V3: RNAi scan of human cells
PhD Bristol 1985
Zhang et al. Cell 139, 199-210 (2009)
WS 2010 – lecture 3
Cellular Programs
RNA world
short name
full name
mRNA, rRNA, tRNA,
function
you know them well ...
oligomerization
Single-stranded
snRNA
snoRNA
small nuclear RNA
small nucleolar RNA
splicing and other functions
nucleotide modification of RNAs
Long ncRNA
Long noncoding RNA
various
miRNA
MicroRNA
gene regulation
single-stranded
siRNA
small interfering RNA
gene regulation
double-stranded
WS 2010 – lecture 3
Biological Sequence Analysis
2
RNA structure
Also single stranded RNA molecules frequently adopt a specific tertiary structure.
The scaffold for this structure is provided by secondary structural elements which are
H-bonds within the molecule.
This leads to several recognizable "domains" of secondary structure like hairpin
loops, bulges and internal loops.
RNA hairpin 2RLU
Stem loop 1NZ1
www.rcsb.org
WS 2010 – lecture 3
Biological Sequence Analysis
3
snRNAs
Small nuclear RNA (snRNA) are found within the nucleus of eukaryotic cells.
They are transcribed by RNA polymerase II or RNA polymerase III and are
involved in a variety of important processes such as
- RNA splicing,
- regulation of transcription factors or RNA polymerase II, and
- maintaining the telomeres.
They are always associated with specific proteins. The complexes are referred to
as small nuclear ribonucleoproteins (snRNP) or sometimes as snurps.
A large group of snRNAs are known as small nucleolar RNAs (snoRNAs).
These are small RNA molecules that play an essential role in RNA biogenesis and
guide chemical modifications of rRNAs, tRNAs and snRNAs.
They are located in the nucleolus and the cajal bodies of eukaryotic cells.
www.wikipedia.org
WS 2010 – lecture 3
Biological Sequence Analysis
4
siRNAs
Small interfering RNA (siRNA), sometimes known as short interfering RNA or
silencing RNA, is a class of
- double-stranded RNA molecules,
- that are 20-25 nucleotides in length (often precisely 21 nt) and play a variety of
roles in biology.
Most notably, siRNA is involved in the RNA interference (RNAi) pathway, where it
interferes with the expression of a specific gene.
In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related
pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of
a genome.
www.wikipedia.org
WS 2010 – lecture 3
Biological Sequence Analysis
5
siRNAs
The enzyme dicer trims
double stranded RNA, to
form small interfering RNA
or microRNA.
These processed RNAs are
incorporated into the RNAinduced silencing complex
(RISC), which targets
messenger RNA to prevent
translation
www.wikipedia.org
WS 2010 – lecture 3
Biological Sequence Analysis
6
miRNAs
RNA interference may involve siRNAs or miRNAs.
Nobel prize in Physiology or Medicine 2006 to
for their on RNAi in C. elegans.
Andrew Fire
Craig Mello
microRNAs (miRNA) are single-stranded RNA molecules of 21-23 nucleotides in
length, which regulate gene expression.
miRNAs are encoded by DNA but not translated into protein (non-coding RNA).
Instead, each primary transcript (a pri-miRNA) is processed into a short stemloop structure called a pre-miRNA and finally into a functional miRNA.
www.wikipedia.org
WS 2010 – lecture 3
Biological Sequence Analysis
7
miRNAs
Mature miRNA molecules are partially complementary to one or more messenger
RNA (mRNA) molecules.
Their main function is to down-regulate gene expression of this mRNA.
miRNAs, especially those in animals, typically have incomplete base pairing to a
target and inhibit the translation of many different mRNAs with similar sequences.
In contrast, siRNAs typically base-pair perfectly and induce mRNA cleavage only
in a single, specific target.
www.wikipedia.org
WS 2010 – lecture 3
Biological Sequence Analysis
8
U2OS cells
U2OS is a human osteosarcoma cell line that was cultivated from the bone tissue of
a fifteen-year-old human female suffering from osteosarcoma.
Established in 1964, the original cells were taken from a moderately differentiated
sarcoma of the tibia (dt. Schienenbein).
...
answers.yahoo.com
WS 2010 – lecture 3
Cellular Programs
Cell transfection with Lipfectamine 2000 (Invitrogen)
For successful transfection, a nucleic acid, which carries a net negative charge under normal
physiological conditions, must come into contact with a cell membrane that also carries a net
negative charge.
Lipofectamine 2000 is a cationic liposome formulation that functions
by complexing with nucleic acid molecules, allowing them to overcome
the electrostatic repulsion of the cell membrane and to be taken up by the cell.
While there is a great deal of structural variation in lipid molecules that are able to mediate
transfection, all have a number of common features:
(1) ... a positively charged head group (one or more positively charged nitrogen atoms) to
allow interaction between the transfection reagent and the negatively charged sugar–
phosphate backbone of a nucleic acid molecule.
(2) A spacer usually links the charged head group to 1-3 hydrocarbon chains ... that are often
14 or more carbon atoms in length and may contain unsaturated carbons and cis- or transforms.
Dalby et al. Methods, 33, 95-103 (2004)
WS 2010 – lecture 3
Cellular Programs
Cell transfection with Lipfectamine 2000 (Invitrogen)
The cationic lipid molecule is often formulated with a neutral co-lipid (helper lipid).
The positive charge on the surface of the liposome generates an
electrostatic interaction with nucleic acids and facilitates contact
with the negatively charged cell membrane.
The neutral co-lipid mediates fusion of the liposome with the
cell membrane effecting entry of the nucleic acid.
To achieve expression of the transgene, DNA must reach the nucleus of the cell and become
accessible to the transcriptional machinery.
In actively dividing cells, transfected DNA may simply become trapped in the nucleus
following the reassembly of the nuclear envelope at the end of mitosis.
However, Lipofectamine 2000 efficiently transfected post-mitotic neurons and rat primary
hepatocytes as well, suggesting that Lipofectamine 2000 may promote penetration of DNA
through intact nuclear envelopes in these cell types.
Dalby et al. Methods, 33, 95-103 (2004)
WS 2010 – lecture 3
Cellular Programs
Gene integration into host cell DNA by lentivirus
Lentivirus (lenti-, Latin for "slow") is a genus of viruses of the Retroviridae family,
characterized by a long incubation period.
HIV, SIV, and FIV are all examples of lentiviruses.
Lentiviruses can deliver a significant amount of genetic information into the DNA of
the host cell.
They have the unique ability among retroviruses of being able to replicate in nondividing cells, so they are one of the most efficient methods of a gene delivery
vector.
www.wikipedia.org
WS 2010 – lecture 3
Cellular Programs
www.wikipedia.org
WS 2010 – lecture 3
Cellular Programs
Bmal1-dLuc + Per2dLuc reporter cell lines
In luminescent reactions, light is produced by
the oxidation of a luciferin (a pigment):
luciferin + O2 → oxyluciferin + light
The most common luminescent reactions
release CO2 as a product.
The rates of this reaction between luciferin and
oxygen are extremely slow until they are
catalyzed by luciferase, sometimes mediated
by the presence of cofactors such as calcium
ions or ATP.
Firefly luciferase
The reaction catalyzed by firefly luciferase takes place in 2 steps:
luciferin + ATP → luciferyl adenylate + Ppi
luciferyl adenylate + O2 → oxyluciferin + AMP + light
www.wikipedia.org
WS 2010 – lecture 3
Cellular Programs
The expanded clock gene network (Fig. 5)
Zhang et al. Cell 139, 199-210 (2009)
WS 2010 – lecture 3
Cellular Programs
NIH DAVID pathway analysis tool
Huang et al. Nature Protocols, 4, 44-57 (2008)
WS 2010 – lecture 3
Cellular Programs
NIH DAVID pathway analysis tool
Huang et al. Nature Protocols, 4, 44-57 (2008)
WS 2010 – lecture 3
Cellular Programs
NIH DAVID pathway analysis tool
Huang et al. Nature Protocols, 4, 44-57 (2008)
WS 2010 – lecture 3
Cellular Programs
NIH DAVID pathway analysis tool
Huang et al. Nature Protocols, 4, 44-57 (2008)
WS 2010 – lecture 3
Cellular Programs
NIH DAVID pathway analysis tool
Huang et al. Nature Protocols, 4, 44-57 (2008)
WS 2010 – lecture 3
Cellular Programs
NIH DAVID pathway analysis tool
Huang et al. Nature Protocols, 4, 44-57 (2008)
WS 2010 – lecture 3
Cellular Programs
NIH DAVID pathway analysis tool
Huang et al. Nature Protocols, 4, 44-57 (2008)
WS 2010 – lecture 3
Cellular Programs
BioGPS
http://biogps.gnf.org/circadian/
Dalby et al. Methods, 33, 95-103 (2004)
WS 2010 – lecture 3
Cellular Programs