Immunologie für Studierende der Biologie & Molekularmedizin. mirna und Immunität. Jürgen Wittmann (jwittman@molmed.uni-erlangen.

Transcription

1 Immunologie für Studierende der Biologie & Molekularmedizin Konzepte der Immunologie Erlangen SS 2008 mirna und Immunität Jürgen Wittmann Division of Molecular Immunology Department of Internal Medicine III Nikolaus-Fiebiger-Center University of Erlangen-Nürnberg Universitätsklinik um Erlangen What is RNA (ribonucleic acid)? Ribonucleotides (Ribose, phosphate and base) DNA RNA 1

2 RNAs have many functions mrna (messenger RNA) rrna (ribosomal RNA) trna (transfer RNA) snrna (small nuclear RNA splicing) grna (guide RNA RNA editing) ribozymes Telomerase RNA (telomere maintenance) genomic RNA (viruses) sirna (small interfering RNA RNA silencing) shrna (short hairpin RNA RNA silencing) rasirna (repeat-associated RNA function unknown) pirna (Piwi-interacting RNA control of transposons) mirna (microrna gene regulation) The pathway to RNAi Source: unknown 2

3 micrornas (mirnas) first discovered in 1993 by Victor Ambros in C. elegans: lin-4 RNA regulates LIN-14 protein levels, but not lin-14 mrna levels important for development and differentiation second mirna (let-7) discovered in 2000 Definition: A mirna is a ssrna of ~22 nucleotides in length, which is generated by the RNase-III-type enzymes Drosha and Dicer from an endogenous transcript that contains a local hairpin structure. originate as a primary RNA (pri-mirna) of several hundred base pairs most pri-mirnas contain cap and poly(a) tail and are transcribed by RNA Polymerase II; some mirnas seem to be transcribed by RNA Polymerase III Mechanism of mirnas mirna Pol II/III pri-mirna nucleus cytoplasm 3

4 Pri-miRNA processing 3 5 Drosha DGCR8 5 3 pri-mirna 5 3 processing pre-mirna Mechanism of mirnas mirna Pol II/III Drosha/ DGCR8 pri-mirna pre-mirna nucleus cytoplasm 4

5 Mechanism of mirnas mirna Pol II/III Drosha/ DGCR8 nucleus pri-mirna pre-mirna Exportin 5 cytoplasm NPC Mechanism of mirnas mirna Pol II/III Drosha/ DGCR8 nucleus pri-mirna pre-mirna Exportin 5 cytoplasm NPC Dicer mirna 5

6 RNase-III-type nucleases involved in RNAi humans possess two members of the RNase-III family, Dicer and Drosha Dicer family proteins are ATP-dependent nucleases cleave dsrna leaving 2 bp 3 overhangs Dicer homologs exist in many organisms including C. elegans, Drosophila, yeast and humans HELICASE PAZ RIIIa RIIIb D Dicer (1922 aa) PRO-RICH RS-RICH RIIIa RIIIb D Drosha (1374 aa) RIII D bacterial RNaseIII (226aa) Role of murine Dicer in hematopoiesis Loss of Dicer in S. pombe: defective chromosome segregation, loss of silencing of centromeric repeats Deletion in M. musculus: embryonically lethal E7.5 embryos T cells: Lck-cre (DN T cells): Cd4-cre (DP T cells): FoxP3-cre (Treg cells): B cells: Mb1-cre (Early B cells): Bernstein et al., 2003 Nat. Genet. 35: Thymocytes reduced, Treg cells lost Peripheral T cells decreased, autoimmune disease Uncontrolled autoimmunity Block of Pro-B to Pre-B cell transition 6

7 Mechanism of mirnas mirna Pol II/III Drosha/ DGCR8 nucleus pri-mirna pre-mirna Exportin 5 cytoplasm NPC Dicer mirna leading strand passenger strand RISC Which is the right strand for incorporation into RISC? Problem: Both strands of a mirna could be used, but only one can be incorporated into RISC and thus be biologically active. Solution: Strand selection generally follows the rule that the strand with the less tightly paired 5 end is preferentially loaded into RISC. mmu-mir-155 mmu-mir-143 mmu-mir-142 7

8 RISC effector complex RISC is a large (~500-kDa) RNA-multiprotein complex components of the RNAi machinery specifically recognize the sirna or mirna duplex and incorporate a single strand into RISC contains a member of the Argonaute protein family minimal RISC: Argonaute protein and mirna WT ago1 T argonaute squid RISC mechanism of action RISC contains a member of the Argonaute protein family: S. pombe: 1 Argonaute H. sapiens: 8 Argonautes A. thaliana: 10 Argonautes C. elegans: 27 Argonautes Important domains: Piwi Argonaute Zwille (PAZ) - responsible for binding to the 3 end of the mirnas PIWI - domain with RNase H homology, slicer activity crystal structure of Pyrococcus furiosus Argonaute protein solved Song et al., 2004 Science 305:

9 Role of murine Ago proteins in hematopoiesis M. musculus contains 8 Argonaute genes: MIWI family (4 members) Ago family (4 members) mago1 KO: mice viable and fertile, no obvious hematopoietic defects mago3 KO: mice viable and fertile, no obvious hematopoietic defects mago4 KO: not reported (yet) mago2 KO: mice die before birth! Phenotype of mago2 knock out Ago2 is a key regulator of erythroid development: splenomegaly and anemia Ago2 is a key regulator of B-lymphoid development: pro-b cells OK, affects pre-b cells differentiation and peripheral B cells pro-b (B220 lo CD43 + IgM ) pre-b (B220 lo CD43 IgM ) O Carroll et al., 2007 Genes Dev. 21: Retroviral rescue experiments revealed that the Slicer endonuclease activity is dispensable for hematopoietic development as well as for mirna biogenesis 9

10 Mechanism of mirnas mirna Pol II/III Drosha/ DGCR8 nucleus pri-mirna pre-mirna Exportin 5 cytoplasm NPC RISC 3 UTR Ribosome mrna RISC AAA 3 UTR RISC 3 UTR Cap mrna mrna C B Dicer leading strand RISC mirna passenger strand RISC A mrna Where does the action take place? P bodies: specific cytoplasmic foci, sites of mrna decapping and degradation 35 proteins known, conserved from yeast to man Also in p bodies: concentration of Argonaute proteins mrna targets of mirnas Presumable Function: degradation of mrnas storage of mrnas Flag-Dcp1a Myc-Ago2 Merge Liu et al., 2005 NatCellBiol 7:

11 Target Prediction is often based on mirna mirna seed mrna seed match perfect pairing of mirna-mrna seed match seed match conservation across different species number of predicted sites for the same mirna on a certain 3 UTR mirna-target pair free energy outside the seed target site accessibility mirna-target pair secondary structure Target Prediction Algorithms Lewis et al., John et al., bibiserv.techfak.uni-bielefeld.de/rnahybrid Rehmsmeier et al., pictar.bio.nyu.edu/ Krek et al., mirna.imbb.forth.gr/microinspector/ Rusinov et al., diana.pcbi.upenn.edu/cgi-bin/micro_t.cgi Kiriakidou et al., microrna.sanger.ac.uk/index.shtmlindex.shtml Griffith-Jones et al.,

12 mirnas play important roles in all aspects of life Brain development (mir-430) Patterning of nervous system (mir-273) Pancreatic islet-cell development (mir-375) Adipocyte differentiation (mir-143) Limb patterning (mir-196) Heart development (mir-1) Programmed cell death (mir-14) Many viruses encode their own mirna genes: Epstein Barr virus, Herpes Simplex Virus 1, Human cytomegalovirus, Human immunodeficiency virus 1, Kaposi sarcoma-associated herpesvirus, Mareks disease virus, Mareks disease virus type 2, Mouse cytomegalovirus, Mouse gammaherpesvirus 68, Rhesus lymphocryptovirus, Rhesus monkey rhadinovirus, Simian virus 40 The number of mirnas increases steadily mirnas are registered at: Homo sapiens: 678 mirnas Mus musculus: 472 mirnas Sanger release Date 12/02 01/03 04/03 05/03 07/03 07/03 09/03 11/03 01/04 04/04 07/04 09/04 12/04 04/05 06/05 10/05 02/06 05/06 07/06 10/06 02/07 05/07 08/07 12/07 04/08 known mirnas

13 Genomic organization of mirnas pri-mir-23a-27a Cap (A) n pri-mir-21 5 Cap (A) n pri-mir-155 in bic ncrna 5 Cap (A) n pri-mir-26b 5 Cap (A) n pri-mir Cap (A) n adapted from VN Kim, 2005 Nat Rev Mol Cell Biol. 6: Detection of mirnas Cloning of mirnas real time RT-PCR analysis Northern blot analysis In situ hybridization mirna microarrays Plasterk, 2006 Cell 124:

14 Cloning of mirnas total RNA extraction small RNAs selection reverse transcription blast search sequence small RNAs cloning (cdna library) stem loop formation 1. Pro-B cells 2. Pre-B cells 3. Immature B cells 4. Marginal B cells 5. Follicular B cells 6. T1 B cells 7. B1 B cells 8. Plasma B cell line Reporter assay for mrna target validation HEK293 cells transiently transfected with: Expression plasmids for: empty vector (control), mmu-mirna-201 (control), mmu-mir (experiment) Renilla luciferase reporter construct: RPS6KA5 3 UTR binding site (experiment) Firefly luciferase normalization plasmid no mirna Renilla Luc Test 3 UTR Expectations: 140 no repression 120 mir-201 Renilla Luc Test 3 UTR cannot bind: no repression % Luciferase activity Luciferase 20 0 mir Renilla Luc Test 3 UTR can bind: repression no mirna mir-201 mir

15 Test of RPS6KA5 mrna for mirna regulation in Luciferase assay no mirna mmu-mirna-201 mmu-mirna mmu-mirna % Luciferase activity E E1378 E E1363 E E2024 E E mirnas in the immune response Lindsay, 2008 TreImm 7:

16 mirnas in Hematopoiesis mirnas in the adaptive immune response mir-181a: Expressed in thymus-derived B cells, regulation of B cell differentiation mir-17~92: Control of Pro- to Pre-B cell transition and apoptosis mir-155: Downregulates PU.1, leading to lower IgG1 levels 16

17 mirnas in the adaptive immune response mir-181a: Inconclusive role during T cell development, positive regulation of TCR-mediated response to antigen in CD4 + T cells mir-17~92: Control of Pro- to Pre-T cell transition and apoptosis mir-155: Influences differentiation decision of Th1/Th2 cells and cytokine production mirnas in the innate immune response mir-146a: Upregulated by LPS through TLRs mir-155: Upregulated by several external stimuli, regulated by proinflammatory transcription factors AP-1 and NF-κB 17

18 mirnas in the innate immune response mir-223: Regulator of neutrophil proliferation and activation, negative regulation of maturation, but not differentiation of granulocytes mir-17~92: Involved in monocyte differentiation and maturation by targeting the transcription factor AML-1 Link of mirnas and cancer mir-15a and mir16-1 negatively regulate BCL-2 Cimmino et al., 2005 PNAS 102: mir-143 and mir-145 are significantly reduced in colorectal tumors and others Michael et al., 2003 Mol. Cancer Res. 1: mir-21 is upregulated in glioblastoma Chan et al., 2005 Cancer Res. 65: mir-15a and mir-16-1 are deleted in more than 65% of B cell chronic lymphocytic leukemia (CLL) and many other tumors Calin et al., 2002 PNAS 99: mirnas regulate e.g. c-myc and ras Johnson et al., 2005 Cell 120: mir-155 is upregulated in Burkitt and Hodgkin lymphoma and acts as an oncogene in cooperation with c-myc Metzler et al., 2004 Genes Chromosomes Cancer 39: Transgenic expression of mir-155 results in pre-b leukemias in the mouse Costinean et al., 2005 PNAS 103: mir-17~92 and c-myc overexpression lead to B cell lymphomas He et al., 2005 Nature 435:

19 Function of mirnas as oncogenes or tumor suppressors Proliferation Invasion Angiogenesis Cell death Oncomirs Caldas and Brenton, 2006 Nat. Med. 11: The mir-17~92 cluster Three related clusters Knock out: lethal viable, no phenotype viable, no phenotype Ventura et al., 2008 Cell 132: KO mice die of lung and heart defects 19

20 mir-17~92 KO mice have a block in early B cell development Ventura et al., 2008 Cell 132: apoptosis B cell phenotype: Block from Pro- to Pre-B cell transition WT: inhibition of cell death in Pre-B cells through downregulation of the proapototic protein Bim KO: no downregulation of Bim possible Transcription Factor P Ribosome Bim mir-17~92 cluster RISC 3 UTR rasirnas tasirnas nat-sirnas pirnas rasirnas: repeat-associated sirnas rasirnas corresponding to repetitive elements repress the repeat sequences at the transcriptional or post-transcriptional level and maintain a centromeric heterochromatic structure; found in lower eukaryotes, C. elegans, D. melanogaster, plants and recently in mouse oocytes tasirnas: trans-acting sirnas processed from dsrnas synthesized by an endogenous RNA-dependent RNA polymerase (RdRp) utilizing endogenous RNAs as template, found only in plants so far nat-sirnas: natural antisense sirnas processed from dsrna formed between endogenous sense and antisense transcripts, found only in plants so far pirnas: Piwi-interacting RNAs a novel class of nt small RNAs in the mammalian testes and somatic cells; pirnas are associated with Miwi and depend on Miwi for their biogenesis and/or stability, pirnas have been linked to transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells; 81% of pirna clones are derived from only 42 genomic regions 20

21 Our interest in mirnas µ fct? L? Stem cell Early pro-b Late pro-b Early pre-b Late pre-b Immature B cell Mature B cell D J H V H DJ H V L J L Are there mirnas which are controlled by pre-bcr signals? What is the effect of those mirnas on B cell differentiation? Which mirnas are induced upon antigen activation? Which mrnas are controlled by the regulated mirnas? Do the identified mirnas play a role during lymphomagenesis? 21