Biological function of RNA interference (RNAi) pathways in the moss Physcomitrella patens (Hedw.) Bruch & Schimp [Elektronische Ressource] / von Basel Khraiwesh

Khraiwesh

Sujets

• Biologie

Informations

• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

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 Δ mutants and wild type………….19
1.2.1.1.5 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………………………………… 146
4.6 Acknowledgments……………………………………………………………... 152
4.7 Erklärung………………………………………………………………………....153
IPublications

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
thDICER-LIKE gene causes silencing of microRNA targets in Physcomitrella patens. 5
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.


IIChapter 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 re