V3: RNAi scan of human cells

Cellular Programs

V3: RNAi scan of human cells

Zhang et al. Cell 139, 199-210 (2009)

WS 2010 – lecture 3

PhD Bristol 1985

Biological Sequence Analysis2

RNA world

short name full name function oligomerization

mRNA, rRNA, tRNA, you know them well ... Single-stranded

snRNA small nuclear RNA splicing and other functions

snoRNA small nucleolar RNA nucleotide modification of RNAs

Long ncRNA Long noncoding RNA various

miRNA MicroRNA gene regulation single-stranded

siRNA small interfering RNA gene regulation double-stranded



RNA structure

www.rcsb.org

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



snRNAs

www.wikipedia.org

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.



siRNAs

www.wikipedia.org

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.



siRNAs

www.wikipedia.org

The enzyme dicer trims

double stranded RNA, to

form small interfering RNA

or microRNA.

These processed RNAs are

incorporated into the RNA-

induced silencing complex

(RISC), which targets

messenger RNA to prevent

translation



miRNAs

www.wikipedia.org

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 stem-

loop structure called a pre-miRNA and finally into a functional miRNA.



miRNAs

www.wikipedia.org

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.


Cellular Programs

U2OS cells

answers.yahoo.com


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). ...

Cellular Programs

Cell transfection with Lipfectamine 2000 (Invitrogen)

Dalby et al. Methods, 33, 95-103 (2004)


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 trans-

forms.

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.

Cellular Programs

Gene integration into host cell DNA by lentivirus

www.wikipedia.org


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 non-dividing cells, so they are one of the most efficient methods of a gene delivery vector.

Cellular Programs

www.wikipedia.org


Cellular Programs

Bmal1-dLuc + Per2dLuc reporter cell lines

www.wikipedia.org


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

Cellular Programs

The expanded clock gene network (Fig. 5)

Zhang et al. Cell 139, 199-210 (2009)


Cellular Programs

NIH DAVID pathway analysis tool

Huang et al. Nature Protocols, 4, 44-57 (2008)


Cellular Programs

BioGPS



http://biogps.gnf.org/circadian/

http://biogps.gnf.org/circadian/

V3: RNAi scan of human cells

Documents

Transcript of V3: RNAi scan of human cells