42528 Bioinformatics and RNA Technologies. mirna overview / 04 / 08

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1 42528 Bioinformatics and RNA Technologies mirna overview / 04 / 08 Dr. Mathieu REDERSTORFF Division of Genomics and RNomics Innsbruck Biocenter, Medical University Innsbruck

2 Examples of guide RNAs: micrornas (mirnas) and small interfering RNAs (sirnas) nt in size found in most eukaryal organisms, including humans

3 mirnas: : the history mirnas were originally found as small RNAs that control development of C. elegans a nematode (worm) Lin-4, a small RNA with a size of 22 nt does not code for a protein, but functions on the level of RNA Lin-4 has antisense complementarity to multiple sites in the 3 -UTR of the lin-14 gene Lin-4 reduces the amount of lin-14 protein without noticeable change in amount of Lin-14 mrna Together these discoveries supported a model, in which the lin-4 RNA pairs to the lin UTR to specify translational repression of the lin-14 message as part of the regulatory pathway that triggers the transition from cell divisions of the first larval stage to the second.

4 mirnas: : the history Lin-4 is now recognized as the founding member of an abundant class of tiny regulatory RNAs called micrornas or mirnas The importance of mirna-directed gene-regulation is coming into focus as more and more mirnas and their regulatory targets and functions are discovered Recently discovered mirna functions include control of cell proliferation, cell death and fat metabolism in flies, neuronal patterning in nematodes, modification of hemapoietic lineage differentiation in mammals and control of leaf and flower development in plants Computational approaches for finding messages indicate that these examples represent a very small fraction of the total

5 mirnas: : Transcription The two candidate polymerases for pri-mirnas transcription are pol II and pol III. Pol II produces the mrnas and some non-coding RNAs, including the small nucleolar RNAs and four of the small nuclear RNAs (snrnas)) of the spliceosome. Pol III produces some of the shorter non- coding RNAs,, including trnas,, 5S ribosomal RNA and the U6 snrna The mirnas processed from the introns of protein-coding host genes are undoubtedly transcribed by pol II It is anticipated that many of the other mirnas are also Pol II products, even though some of the metazoan mirna genes do not have the classical signals for polyadenylation: : 1) the pri-mirnas can be quite long, more than 1 kb, 2) the presumed mirnas have often internal U-runs, which would be expected to prematurely terminate pol III transcription, 3) many mirnas are differentially expressed during development, as is observed for pol II but not for pol III products and 4) fusions that place the open reading frame of a reporter protein downstream of a mirna gene lead to robust reporter protein expression, suggesting that mirna primary transcripts are capped pol II transcripts

6 Classical Pol II transcripts: pri-mirna polycistronic monocistronic intronic

7 Pre-miRNAs In 2001, three labs cloning small RNAs from flies, worms, and human cells reported a total of over one hundred additional genes for tiny non-coding RNAs, approximately 20 new genes in D. melanogaster,, 30 new genes in humans and about 60 in worms (experimental RNomics: : procedure and strategy) C. elegans The RNA products of these genes resembled the lin-4 and let-7 strnas in that they were 22 nt endogenously expressed RNAs,, potentially processed from one arm of a stem loop structure Metazoan mirnas Plant mirnas

8 mirna biogenesis and function

9 mirna biogenesis and function: mirtrons

10 RISC targets mrnas to P-bodies

11 sirna VS mirna

12 Drosha Processes pri-mirna into pre-mirna Leaves 3 overhangs on pre-mirna Nuclear RNAse-III enzyme [Lee at al., 2003] Tandem RNAse-III domains How does it identify pri-mirna? Hairpin terminal loop size Stem structure Hairpin flanking sequences Not yet found in plants Maybe Dicer does its job?

13 Dicer Cleaves dsrna or pre-mirna Leaves 3 overhangs and 5 P Cytoplasmic RNAse-III enzyme Functional domains in Dicer [Bernstein et al., 2001] Putative helicase PAZ domain Tandem RNAse-III domains dsrna binding domain Multiple Dicer genes in Drosophila and plants [He and Hannon, 2004] Functional specificity?

14 Dicer

15 RNA Induced Silencing Complex (RISC) RNAi effector complex Critical for target mrna degradation or translation inhibition Not well characterized: 4 subunits? More? Activities associated with RISC Helicase Endonuclease and exonuclease Slicer (or is it Dicer?) homology seeking /RNA binding Preferentially incorporates one strand of unwound RNA [Khvorova et al., 2003] Antisense How does it know which is which?

16 RISC Preference for Antisense RNA Helps ensure specificity for target 5 stability of sirna and mirna duplex strands often different The strand with less 5 stability usually incorporated into RISC [Schwarz et al., 2003] Due to easier unwinding from one end? If strand stability is similar (rare), strands incorporated at similar frequency [He and Hannon, 2004]

17 Argonaute (Ago) Consistently co-purifies with RISC [Hammond et al., 2001] Homology seeking activity? Binds sirna and mirna [Ekwall, 2004] Distinguishes antisense strand [Novina and Sharp, 2004] Multiple Ago family proteins Different RISCs? Tissue specific? Developmentally regulated? Evidence for different RISCs [Tijsterman et al., 2004] Drosophila Dicer1 vs Dicer2/R2D2 Inhibition vs. degradation [Lee et al., 2004]

18 micro RNAs (mirnas) regulate gene expression about mirnas are thought to exist in the human genome regulating 1/3 of all protein genes (i. e genes)

19 mirnas inhibit translation of an mrna Inhibition of translation Exon 1 Exon 2 RISC mirna Watson-Crick base pairing At which level? Initiation, elongation?

20 Target recognition

21 Target recognition

22 Post-transcriptional control: different levels 5 UTR 3 UTR

23 Riboswitches

24 Post-transcriptional control: different levels 5 UTR 3 UTR

25 Selenocysteine incorporation AAA Nucleolin SBP2 SECIS?? L30 ARNm 3'UTR m7gppp AUG UGA Stop

26 Translation initiation: how does it work?

27 Examples of constructs to study the mode of repression

28 The different theories

29 Toward more functional studies of mirnas...

30 (Lagos-Quintana et al, 2002, Current Biology, 12: 1-20)

31 mirnas : tissue-specific expression in zebrafish Expression in: muscle blood vessels and the heart lateral-line system (a mechano-sensory system detecting water motion) kidney precursor

32 mirna-function: : regulatory roles The most pressing question to arise from the discovery of the hundreds of different mirnas is, what all these tiny non-coding RNAs are doing? For lin-4, let-7, and several other mirnas identified by forward genetics, crucial clues to their function and regulatory targets came even before their status as non-coding RNA genes was discovered. These and other mirnas that have reported functions:

33 mirnas and the immune system Regulation of the Germinal Center Response by MicroRNA-155 To-Ha Thai,1 Dinis Pedro Calado,1 Stefano Casola,2 K. Mark Ansel,1 Changchun Xiao,1 Yingzi Xue,3 Andrew Murphy,3 David Frendewey,3 David Valenzuela,3 Jeffery L. Kutok,4 Marc Schmidt-Supprian,1 Nikolaus Rajewsky,5 George Yancopoulos,3 Anjana Rao,1 Klaus Rajewsky1* Requirement of bic/microrna-155 for Normal Immune Function Antony Rodriguez,1* Elena Vigorito,2* Simon Clare,1 Madhuri V. Warren,1,3 Philippe Couttet,1 Dalya R. Soond,2 Stijn van Dongen,1 Russell J. Grocock,1 Partha P. Das,4 Eric A. Miska,4 David Vetrie,1 Klaus Okkenhaug,2 Anton J. Enright,1 Gordon Dougan,1 Martin Turner,2 Allan Bradley1 MicroRNAs are a class of small RNAs that are increasingly being recognized as important regulators of gene expression. Although hundreds of micrornas are present in the mammalian genome, genetic studies addressing their physiological roles are at an early stage. We have shown that mice deficient for bic/microrna-155 are immunodeficient and display increased lung airway remodeling. We demonstrate a requirement of bic/microrna-155 for the function of B and T lymphocytes and dendritic cells. Transcriptome analysis of bic/microrna-155 deficient CD4+ T cells identified a wide spectrum of microrna-155 regulated genes, including cytokines, chemokines, and transcription factors. Our work suggests that bic/microrna-155 plays a key role in the homeostasis and function of the immune system. A viral microrna functions as an orthologue of cellular mir-155 Eva Gottwein1, Neelanjan Mukherjee2, Christoph Sachse4, Corina Frenzel4, William H. Majoros5, Jen-Tsan A. Chi1,5, Ravi Braich7, Muthiah Manoharan7, Jürgen Soutschek7, Uwe Ohler3,5,6 & Bryan R. Cullen

34 mirnas and cancer Regulation of the p27kip1 tumor suppressor by mir-221and mir-222 promotes cancer cell proliferation Carlosle Sage1,5, Remco Nagel1,5, David A Egan2, Mariette Schrier1, Elly Mesman1, Annunziato Mangiola3, Corrado Anile3, Giulio Maira3, Neri Mercatelli4, Silvia Anna Ciafre`4, Maria Giulia Farace4 and Reuven Agami1,* Endogenous human micrornas that suppress breast cancer metastasis Sohail F. Tavazoie1,2, Claudio Alarco n1, Thordur Oskarsson1, David Padua1, Qiongqing Wang1, Paula D. Bos1, William L. Gerald3 & Joan Massague 1 A search for general regulators of cancer metastasis has yielded a set of micrornas for which expression is specifically lost as human breast cancer cells develop metastatic potential. Here we show that restoring the expression of these micrornas in malignant cells suppresses lung and bone metastasis by human cancer cells in vivo. Of these micrornas, mir-126 restoration reduces overall tumour growth and proliferation, whereas mir-335 inhibits metastatic cell invasion. mir-335 regulates a set of genes whose collective expression in a large cohort of human tumours is associated with risk of distal metastasis. mir-335 suppresses metastasis and migration through targeting of the progenitor cell transcription factor SOX4 and extracellular matrix component tenascin C. Expression of mir-126 and mir-335 is lost in the majority of primary breast tumours from patients who relapse, and the loss of expression of either microrna is associated with poor distal metastasis-free survival. mir-335 and mir-126 are thus identified as metastasis suppressor micrornas in human breast cancer.