAUAGUaguaa3
5gggaUGAGGUAGUAGGUUGUAUAGUuuuag3
5gggaUGAGGUAGUAGGUUGUAUAGUuuuag3
5gggaUGAGGUAGUAGGUUGUAUAGUuuuag3
5agguUGAGGUAGUAGGUUGUAUAGUuuaga3
5ggggUGAGGUAGUAGGUUGUAUAGUuuggg3
(Pasquinelli et al., 2000)
lin-4 RNA
C. elegans
brain
heart
kidney
liver
lung
trachea
bone marrow
let-7 RNA
5S rRNA
Homo sapiens
colon
small intestine
spleen
stomach
thymus
let-7 RNA is expressed in human tissues!
Downregulation of let-7 in cancer cells
Healthy differentiated tissue
let-7
Cancerous tissue
let-7
MicroRNAs (miRNAs)!
A. thaliana/
O. sativa
C. elegans
D. melanogaster
H. sapiens/
M. musculus
>80 miRNAs
>100 miRNAs
>100 miRNAs
>200 miRNAs
See the RNA Family database - [Link]
A microRNA controlling left/right neuronal asymmetry
in C. elegans (Johnston & Hobert, 2003)
The Drosophila microRNA miR-14 suppresses cell death
and is required for normal fat metabolism (Xu et al., 2003)
Control of leaf morphogenesis by
miRNAs (Palatnik et al., 2003)
MicroRNAs regulate hematopoietic lineage
differentiation (Chen et al., 2004)
Summary of miRNAs!
From genes transcribed by RNA polymerase II! Stem-loop precursor miRNA ! Processing by drosha, dicer, RISC ! Hybridize to 3 UTR! Imperfect base-pairing to target mRNA! Inhibit translation and mediate RNA degradation!
Genomics!
Genome-wide analysis of gene structure and expression:! evolutionary relationships between species! the presence of functional elements in the genome! functions of particular proteins with identiable motifs! relationships between proteins from different species! global patterns of gene expression!
Once you have the sequence of DNA of interest!
How to compare nucleotide or ! protein sequencesBLAST!
http:/ /[Link]/BLAST/!
blastp = protein query compared to protein databases! blastn = nucleotide query compared to other nucleotide databases !
Blast result: Human NF1 sequence similar ! to the yeast protein Ira!
IDENTICAL amino acids!
SIMILAR amino acids!
Yeast Ira is a GTPase accelerating protein (GAP) that ! regulates the Ras protein to control cell division.!
Lodish Fig. 6-25
What yeast can teach us about a human disease gene
Yeast Ira
-GTPase modulating protein (GAP) that regulates Ras activity -Ras is an oncogene -Ras and GAP modulate cell growth
Neurofibromatosis 1
-Genetic disease associated with mutations in the NF1 gene -NF1 regulates the protein Ras -Mutant NF1 misregulates Ras -Tumors develop from the peripheral nervous system
Joseph Merrick aka The Elephantman (1862-1890) Thought to have had NF1 mutation
Evolutionary conservation of core processes!
Based on protein similarity, about 1500 common! genes between humans, ies, and worms!
>20%! of genes!
Wow!!
Analyzing gene expression! on a LARGE scale!
DNA Microarrays: The analysis of! thousands of genes expressed simultaneously!
We have discussed ways to look at expression of a single gene! (under different conditions):! e.g. Northern blot!
What if we want to look at the expression of MANY genes in ! response to a change of conditions?! Microarray preparation:! 1.~1kb region of each gene is amplied by PCR, attached to the ! slide and denatured ~6000 spots in a 2x2 cm slide! 2. Chip method--DNA oligos (~20 nt) of specic sequence! are synthesized on the slide; several oligos/gene represented! 3. Some chips are commercially available!!
Then take total RNA from a sample and see what RNAs are ! highly expressed such that they anneal to the ChIP!
Remember: The position of ea. gene on the slide is known!
QUESTION:!
How does gene expression change when cells ! Are grown under different environmental conditions!
DNA microarray analysis of gene expression!
Reference! Experimental!
X! Y! Z!
X! Y! Z!
Small region of a microarray ! representing expression !
(using a uorescence scanner)!
Yellow indicates no difference! upon addition of serum!
Interpreting the results: Analyzing relationships! between genes!
Example: Human broblasts treated with serum!
500/8613 genes examined change substantially ! with prolonged treatment!! !
Interestingbut what is the relationship! between these different genes???!
Interpreting the results by clustering ! genes with similar expression proles !
Example: Human broblasts treated with serum!
- Each column is one gene analyzed over time! - 5 groups (A-E) of genes with related biological! functions show similar expression patterns !
INCREASED AFTER FEEDING! DECREASED AFTER FEEDING! NO SIGNIFICANT CHANGE!
Lodish Fig. 5-30
Cholesterol biosyn!
Cell cycle!
Wound healing!
Microarrays in medicine!
Gene expression in disease tissue! Diagnosis! Molecular basis of prognosis! (certain gene expression patterns! may correlate with a good or bad! prognosis)!
D. Jones U. of Utah!
Microarray analysis ! colon tumor vs. ! normal colon tissue!
How to study histone modication! Chromatin IP!
Question: Does a protein (e.g. Rap1 or Sp1 ) bind to a particular region of DNA ! in vivo?!
First, ! crosslink protein ! to DNA using ! formaldehyde!
These are the fragments! Of DNA that are associated! With a particular protein.!
ChIP-chip can be used to sh for in vivo association ! of a protein with DNA throughout the genome!
What if we want to nd where in the genome a protein binds in vivo ! under some set of conditions. !
Example: Where in the genome does SP1 bind upon serum ! Stimulation in vivo?!
Large scale sequencing!
