Biological function of RNA interference (RNAi) pathways in the moss Physcomitrella patens (Hedw.) Bruch & Schimp. Inaugural-Dissertation zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau von Basel Khraiwesh aus Jinin Camp - Palästina Freiburg im Breisgau, 2009 Dekan: Prof. Dr. Ad Aertsen Promotionsvorsitzender: Prof. Dr. Eberhard Schäfer Betreuer: Prof. Dr. Ralf Reski, PD Dr. Wolfgang Frank Referent: Prof. Dr. Ralf Reski, PD Dr. Wolfgang Frank Koreferent: Prof. Dr. Wolfgang R. Hess Tag der Verkündigung des Ergebnisses: 24. April 2009 This work has been created in the Department of Plant Biotechnology Institute of Biology II Faculty of Biology Albert-Ludwigs University of Freiburg under the guidance of Prof. Dr. Ralf Reski and PD Dr. Wolfgang Frank To my marvelous mother and dear family To my wife and my lovely boys, For your support, understanding and always being there for me… Index Index List of contents I Publications and manuscripts related to this work II 1 Chapter Ι: Introduction and Overview……………………………….. 1 1.1 Background………………………………………………………………………… 1 1.1.1 RNA Interference: function and technology…………………………………………… 1 1.1.2 Small RNAs and gene silencing………………………………………………………… 2 1.1.2.1 MicroRNAs (miRNAs)…………………………………………………………………3 1.1.2.2 Trans-acting short interfering RNAs (ta-siRNA)…………………………………… 5 1.1.2.3 Repeat-associated RNAs (ra-siRNA)………………………………………………. 6 1.1.2.4 Natural antisense transcript-derived small interfering RNAs (nat-siRNA)……… 6 1.1.2.5 Piwi-associated RNAs (piRNAs)……………………………………………………. 7 1.1.2.6 Secondary transitive siRNA…………………………………………………………. 7 1.1.3 Dicer proteins……………………………………………………………………………... 9 1.1.4 Physcomitrella patens as a model system…………………………………………… 11 1.2 Results and Discussion………………………………………………………… 14 1.2.1 DICER-LIKE genes in Physcomitrella patens………………………………………...14 1.2.1.1 Generation and molecular analysis of ΔPpDCL1b knockout mutants…………. 16 1.2.1.1.1 Knockout of PpDCL1b causes developmental disorders…………………….. 17 1.2.1.1.2 MiRNA biogenesis is not affected and miRNA-directed cleavage of mRNA- targets is abolished in ΔPpDCL1b mutant lines………………………………..17 1.2.1.1.3 Generation of transitive siRNA in ΔPpDCL1b mutant lines…………………...18 1.2.1.1.4 Analysis of DNA methylation in ΔPpDCL1b mutants and wild type………….19 1.2.1.1.5 Analysis of the ta-siRNA pathway in ΔPpDCL1b mutants…………………….20 1.2.1.1.6 Analysis of ΔPpDCL1b mutants and wild type lines expressing amiR-GNT1…………………………………………………………………………21 1.2.1.1.6.1 Specific methylation of a miRNA1026 target gene in response to the phytohormone abscisic acid (ABA)………………………………………….. 21 1.2.1.1.7 Expression profiling of transcription factor genes in ΔPpDCL1b mutant lines…………………………………………………………………………………22 1.2.2 Highly specific gene silencing by artificial miRNAs in Physcomitrella patens……. 24 1.3 Conclusion………………………………………………………………………… 27 1.4 References………………………………………………………………………… 29 2 Chapter II: Manuscript 1……………………………………………..34 Transcriptional control of gene expression by microRNAs………………35 3 Chapter III: Publication 1…………………………………………..121 Specific gene silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted gene knockout……………………….122 4 Chapter IV: Appendices……………………………………………. 136 4.1 Flow cytometric measurements (FCM)……………………………………...136 4.2 Physcomitrella patens DCL1b (PpDCL1b) mRNA……………………….. 137 4.3 DNA vectors……………………………………………………………………... 140 4.4 Genes downregulated in ΔPpDCL1b mutants…………………………….. 141 4.5 Genes upregulated in ΔPpDCL1b mutants………………………………… 146 4.6 Acknowledgments……………………………………………………………... 152 4.7 Erklärung………………………………………………………………………....153 I Publications Publications and manuscripts related to this Work: Manuscript #1 - Khraiwesh, B., M. A. Arif, G. I. Seumel, S. Ossowski, D. Weigel, R. Reski, W. Frank. (2009): Transcriptional control of gene expression by microRNAs. Submitted. Publication #1 - Khraiwesh, B., S. Ossowski, D. Weigel, R. Reski, W. Frank (2008): Specific gene silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted gene knockouts. Plant Physiology, 148: 684–693. This work has been presented at the following conferences: Talks (presented by W. Frank) − Frank, W., Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R. (2007): Specific epigenetic control of microRNA target genes to compensate for RNAi dysfunctions in a Physcomitrella patens DICER-LIKE mutant. Botanical Congress, September 3-7, 2007, University of Hamburg, Germany. − Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R., Frank, W. (2007): Specific epigenetic control of microRNA target genes to compensate for RNAi dysfunctions in a Physcomitrella patens DICER-LIKE mutant. The Annual International Conference for Moss Experimental Research, August 2-5, 2007, Korea University, Seoul, Korea. Posters − Khraiwesh, B., Ossowski, S., Weigel, D., Reski, R., Frank, W. (2008): Specific gene silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted gene knockouts. Annual Meeting of the RNA Society, July 28-August 3, 2008, Free University Berlin, Germany. − Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R., Frank, W. (2007): Knockout of a DICER-LIKE gene causes silencing of microRNA targets in Physcomitrella patens. 5th Colmar Symposium: The New RNA Frontiers, November 8-9, 2007, Colmar, France. − Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R., Frank, W. (2007): Knockout of a DICER-LIKE gene causes silencing of microRNA targets in Physcomitrella patens. The Annual International Conference for Moss Experimental Research, August 2- 5, 2007, Korea University, Seoul, Korea. II Chapter I Background 1 Chapter Ι: Introduction and Overview 1.1 Background 1.1.1 RNA Interference: function and technology RNA interference (RNAi) is a mechanism regulating gene transcript levels by either transcriptional gene silencing (TGS) or by posttranscriptional gene silencing (PTGS), which acts in genome maintenance and the regulation of development (Hannon, 2002; Agrawal et al., 2003). Since the discovery of RNAi in Caenorhabditis elegans (Lee et al., 1993; Fire et al., 1998) extensive studies have been performed focusing on the different aspects of RNAi. In particular, the elucidation of the essential components of RNAi pathways has advanced extensively (Tomari and Zamore, 2005). RNAi has been discovered in a wide range of organisms from plants and fungi to insects and mammals suggesting that it arose early in the evolution of multicellular organisms (Sharp, 2001; Hannon, 2002). The RNAi pathway is typically initiated by ribonuclease III-like nuclease enzymes, called Dicer, that cleave double stranded RNA molecules (dsRNAs; typically >200 nt) into small fragments bearing a 3’ overhang of two nucleotides. One of these two strands is coupled to a second endonuclease enzyme called Argonaute (AGO) and then integrated into a large complex (RNA-induced silencing complex, RISC). Subsequently, it has been shown that RISC contains at least one member of the AGO protein family, which is likely to act as an endonuclease and cuts the mRNA. In Drosophila and humans, AGO2 has been identified as being responsible for this cleavage and the catalytic component of the RISC complex. It was proposed that small interfering RNA (siRNA) guide the cleavage of mRNA. SiRNAs are key to the RNAi process and they have complementary nucleotide sequences to the targeted RNA strand. In certain systems, in particular plants, worms and fungi, an RNA dependent RNA polymerase (RdRP) plays an important role in generating siRNA (Cogoni and Macino, 1999). Another outcome are epigenetic changes such as histone modification and DNA methylation (Matzke and Matzke, 2004; Schramke and Allshire, 2004) (Figure1). In medical research, RNAi is on the way to becoming an important tool to treat HIV, hepatitis C, and cancer (Hannon and Rossi, 2004) and in plants RNAi technology has been used to improve their nutritional value (Tang and Galili, 2004). For science in general it is already a tool of large scale reverse genetic approaches and aids in unravelling gene functions in many species. 1 Chapter I Background Figure 1: Overview of RNA interference (adapted from Matzke and Matzke, 2004). The Dicer enzymes produce siRNA from dsRNA and mature miRNA from hairpin-like miRNA precursor transcripts. MiRNA or siRNA is bound to an AGO enzyme and an effector complex is formed, either a RISC or RITS (RNA-induced transcriptional silencing) complex. RITS affects the rate of transcription by histone and DNA modifications whereas RISC cleaves mRNA or inhibits its translation. 1.1.2 Small RNAs and gene silencing Small non-coding RNAs (20-24 nucleotides in size) have been increasingly investigated and they are important regulators of PTGS in eukaryotes (Hamilton and Baulcombe, 1999; Mello and Conte, 2004; Baulcombe, 2005). They were first discovered in the nematode Caenorhabditis elegans (Lee et al., 1993) and are responsible for phenomena described as RNAi, co-suppression, gene silencing or quelling (Napoli et al., 1990; de Carvalho et al., 1992; Romano and Macino, 1992). Shortly after these reports were published, it was shown that PTGS in plants is correlated to small RNAs (Hamilton and Baulcombe, 1999). These small RNAs regulate various biological processes, often by interfering with mRNA translation. Based on their biogenesis and function small RNAs are classified as repeated- associated small interfering RNAs (ra-siRNAs), trans-acting siRNAs (ta-siRNAs), natural- antisense transcript-derived