Functional and Biomedical Aspects of Genome Research

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1 Functional and Biomedical Aspects of Genome Research Vorlesung SS 04 Bartsch, Jockusch & Schmitt-John Mi. 9:15-10:00, in W Functional RNAs Thomas Schmitt-John micro RNAs small interfering RNAs RNA interference

2 Non protein-coding RNAs rrnas (ribosomal RNAs): Function of Ribosomes trnas (transfer RNAs): AA transfer for protein synthesis Ribozymes: RNAs with enzymatic function mirnas (micro RNAs): Putative translational regulatory RNAs strnas (small temporally regulated RNAs): let-7 and lin-4 heterochromatic mirnas (control developmental programs). sirnas (small interfering RNAs): Generated out of cleavage of long dsrnas; antiviral, defense mechanism snrnas (small nulear RNAs): Splicing of pre-mrnas snornas (small nucloelar RNAs): modification of other RNAs.

3 Ribozymes ribonucleic acid with catalytic characteristics (cleavage of other ribonucleic acids); RNase P hammerhead ribozyme

4 snrnas snrnas are incorperated in multi protein complexes (snrnp) and play a crucial role in splicing of primary mrna transcripts MPI Göttingen

5 snornas snornas are also incorperated in multi protein complexes (snornp); most frequently located in the nucleolus; two functional classes of snorna C/D box containin snornps are associated with methylation of mrna (stability), snrna (structure), DNA (imprinting) Hüttenhofer et al HACA box containin snornps are associated to pseudourilation of e.g. trna

6 strnas

7 mirnas

8 mirna biogenesis mirna genes are typically found in intergenic areas but can also be found in sense or antisense orientation within introns of known genes; some mirnas are cluster and transcribed as polycystronic genes the RNA polymerase(s) or regulatory elements that control mirna transcription are unknown the primary transcript of mirnas are named pri-mirnas pri-mirna is processed to pre-mirna by DROSHA; pre-mirnas always occur as hairpin intermediates (60 80 nt) Nelson et al export of pre-mirna (but not pri-mirna) is mediated by Exportin 5 which is Ran-GTP dependent and recognizes the hairpin structure; sequence-motif independent procedure

9 mirna biogenesis Dicer and mirnp in the cytosol pre-mirnas are processed by DICER to form the mature mirna the mirna is located in a Protein complex called mirnp or RISC (RNAinduced silencing complex) mirnps regulate translation of target mrnas and RISC induces degradation

10 mirnas and sirnas (small interfering RNAs) recently discoverd new class of endogenous mirna based posttranscriptional gene regulation was awarded by Science in 2002 as the Breakthrough of the Year nt mirna CAP pa CAP pa endonucleolytic cleavage inhibit translation CAP degradation pa imperfect binding of mirnas to specific sites in the 3 UTR of the target mrna leads to repression of the translation perfect binding of sirnas to the coding region of the target mrna leads to gene silencing by degradation of the mrna

11 mirnas and sirnas (small interfering RNAs) imperfect binding of mirnas to specific sites in the 3 UTR of the target mrna leads to gene silencing by repression of translation perfect binding of sirnas to the coding region of the target mrna leads to gene silencing by degradation of the mrna Nelson et al What is the origin of mirnas and sirnas and what do they have in common?

12 mirna sirna mirnp incorporated in RISC imperfect binding to 3 UTR of the target leads to inhibition of translaition Function? Gene Regulation perfect binding to coding region leads to cleavage of the mrna at the RNA: RNA hybrid location Function? Virus Defence mirnp: ribonucleoprotein complex containing as known an Argonaute protein (eif2c2), Gemin 3 (helicase), Gemin 4 RISC: RNA-induced silencing complex, multiprotein complex also containing the Argonaute proteins eif2c1 and eif2c2, further proteins like a multisubunit nuclease

13 up to now ~900 mirnas have been found in different species 207 mirnas 147 intergenic 55 intronic (+) 9 intronic (-) 32 cluster 2miRNA-cluster 21 3miRNA-cluster 9 6miRNA-cluster 2

14 mirna cel-lin-4 cel-lin-7 cel-lin-6 Dme-batam Dme-mir2 Dme-mir-7 Hsa-181 mirna targets Target lin-14 lin 28 lin 41 hid pro-apopt. Reaper/grim/sickle E(spl);BRD,hairy? Function of Target larval developmental timing larval developmental timing neural developmental timing asymmetry chemor. expr. apoptose notch signaling pathway hematopoetic differentiation Hsa-mir-15/16/142/155 Ath-165/166 Ath-172 Ath-JAW Ath-159? Phabulosa/Phavoluta APATALA2 TCP MYB33 oncogenesis meristem init. and leaf morphogenese flower development leaf morphogenese leaf morphogenese

15 Validation of predicted of mirna targets predicted mirna target site Luciferase constitutive promotor 3 UTR Luciferase Luciferin Luciferase

16 Biomedical aspects of the mirna system pre-mirna Dicer complex mirna mirna + mirnp genesilencing by inhibition of translation dsrna sirna duplex sirna duplex + RISC genesilencing by mrna degradation Dicer-KO in mice is lethal during early embryonic development Let7 and lin4 regulate the timing of C. elegans development Fragile X-Syndrome is caused by mutations in FMR1, the Drosophila homolog of which is a component of the RISC complex SMN1 (survival of motoneuron 1) mutated in SMA patients is associated with Gemin 3 and might be associated with the mirnp

17 RNA-interference (RNAi) Long double-stranded RNAs (dsrnas; typically >200 nt) can be used to silence the expression of target genes in a variety of organisms and cell types (e.g., worms, fruit flies, and plants). Upon introduction, the long dsrnas enter a cellular pathway that is commonly referred to as the RNA interference (RNAi) pathway. First, the dsrnas get processed into nucleotide (nt) small interfering RNAs (sirnas) by an RNase III-like enzyme called Dicer (initiation step). Then, the sirnas assemble into endoribonucleasecontaining complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process. The sirna strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step). Cleavage of cognate RNA takes place near the middle of the region bound by the sirna strand. In mammalian cells, introduction of long dsrna (>30 nt) initiates a potent antiviral response, exemplified by nonspecific inhibition of protein synthesis and RNA degradation. The mammalian antiviral response can be bypassed, however, by the introduction or expression of sirnas.

18 RNA-interference (RNAi) as used for functional Genomics in C. elegans and Drosophila PCR a cdna flanked by T7 and Sp6 Promoters in vitro transcription of both strands anneal transcripts to dsrna microinject dsrna into C. elegans embryos analyze the phenotype of the developing worm

19 sirna for functional gene analyis in mammalian cells Effects of GAPDH sirna on GAPDH mrna and Protein Levels. HeLa cells were plated at 200,000 cells/well into a 6 well culture plate. 24 hours later they were transfected with either a chemically synthesized sirna at a final concentration of 100 nm or with psilencer 2.0-GAPDH; both target human GAPDH. Samples were harvested 48 hours after transfection, and both RNA and protein were isolated using the PARIS Kit. mrna knockdown was evaluated by Northern blot using an antisense radiolabeled RNA probe transcribed from ptri-gapdh human (Ambion Cat #7430). For the Western blot, anti-gapdh antibody (Ambion Cat #4300) was used.

20 Vector based sirna insertion of chemically synthezised sirna-target sequence transfection of the expression vector in target cells transcription in situ by RNA pol III spontaneous folding of the transcript processing of the loop RNA by endogenous DICER and RISC revealing a knock-down of the target gene

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22 Biomedical perspectives sirna Functional analysis of the mammalian genome Analysis of disease genes Therapeutic knock-down of disease genes Antiviral therapy launched for HIV and Hepatitis Virus