Presentation on theme: "Cellular Programs V3: RNAi scan of human cells Zhang et al. Cell 139, 199-210 (2009) WS 2010 – lecture 3 PhD Bristol 1985."— Presentation transcript:
Cellular Programs V3: RNAi scan of human cells Zhang et al. Cell 139, (2009) WS 2010 – lecture 3 PhD Bristol 1985
Biological Sequence Analysis 2 RNA world short namefull namefunctionoligomerization mRNA, rRNA, tRNA, you know them well...Single-stranded snRNAsmall nuclear RNAsplicing and other functions snoRNAsmall nucleolar RNAnucleotide modification of RNAs Long ncRNALong noncoding RNAvarious miRNAMicroRNAgene regulationsingle-stranded siRNAsmall interfering RNAgene regulationdouble-stranded WS 2010 – lecture 3
Biological Sequence Analysis 3 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 2RLUStem loop 1NZ1 WS 2010 – lecture 3
Biological Sequence Analysis 4 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. WS 2010 – lecture 3
Biological Sequence Analysis 5 siRNAs Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of - double-stranded RNA molecules, - that are 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. WS 2010 – lecture 3
Biological Sequence Analysis 6 siRNAs 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 WS 2010 – lecture 3
Biological Sequence Analysis 7 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 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. WS 2010 – lecture 3
Biological Sequence Analysis 8 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. WS 2010 – lecture 3
Cellular Programs U2OS cells answers.yahoo.com WS 2010 – lecture 3 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, (2004) WS 2010 – lecture 3 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) Dalby et al. Methods, 33, (2004) WS 2010 – lecture 3 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 WS 2010 – lecture 3 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 WS 2010 – lecture 3
Cellular Programs Bmal1-dLuc + Per2dLuc reporter cell lines WS 2010 – lecture 3 In luminescent reactions, light is produced by the oxidation of a luciferin (a pigment): luciferin + O 2 → oxyluciferin + light The most common luminescent reactions release CO 2 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 + Pp i luciferyl adenylate + O 2 → oxyluciferin + AMP + light