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

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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.

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Published 01 January 2009
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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 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.


1Chapter 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 siRNAs (nat-siRNAs) and microRNAs (miRNAs) (Vazquez,
2006) (Table 1).
2Chapter I Background

Table 1: Classes of small RNAs identified in eukaryotes (Chapman and Carrington,
2007)
Class Description Biogenesis and genomic origin Function
miRNA MicroRNA Processing of foldback miRNA gene Posttranscriptional regulation of
transcripts by members of the Dicer and transcripts from a wide range of
RNaseIII-like families genes
Primary Small interfering Processing of dsRNA or foldback RNA by Binding to complementary target
siRNA RNA members of the Dicer family RNA; guide for initiation of
RdRP dependent secondary
siRNA synthesis
Secondary Small interfering RdRP activity at silenced loci Posttranscriptional regulation of
siRNA RNA (Caenorhabditis elegans) processing of transcripts; formation and
RdRP derived long dsRNA or long maintenance of heterochromatin
foldback RNA by members of the Dicer
family (Arabidopsis thaliana)
tasiRNA Trans-acting miRNA-dependent cleavage and RdRP Posttranscriptional regulation of
siRNA dependent conversion of TAS gene transcripts
transcripts to dsRNA, followed by Dicer
processing
natsiRNA Natural antisense Dicer processing of dsRNA arising from Posttranscriptional regulation of
transcript- sense and antisense transcript pairs genes involved in pathogen
derived defense and stress responses in
siRNA plants
piRNA Piwi-interacting A biogenesis mechanism is emerging Suppression of transposons and
RNA which is Argonaute dependent but Dicer- retroelements in the germ lines
independent of flies and mammals
1.1.2.1 MicroRNAs (miRNAs)
MiRNAs are ~21nt small RNAs which are encoded by endogenous MIR genes. Their primary
transcripts form precursor RNAs exhibiting a partially double-stranded stem-loop structure
which are processed by DICER-LIKE proteins to release mature miRNAs (Bartel, 2004). In
animals, the primary miRNAs (pri-miRNAs) are cleaved in the nucleus by an enzyme named
Drosha to form the pre-miRNAs (Lee et al., 2003). The pre-miRNAs are then transported
into the cytoplasm where they are processed into the mature double stranded miRNAs,
through cleavage by a second enzyme, Dicer (Bartel, 2004). The first enzyme, Drosha,
required for processing of pri-miRNAs in animals, does not exist in plants, so the precursor
miRNA is directly cleaved within the nucleus to generate the mature miRNA (Baulcombe,
2004) (Figure 2a).
Computational analysis of miRNAs and their potential target mRNAs revealed that many of
the miRNA targets belong to the group of transcription factors (Palatnik et al., 2003; Wang
et al., 2004). In addition to the control of targets at the post-transcriptional level miRNAs
regulate gene expression by epigenetic changes such as DNA and histone methylation (Bao et
al., 2004; Lippman and Martienssen, 2004). Overexpression or knockdown of miRNA genes
can lead to abnormalities during development (Palatnik et al., 2003; Chen, 2004). For
example, plants expressing MIR159, which targets members of MYB transcription factors,
exhibit delayed flowering time and male sterility (Achard et al., 2004; Schwab et al., 2005).
Plants expressing MIR160, which targets members of the ARF transcription factor family,
exhibits agravitropic roots with disorganized root caps and increased lateral rooting (Wang
3Chapter I Background

et al., 2005). Plants expressing MIR166, which targets members of HD-ZIP transcription
factors, are arrested in seedling development, and show fasciated apical meristems and femal
sterility (Williams et al., 2005).
(g) Piwi-interacting RNA (piRNA)

Figure 2: Small RNA pathways (modified after Vazquez, 2006; Chapman and
Carrington, 2007). (a) The miRNA (b) trans-acting siRNA precursors are non-coding RNAs (c)
nat-siRNA precursors derive from cis-antisense overlapping coding transcripts. All three precursors
are transcribed by RNA polymerase Pol II. (e) Two miRNAs can guide AGO1-mediated cleavage of
TAS precursors. The resultant 5’ fragment (TAS1a, 1b, 1c and 2) or 3’ fragment (TAS3) is used by
RDR6 and SGS3 as a template for the production of a long dsRNA , which is then cleaved in a phased
fashion every 21-nt by DCL4. (f) ta-siRNAs are then 2’-O-methylated at their 3’ ends by HEN1 and
guide a slicer-competent AGO protein (AGO7 for TAS3 siRNAs or an unidentified AGO protein for
other ta-siRNAs) to their targets for cleavage. (d) A self-amplifying loop believed to depend on RNA
Pol IVa is involved in maintaining ra-siRNA-guided methylation of certain DNA repeats. (g) Piwi-
interacting RNA (piRNA) biogenesis. Black bars represent genes, with their transcription initiation
sites indicated by arrows. Thin black strands represent transcripts of genes encoding small RNAs, and
thin blue strands represent target mRNAs. Boxes with broken lines indicate parts of the ta-siRNA and
nat-siRNA pathways for which the cellular location is not well established. In small RNA duplexes, the
red strands correspond to the guide strand and the black strands correspond to the passenger strand
to show that, in the case of siRNAs, the guide strand can originate from either the original sense
strand or the newly RDR-synthesized complementary strand.

Target sites in plant miRNAs normally share perfect or nearly perfect complementarity with
their target sequence and are often in coding regions (Schwab et al., 2005), whereas in
4Chapter I Background

animals, target sites are often only partially complementary to their miRNAs and are mostly
located in the 3'UTR of target genes (Filipowicz, 2005). Currently, hundreds of miRNAs have
been identified in plant species and deposited in the miRBase database
(http://microrna.sanger.ac.uk/sequences/index.shtml) (Table 2).

Table 2: Plant species and number of miRNAs deposited in miRBase database
(Version 12.0, 2008)
Species Group Number of miRNAs
Arabidopsis thaliana Eudicots 184
Medicago truncatula Eudicots 30
Populus trichocarpa Eudicots 215
Oryza sativa Monocots 243
Zea mays Monocots 96
Physcomitrella patens Mosses 220

1.1.2.2 Trans-acting short interfering RNAs (ta-siRNA)

MiRNAs are required for the biogenesis of ta-siRNAs, and both miRNAs and ta-siRNAs
regulate mRNA stability and translation (Baulcombe, 2004). Ta-siRNAs arise in plants from
specific TAS loci (Figure 2b). TAS transcripts are RNA polymeraseII-dependent and function
as highly specialized precursors that feed into an RdRP-dependent siRNA biogenesis
pathway. They are targets for cleavage by miRNA-guided mechanisms and yield siRNAs that
are in a 21-nt register with the cleavage site (Allen et al., 2005; Rajagopalan et al., 2006;
Chapman and Carrington, 2007).
Arabidopsis contains different characterized TAS gene families. TAS1a-c and TAS2 ta-siRNA
biogenesis is initiated by miR173-guided cleavage on the 5 ′ side of the ta-siRNA generating
region, while TAS3 ta-siRNAs form by miR390-guided cleavage on the 3 ′ side. MiR390 also
interacts in a non cleavage mode with a second site near the 5 ′ end (Axtell et al., 2006;
Montgomery et al., 2008). The resultant 5’ fragment (TAS1a-c and TAS2) or 3’ fragment
(TAS3) is used by RDR6 and SGS3 as a template for the production of a long dsRNA, which
is then cleaved in a phased fashion every 21-nt by DCL4. This processing involves an
interaction between DCL4 with DRB4 for TAS3. Ta-siRNAs are later incorporated into the
RISC-like complex and guide cleavage of the complementary mRNAs. However, in
Physcomitrella patens, miR173 is absent and therefore miR390 is responsible for the
generation of TAS precursors (Axtell et al., 2006). TAS3 ta-siRNAs, but not those from
TAS1a-c or TAS2, are dependent on a specialized AGO7 (also called ZIP) (Montgomery et al.,
2008). The mechanisms for recognition and routing of transcripts through the ta-siRNA or
RDR6/DCL4-dependent pathway are not well understood. Axtell et al. (2006) proposed a
5