Download Serine/Arginine-rich proteins Physcomitrella patens Andreas Ring

Department of Physics, Chemistry and Biology
Master Thesis
Serine/Arginine-rich proteins
in Physcomitrella patens
Andreas Ring
LITH-IFM-A-EX--11/2447—SE
Supervisor: Johan Edqvist, Linköpings universitet
Examiner: Matthias Laska, Linköpings universitet
Department of Physics, Chemistry and Biology
Linköpings universitet
SE-581 83 Linköping, Sweden
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Molecular Genetics, IFM
2011-06-03
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LITH-IFM-A-Ex— 11/2447 —SE
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Titel
Title
Serine/Arginine-rich proteins in Physcomitrella patens
Författare
Author
Andreas Ring
Sammanfattning
Abstract
Serine/Arginine-rich proteins (SR-proteins) have been well characterized in metazoans and in the flowering plant
Arabidopsis thaliana. But so far no attempts on characterizing SR-proteins in the moss Physcomitrella patens have
been done. SR-proteins are a conserved family of splicing regulators essential for constitutive- and alternative
splicing. SR-proteins are mediators of alternative splicing (AS) and may be alternatively spliced themselves as a form
of gene regulation. Three novel SR-proteins of the SR-subfamily were identified in P. patens. The three genes show
conserved intron-exon structure and protein domain distribution, not surprising since the gene family has evidently
evolved through gene duplications. The SR-proteins PpSR40 and PpSR36 show differential tissue-specific
expression, whereas PpSR39 does not. Tissue-specific expression of SR-proteins has also been seen in A. thaliana.
SR-proteins determine splice-site usage in a concentration dependent manner. SR-protein overexpression
experiments in A. thaliana and Oryza sativa have shown alteration of splicing patterns of endogenous SR-proteins.
Overexpression of PpSR40 did not alter the splicing patterns of PpSR40, PpSR36 and PpSR39. This suggests that
they might not be a substrate for PpSR40. These first results of SR-protein characterization in P. patens may provide
insights on the SR-protein regulation mechanisms of the common land plant ancestor.
Nyckelord
Keyword
Physcomitrella patens, SR protein, gene expression analysis, overexpression
Contents
1. Abstract .................................................................................................................................. 2
2. List of abbreviations ............................................................................................................... 2
3. Introduction ............................................................................................................................ 2
4. Materials and methods ........................................................................................................... 4
4.1 Plant material and standard growth conditions ................................................................ 4
4.2 Sequence data mining....................................................................................................... 4
4.3 Phylogenetic analysis ....................................................................................................... 4
4.4 Primer design.................................................................................................................... 5
4.5 Gene expression analysis ................................................................................................. 5
4.6 Overexpression of PpSR40 in P. patens .......................................................................... 5
4.6.1 Generation of pCMAK1:PpSR40 construct .............................................................. 5
4.6.2 Transformation of P. patens ...................................................................................... 6
4.6.3 Gene expression analysis of pCMAK1:PpSR40 transformants ................................ 8
5. Results .................................................................................................................................... 8
5.1 Sequence Data Mining ..................................................................................................... 8
5.1.1 Identification of SR proteins in P. patens ..................................................................... 8
5.1.2 SR-protein transcripts .................................................................................................. 10
5.2 Phylogenetic analysis ..................................................................................................... 10
5.3 Gene expression analysis ............................................................................................... 12
5.4 Overexpression of PpSR40 in P. patens ........................................................................ 12
5.4.1 Gene expression analysis of pCMAK1:PpSR40 transformants .............................. 13
6. Discussion ............................................................................................................................ 14
6.1 Protein identification and characterization ..................................................................... 14
6.2 SR-protein transcripts in P. patens ................................................................................. 15
6.3 Gene expression analysis ............................................................................................... 15
6.4 Overexpression of PpSR40 in P. patens ........................................................................ 15
6.4.1 Transformation of P. patens .................................................................................... 15
6.4.2 Gene expression analysis of P. patens transformants ............................................. 16
6.5 Conclusion...................................................................................................................... 17
7. Acknowledgements .............................................................................................................. 17
8. References ............................................................................................................................ 17
1
1. Abstract
Serine/Arginine-rich proteins (SR-proteins) have been well characterized in metazoans and in
the flowering plant Arabidopsis thaliana. But so far no attempts on characterizing SRproteins in the moss Physcomitrella patens have been done. SR-proteins are a conserved
family of splicing regulators essential for constitutive- and alternative splicing. SR-proteins
are mediators of alternative splicing (AS) and may be alternatively spliced themselves as a
form of gene regulation. Three novel SR-proteins of the SR-subfamily were identified in P.
patens. The three genes show conserved intron-exon structure and protein domain
distribution, not surprising since the gene family has evidently evolved through gene
duplications. The SR-proteins PpSR40 and PpSR36 show differential tissue-specific
expression, whereas PpSR39 does not. Tissue-specific expression of SR-proteins has also
been seen in A. thaliana. SR-proteins determine splice-site usage in a concentration dependent
manner. SR-protein overexpression experiments in A. thaliana and Oryza sativa have shown
alteration of splicing patterns of endogenous SR-proteins. Overexpression of PpSR40 did not
alter the splicing patterns of PpSR40, PpSR36 and PpSR39. This suggests that they might not
be a substrate for PpSR40. These first results of SR-protein characterization in P. patens may
provide insights on the SR-protein regulation mechanisms of the common land plant ancestor.
2. List of abbreviations
Polymerase chain reaction - PCR
Polyethylene glycol – PEG
Regulated unproductive splicing and
translation - RUST
Reverse transcriptase polymerase chain
reaction – RT-PCR
RNA recognition motif – RRM
Small nuclear ribonucleoprotein – snRNP
Untranslated region - UTR
Alternative splicing – AS
Cauliflower mosaic virus – CaMV
Coding sequence - CDS
Complementary DNA - cDNA
Exonic splicing enhancer - ESE
Expressed sequence tags – EST
Heterogeneous nuclear ribonucleoprotein hnRNP
Nonsense-mediated decay - NMD
Nuclear localization signal - NLS
Keywords:
Physcomitrella patens, SR protein, gene expression analysis, overexpression
2
3. Introduction
Traditionally Arabidopsis thaliana has been regarded as the undisputed premier model to
study plant genetics. Recently, a plant model contender in the form of the modest moss
Physcomitrella patens has emerged. However, the potential of moss in genetics was already
recognized in the early 1920’s by von Wettstein (Cove et al., 1997; von Wettstein, 1924). P.
patens belongs to the division Bryophyta (mosses) and characteristic to all plants, have an
alternation of generations in their life-cycle. The moss has a dominant haploid gametophytic
and a diploid sporophytic generation. The gametophytes of P. patens are monoecious, that is
with both male and female gamete-producing organs, called antheridia and archegonia,
respectively. Upon gamete fertilization, a diploid zygote is produced, which undergo cell
division until a multicellular mature sporophyte emerges. The mature sporophyte produces
haploid spores through meiosis that in turn will grow in to a filamentous juvenile
gametophyte stage called protonema. Protonema consists of two cell-types, chloronema that
have densely packed cells with large chloroplasts and caulonema that have less chloroplasts
with less chlorophyll (Cove et al., 1997). After spore-germination or protoplast regeneration,
chloronemal filaments are produced, from which caulonemal filaments can be branched. Buds
originate from caulonema, which later develops into a leafy gametophyte and thus completing
the life cycle of P. patens.
The moss P. patens has the key attributes for being a good model system, namely it is easy
to grow and maintain, it is open for genetic manipulation and mutant phenotypes can be
selected for (Cove, 2005; Wood, 2000). P. patens has in the field of molecular genetics
become increasingly popular over the past 10 years which can be reflected in the 5-fold
increased number of publication hits for “Physcomitrella patens” in well-used article
databases Pubmed, Scopus and Web of knowledge. This can be explained by a number of
reasons. P. patens has (and other mosses) emerged as a valuable organism to study cell
polarity (Cove et al., 1996; Sievers et al., 1996) and other complex biological processes (Cove
et al., 2006; Cove et al., 1997; Reski, 1998; Wood, 2000). Mosses hold an interesting
evolutionary position, between photosynthetic algae and vascular land plants. P. patens
diverged from the vascular land plant lineage some 430 million years ago, thus making it one
of the earliest plants that conquered land (Kenrick and Crane, 1997). Another feature that
stands out from the plant model organism A. thaliana is that efficient gene targeting can be
achieved (Schaefer, 2001, 2002; Schaefer and Zrÿd, 1997). Because P. patens consists
predominantly of haploid gametophytes, this makes homologous recombination a viable
option but also makes selection for a preferred genotype easier. Moss protoplasts can be
transformed efficiently using a Polyethylene glycol (PEG)-mediated method (Schaefer et al.,
1991). The most important reason for the increased popularity of P. patens is very likely to be
the sequencing of the genome (Rensing et al., 2008).
Most eukaryotic protein coding nuclear genes are interrupted by non-coding sequences
called introns (Sharp and Burge, 1997). Following gene transcription, introns are excised out
of the precursor-mRNA in a process called splicing. This is achieved by a macromolecule
called the spliceosome, which is made up of the U1, U2, U4/U6, and U5 small nuclear
ribonucleoproteins (snRNPs) (Sharp and Burge, 1997). Introns are typically identified by the
consensus dinucleotides GT and AG at the 5’ and 3’ intron boundaries, respectively. However
it was discovered that exceptions to the GT-AG rule exists, introns denoted minor class
introns which used the AT and AC dinucleotides (Hall and Padgett, 1994; Jackson, 1991).
After a series of publications from different groups a novel spliceosome was unveiled,
consisting of the snRNPs denoted U11 (Kolossova and Padgett, 1997; Tarn and Steitz,
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1996b), U12 (Hall and Padgett, 1996; Tarn and Steitz, 1996b), U5 (Tarn and Steitz, 1996b),
U4atac and U6atac (Tarn and Steitz, 1996a).
There are two main splicing mechanisms, constitutive splicing (CS) and alternative
splicing (AS). In CS all introns are removed from the pre-mRNA, leaving all exons to form a
mature mRNA. AS is a mechanism in which pre-mRNA can form several structurally and
functionally different protein products from a single gene (Graveley, 2001; Manley and
Tacke, 1996). This makes AS a major contributor to protein diversity, but AS is also a form of
post-transcriptional gene regulation. AS of pre-mRNA transcripts in which introns or “poison
exons” are spliced into an mRNA can lead to changes in the coding sequences. Any change to
the coding sequence could potentially lead to a premature termination codon (PTC). Proteins
containing PTCs are often not translated; they are rather the target of nonsense-mediated
decay (NMD) which is a RNA surveillance system that prevents the synthesis of truncated
and potentially hazardous protein products (Belgrader et al., 1994; Maquat, 2004). A recent
survey of alternatively spliced SR-protein transcripts containing PTCs shows that about half
of them were targeted by NMD (Palusa and Reddy, 2010). It is proposed that AS and NMD
play important roles in post-transcriptional gene regulation through a prevalent process named
regulated unproductive splicing and translation (RUST) (Lewis et al., 2003). It has been
shown that RUST is highly conserved in mammalian Serine/Arginine-rich proteins (SRproteins) (Lareau et al., 2007) and has been suggested to be important for the autoregulation
of SR-protein expression (Ni et al., 2007).
The SR-proteins are a family of conserved essential splicing factors for the intronrecognition, spliceosome assembly and for the regulation of AS (Xiao-Qin et al., 2007). SRproteins contain one or two N-terminal RNA recognition motif (RRM) and a variable length
C-terminal region of SR-dipeptides constituting an SR-domain (Manley and Tacke, 1996; Wu
and Maniatis, 1993). The RRM domains have high affinity for splicing enhancer elements in
the mRNA and the RRM domains are vital for in vitro splicing (Chandler et al., 1997; Tacke
and Manley, 1995). The SR-domain, that is highly phosphorable, is important for mediating
protein-protein interactions that is necessary for proper splicing (Kohtz et al., 1994; Wu and
Maniatis, 1993; Xiao and Manley, 1997).
It has been shown that the AS of SR-proteins in A. thaliana is controlled in a
developmental and tissue-specific manner (Golovkin and Reddy, 1998; Kalyna et al., 2003;
Lazar et al., 1995; Lopato et al., 1999; Lopato et al., 1996; Palusa et al., 2007). Alternatively
spliced SR-proteins with even subtle differences have been linked to distinct biological
functions (Zhang and Mount, 2009). SR-proteins determine splice-site usage in a
concentration-dependent manner (Lazar and Goodman, 2000; Smith and Valcárcel, 2000).
Generally, an increase in SR-protein concentration leads to the alternative usage of a proximal
5’-splice site (Cáceres et al., 1994; Fu et al., 1992; Screaton et al., 1995; Wang and Manley,
1995; Zahler et al., 1993) but individual SR-proteins have been proven to have opposite effect
on splice site election namely the use of a distal 5’ splice-site (Dauksaite and Akusjärvi,
2004). The usage of a proximal 5’ splice site is counteracted by the heterogeneous nuclear
ribonucleoprotein (hnRNP) A/B family of proteins (Mayeda and Krainer, 1992; Mayeda et al.,
1994).
All in all, this suggests that, ultimately, splice site selection in vivo is determined by
relative concentrations of splicing factors.
The aim of this project was to identify and characterize putative SR-proteins in P. patens.
Three P. patens SR-proteins of the SR-subfamily were identified. The SR-proteins PpSR40
and PpSR36 show tissue-specific expression in protonema. Overexpression of PpSR40 did
not significantly alter splicing patterns of PpSR40, PpSR36 and PpSR40. These first results of
3
SR-protein characterization in P. patens may provide insights on the SR-protein regulation
mechanisms of the common land plant ancestor.
4. Materials and methods
4.1 Plant material and standard growth conditions
P. patens ssp. patens (strain Gransden 2004) were grown axenically on solid BCD-medium (1
mM MgSO4, 1.85 mM, KH2PO4, 10 mM KNO3, 45 µM FeSO4, 0.22 µM CuSO4, 0.19 µM
ZnSO4, 10 µM H3BO4, 0.10 µM Na2MoO4, 2 µM MnCl2, 0.23 µM CoCl2 and 0.17 µM KI)
supplemented with 5 mM ammonium tartrate, 1 mM CaCl2 and 0.8 % (w/v) plant agar in petri
dishes (90 mm diameter) (Ashton and Cove, 1977). The moss were grown in standard
conditions, that was 24 light hour cycle, 25 °C and continuous photosynthetic photon flux of
155 µmoles m-2 sec-1 in a growth chamber (CLF Plant Climatics, Emersacker, Germany,
Percival CU-36L/5). The moss were routinely sub cultured monthly to fresh supplemented
BCD-medium.
Protonema from P. patens was generated by homogenization of gametophyte colonies in
100 ml liquid supplemented BCD-medium by an Ultra-Turrax® T18 basic (IKA, Staufen,
Germany). The liquid culture was grown for 5 days, and then re-homogenized and
subsequently transferred to cellophane covered supplemented solid BCD-medium.
4.2 Sequence data mining
From the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) the
protein sequences of four A. thaliana SR-proteins (AtSR30; AtSR34; AtSR34a; AtSR34b)
were used as queries for homologous proteins in P. patens (taxid: 3218) using NCBI BLASTp
with default settings (Altschul et al., 1997). Protein sequence hits below E-value e-20 were
rejected. The proteins found were built on pre V.1.6 gene models, so subsequently the
proteins were used as queries in a BLASTp with default settings in P. patens database at
http://www.phytozome.net to obtain V.1.6 gene models.
To find expressed sequence tags (ESTs) in P. patens transcriptome BLAST at
http://www.cosmoss.org were used with default settings for each gene. Genomic DNA was
used as query in a BLASTn search in the pp0409_fil database, with an E-value threshold of
1e-2.
Multiple sequence alignment of EST’s was done using MUSCLE with default settings at
the European Bioinformatics Institute (EBI) (http://www.ebi.ac.uk/Tools/msa/muscle/)
(Edgar, 2004).
Protein masses were calculated with the PeptideMass tool at http://www.expasy.ch
(Wilkins et al., 1997).
4.3 Phylogenetic analysis
A.thaliana and P. patens SR-protein sequences from the SR-subfamily were aligned with
ClustalW2 (Larkin et al., 2007) at EBI (http://www.ebi.ac.uk/Tools/msa/clustalw2/) using
default settings; the alignment was saved in the PHYLogeny Inference Package (PHYLIP)
(Felsenstein, 1989) format. A neighbor-joining (Saitou and Nei, 1987) tree was created using
PHYLIP v.3.6.9 programs in order: Seqboot, Protdist, Neighbor and Consense all with default
settings and with the option multiple datasets 100 when possible. A maximum likelihood tree
was created using PhyML 3.0 online (http://www.atgc-montpellier.fr/phyml/) (Guindon and
Gascuel, 2003; Guindon et al., 2005) using default settings and with 100 bootstraps. All
phylogenetic trees were viewed in FigTree v.1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/).
4
4.4 Primer design
Gene specific primers, pre v1.6 gene model, were designed using the following pattern
XXXXYYYYYYZZZZZZZZZZZZZZZ where X is random base pairs, Y is restriction site
consensus for Xho1 (CTCGAG) for forward primer and Apa1 (GGGCCC) for reverse primer
and Z is gene specific untranslated region sequence upstream of the start codon and
downstream of the termination codon. For RT-PCR, gene specific forward primers were used
and reverse primers were designed so that they would span several exons, in which the
resulting amplicons would have evidence for alternate splicing. The housekeeping gene betatubulin 1 was used as an endogenous control (Holm et al., 2010). The full list of primers used
can be viewed in Table 1.
Table 1. Primer sequences for cDNA constructs, cDNA-synthesis, RACE-PCR and RT-PCR.
Primer
Sequence 5' to 3'
dT-adaptor
Apa1-adaptor
PpSR40_f
PpSR40_r
PpSR36_f
PpSR36_r
PpSR39_f
PpSR39_r
PpSR40_r_RT-PCR
PpSR36_r_RT-PCR
PpSR39_r_RT-PCR
Beta-tubulin 1_f
Beta-tubulin 1_r
GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT T
ACT TTG GGC CCG GCC ACG CGT CGA CTA GTA C
AGA ACT CGA GAA CCC ATC GAA ACC AGC ACG AT
GAA CGG GCC CAC TGA GAT TCG CGT GAC GGA AC
TAA GCT CGA GAG ACG ATA TTG TAA CCA GCA CA
CAA CGG GCC CTA CAT AAT TAA CAA GCA GAA AC
GGA ACT CGA GGT TGC TTA TCA CAT TCA ACA AC
ATA TGG GCC CTT AGC AGT CAG AAG GCT ATG AC
GTC CAC GAT GCC CAT TGT TCC C
GGC GTA TTT CAT ATC GTC GTA G
TTC CAG CTG AGC CAT CAC GAA A
GAC TGC TTG CAA GGT TTC CAA G
TTT AGC TGC CCA GGG AAT CGG A
4.5 Gene expression analysis
Total RNA was isolated from ten days old protonema and the upper half-part of five weeks
old gametophytes in an RNAse-free environment using RNeasy plant mini kit (Qiagen
GmbH, Hilden, Germany). Total RNA-isolations were treated with DNase 1 (Fermentas,
Vilnius, Lithuania). cDNA was synthesized from DNA-free total RNA using RevertAid Hminus Reverse Transcriptase (Fermentas) using random hexamer primers. RT-PCR was
performed with DreamTaq DNA polymerase (Fermentas) using 1 µg of cDNA as template
and gene specific primers for the genes PpSR40, PpSR36 and PpSR39 and beta-tubulin 1,
respectively. PCR was run with the settings: 3 min at 94 °C; 35 cycles of 30 sec at 94 °C, 30
sec at 55 °C, 1 min at 72 °C; and 7 min at 72 °C. The resulting amplicons were visualized
with 1x SYBR safe™ (Invitrogen, Carlsbad, USA) and separated on 1.2 % agarose (Saveen &
Werner, Limhamn, Sweden) gels with 0.5xTBE running buffer (Fermentas). Gene expression
data were corrected for background luminescence and normalized using beta-tubulin 1 as
endogenous control using ImageJ (version 1.43u) (Abramoff et al., 2004). The gene
expression of PpSR40, PpSR36 and PpSR39 was analyzed using a One-way ANOVA with
Tukey and Fisher method post-hoc analysis.
4.6 Overexpression of PpSR40 in P. patens
4.6.1 Generation of pCMAK1:PpSR40 construct
Total RNA was isolated from three weeks old gametophytes in an RNAse-free environment
using RNeasy plant mini kit (Qiagen). Total RNA-isolations were treated with DNase 1
(Fermentas). Full-length cDNA was synthesized from DNA-free total RNA using RevertAid
H-minus Reverse Transcriptase (Fermentas) using primer dT-adaptor primer.
5
RACE-PCR was performed with DreamTaq DNA polymerase (Fermentas) using 1 µg of
cDNA as template and gene specific forward primers for PpSR40, PpSR36 and PpSR39
respectively and Apa1-adaptor as reverse primer for all reactions. PCR was run with the
settings: 3 min at 94 °C; 35 cycles of 30 sec at 94 °C, 30 sec at 55 °C, 1 min 30 sec at 72 °C;
and 7 min at 72 °C. One µl of the resulting PCR-product was used as template for PCR using
gene specific primers for PpSR40, PpSR36 and PpSR39, respectively. PCR was run with the
settings: 3 min at 94 °C; 35 cycles of 30 sec at 94 °C, 30 sec at 55 °C, 1 min 30 sec at 72 °C;
and 7 min at 72 °C. The resulting amplicons were visualized with 1x SYBR safe™
(Invitrogen) and separated on 1.2 % agarose (Saveen & Werner) gels with 0.5xTBE running
buffer (Fermentas). A ~900 bp amplicon, corresponding to the gene PpSR40 full-length
cDNA was excised and purified using QIAEX II Gel Extraction Kit (Qiagen). The pCMAK1
plasmid (Fig. 1) was kindly supplied by National Institute of Agrobiological Resources,
Tsukuba, Japan. The pCMAK1 plasmid and the PpSR40 full-length cDNA was digested with
Xho1 (New England Biolabs, Ipswich, USA) and Apa1 (New England Biolabs). The resulting
fragments were purified in between Xho1 and Apa1 digestion and after Apa1 digestion, using
GeneJET™ PCR Purification Kit (Fermentas). The digested pCMAK1 plasmid and the
digested PpSR40 full-length cDNA-amplicon was ligated using T4 DNA ligase (Invitrogen).
The pCMAK1:PpSR40 plasmid was amplified using Subcloning Efficiency™ DH5α™
Chemically Competent E. coli (Invitrogen) and isolated using Maxi Plasmid Kit (Qiagen).
Figure 1. The plasmid pCMAK1 is suitable for bacterial and P. patens
transformation. The plasmid is a pCR4-topo vector which has a pUC replication
origin, lac promotor coupled to LacZα-ccdb lethal gene, kanamycin and
ampicillin resistance for bacterial selection. The plasmid contains an expression
cassette that can be released by a number of restriction enzymes. The expression
cassette is directed to the locus BS213. It uses a variant of the Cauliflower
mosaic virus (CaMV) 35S-promotor, namely the P7113 promotor that is a strong
constitutive promotor. Between the promotor and the nopalin synthase terminator
there are multiple cloning sites for the insertion of fragments. Furthermore, the
cassette has a Zeocin cassette for selection in eukaryotic cells.
4.6.2 Transformation of P. patens
Transformation was performed by the PEG-mediated method (Schaefer et al., 1991). Prior to
the transformation, MMM solution was made by dissolving 8.5 g D-mannitol, 0,305 g
MgCl26H2O and 10 ml 1 % MES pH 5.6 in 100 ml distilled water. The MMM solution was
6
then autoclaved. PEG solution was made by dissolving 80 g PEG-4000, 2 ml 1 M Tris pH 7.5
and autoclaved 0.38 M D-mannitol/0.1 M Ca(NO3)2 solution up to 200 ml. The solution was
heated up to 45 °C to dissolve the PEG and pH was adjusted to 8 and was subsequently filter
sterilized. On the day of the transformation, 1% Driselase solution was prepared by dissolving
500 mg Driselase (Sigma, St. Louis, USA) in 50 ml autoclaved 8.5 % D-mannitol. It was
incubated for 15 minutes in RT and centrifuged at 2500 x g for 5 minutes. The clear
supernatant was collected and adjusted to pH 5.6 and was then filter sterilized. In a sterile
hood, 10 day old protonema from 5-10 plates was collected and treated with 1 % Driselase for
30 minutes in room temperature in order to obtain protoplasts. The tube was turned from time
to time to facilitate mixing. The moss protoplasts were poured through a large meshed filter
into a petri dish. The filtrate was incubated for ten minutes and then poured through a fine
meshed filter into another petri dish. The protoplast solution was then transferred to a 50 ml
falcon tube and was washed by centrifugation at 700 rpm in a Universal 320 centrifuge
(Hettich, Tuttlingen, Germany) at 5 °C for 5 minutes. The supernatant was poured off and the
resulting pellet was re-dissolved in the original volume of 8.5 % D-mannitol. The wash was
repeated and the protoplast pellet was dissolved in 2 ml of 8.5 % D-mannitol. Ten µl of the
protoplast solution was used in a Bürcher chamber to estimate the amount of intact
protoplasts. Three instances of ten µg of pCMAK1:PpSR40 plasmid, where the expression
cassette (Fig. 2) had been released by Not1 (Fermentas) digestion were pipetted into 15 ml
falcon tubes.
Figure 2. The expression cassette released from the pCMAK1:PpSR40 plasmid with Not1 restriction enzyme.
The cassette uses the locus BS213 as target for homologous recombination. The P7113 promoter is a strong
constitutive promoter; Tnos is the Agrobacterium tumefaciens nopaline synthase transcription terminator. The
cDNA-clone is cloned in the cassette between the promotor and the terminator. Transgenic plants are selected for
growth on zeocin.
The protoplast suspension was centrifuged and the supernatant was pipetted off as much as
possible, and then re-suspended to a density of 1.2 x 106 cells in MMM solution. 300 µl of the
protoplast suspension was pipetted to the three tubes containing pCMAK1:PpSR40 plasmid
and were mixed gently. Three µl of PEG solution was added to the tubes and was gently
mixed and then heat shocked at 45°C for 5 minutes. After the heatshock the tubes were
incubated in room temperature for 10 minutes. The transformation mix was progressively
mixed with regeneration media (BCD medium supplemented with 5 mM ammonium tartrate,
1 mM CaCl2, 6.6 % (w/v) D-mannitol and 0.5 % (w/v) D-glucose) 5 x 300 µl drop wise and
then 5 x 1 ml slowly. The tubes were incubated in darkness and room temperature overnight.
From the tubes containing the transformation mix, regeneration media was removed until 3 ml
was left without disturbing the sedimented protoplasts. The protoplasts were resuspended
carefully and 3 ml of 42°C liquid top layer of regeneration media (BCD medium
supplemented with 5 mM ammonium tartrate, 1 mM CaCl2, 6.6 % (w/v) D-mannitol, 0.5 %
(w/v) D-glucose and 1.2 % (w/v) plant agar) was added to each tube and were quickly mixed.
Two ml of the solution was poured onto cellophane covered regeneration plates (BCD
medium supplemented with 5 mM ammonium tartrate, 1 mM CaCl2, 6.6 % (w/v) D-mannitol,
0.5 % (w/v) D-glucose and 0.8 % (w/v) plant agar), three plates per tube total of 9 plates. The
plates were sealed with micropore tape and were grown in standard conditions. After 5 days
the cellophane with the moss on top of it, was transferred to selective BCD-medium
containing 50 µg / ml zeocin (Invitrogen, Carlsbad, USA). Moss was selected for two weeks
and surviving colonies were transferred to BCD-medium supplemented with 5 mM
ammonium tartrate, 1 mM CaCl2 and 0.8 % (w/v) plant agar. After two weeks of non7
selective growth parts of the moss colonies were transferred to selective medium. The
stability of the transformants was assayed by looking at the moss growth on the second
selection stage after two weeks. Moss colonies that showed significant growth were regarded
as stable transformants.
4.6.3 Gene expression analysis of pCMAK1:PpSR40 transformants
Total RNA was isolated from three week old moss colonies in an RNAse-free environment
using RNeasy plant mini kit (Qiagen). Total RNA-isolations were treated with DNase 1
(Fermentas). cDNA was synthesized from DNA-free total RNA using RevertAid H-minus
Reverse Transcriptase (Fermentas) using random hexamer primers. RT-PCR was performed
with DreamTaq DNA polymerase (Fermentas) using 1 µg of cDNA as template and gene
specific primers for the genes PpSR40, PpSR36 and PpSR39 and beta-tubulin 1, respectively.
PCR was run with the settings: 3 min at 94 °C; 35 cycles of 30 sec at 94 °C, 30 sec at 55 °C, 1
min at 72 °C; and 7 min at 72 °C. The resulting amplicons were visualized with 1x SYBR
safe™ (Invitrogen) and separated on 1.2 % agarose (Saveen & Werner) gels with 0.5xTBE
running buffer (Fermentas). Gene expression data were corrected for background
luminescence and normalized using beta-tubulin 1 as endogenous control using ImageJ
(version 1.43u).
5. Results
5.1 Sequence Data Mining
5.1.1 Identification of SR proteins in P. patens
In order to identify SR-proteins in P. patens, four A. thaliana SR-proteins (AtSR30; AtSR34;
AtSR34a; AtSR34b) were used as query in a BLASTp which revealed three homologous
predicted SR-proteins in P. patens. The query proteins show high similarity to
Phypa_205547, Phypa_140978 and Phypa_113630 respectively as can be seen in Table 2.
The three homologous proteins Phypa_205547, Phypa_140978 and Phypa_113630 are 355,
333 and 349 amino acids long respectively. Furthermore, Phypa_205547, Phypa_140978 and
Phypa_113630 has an RRM1 domain (prosite:PS50102) at 7-82, 7-82 and 7-82 respectively,
an RRM2 domain with the conserved signature motif SWQDLKD (prosite:PS50102) at 123202, 127-206 and 125-204 respectively and an RS-rich domain at the C-terminal of the
protein. The proteins all contain a nuclear localization signal (NLS) when analyzed by
TargetP 1.1 (Emanuelsson et al., 2007) and WoLF PSORT (Horton et al., 2007).
The three identified proteins can be classified as proteins in the SR-subfamily of the SRproteins (Barta et al., 2010). The calculated molecular weights of the Phypa_205547,
Phypa_140978 and Phypa_113630 proteins were 39534, 36275 and 38519 daltons. The novel
proteins were assigned appropriate names according to the suggested nomenclature of SRproteins (Barta et al., 2010). Phypa_205547, Phypa_140978 and Phypa_113630 were denoted
as PpSR40, PpSR36 and PpSR39, respectively.
Table 2. The four query proteins of A. thaliana and the relative amino acid sequence identity/similarity in
percentages to the proteins of P. patens. The table also shows the relative amino acid sequence identity/similarity
in percentages between novel proteins.
PpSR40
PpSR36
PpSR39
AtSR30
65 / 77
61 / 74
65 / 76
AtSR34
62 / 80
62 / 80
63 / 81
AtSR34a
65 / 81
68 / 82
66 / 83
AtSR34b
66 / 80
64 / 78
67 / 83
8
PpSR40
81 / 86
84 / 89
PpSR36
81 / 86
83 / 87
PpSR39
84 / 89
83 / 87
-
Figure 3. Multiple sequence alignment of A. thaliana and P. patens SR-proteins in the SR-subfamily. The
protein domains RRM1 and RRM2 show high sequence conservation. The NLS and SWQDLKD motifs are
indicated. Black backgrounds represent amino acid residues that show identity in at least 4 sequences. Gray
backgrounds represent amino acids that show similar characteristics, or if the amino acid residues show
consensus with similar characteristics.
The protein sequence hits were then used in a BLASTp for corresponding genes at
www.phytozome.net using default settings. The three protein sequences correlated to the V1.6
gene models Pp1s28_193V6 (PpSR40), Pp1s173_12V6 (PpSR36) and Pp1s7_8V6 (PpSR39).
As can be seen in figure 4, the three genes are highly similar in the intron-exon structure. The
protein PpSR40 is encoded by a gene that is 4596 bp long, consisting of 13 exons and 12
9
introns. The protein PpSR36 is encoded by a gene that is 5438 bp long consisting of 14 exons
and 13 introns. The protein PpSR39 is encoded by a gene that is 4438 bp long, consisting of
13 exons and 12 introns. The gene lengths signify transcription start to transcription end.
Figure 4. The intron-exon structure of the genes is highly
conserved. The distribution of RRM domains over the intronexon structure is also highly conserved.
5.1.2 SR-protein transcripts
SR-proteins are known to be alternatively spliced, so to investigate AS-events, the genomic
sequences from PpSR40, PpSR36 and PpSR39 were used as queries in transcriptome BLAST
at www.cosmoss.org. The search yielded 13, 16 and 8 redundant EST’s for PpSR40, PpSR36
and PpSR39, respectively. The EST hits were aligned with the PpSR40, PpSR36 and PpSR39
full-length transcript. The PpSR40 gene has two mRNA-isoforms, differing in the third UTR
exon. For the gene PpSR36, only one distinct isoform is recognized (Fig. 5). PpSR39
however, have two very distinct transcriptional (Fig. 4) starts but no other variations in the
transcripts were seen in the EST analysis. PpSR40 is the only gene that shows signs of
alternative splicing.
Figure 5. Intron-exon structure of SR-protein mRNA-isoforms in P. patens. The mRNA isoforms are built on
the V1.6 gene models.
5.2 Phylogenetic analysis
Protein sequences from A. thaliana and P. patens were aligned using ClustalW and a
neighbor-joining and a maximum likelihood were created with 100 bootstraps. The proteins
from P. patens form a separate clade that is supported with high boot strap. Seemingly, the P.
10
patens gene family has evolved from two gene duplications occurring after the separation of
mosses. The SR-protein ancestor in P. patens was subject to gene duplication, resulting in the
PpSR39 protein and the PpSR40 / PpSR36-ancestor that was duplicated and subsequently
diverged to the proteins PpSR40 and PpSR36.
Figure 6. Two phylogenetic trees, (A) Neighbor-joining and (B) maximum likelihood were created of SRsubfamily protein sequences from A. thaliana and P. patens. Numbers indicate the percent of bootstraps that
support the topology; bootstrap values under 50 are not shown. The first two characters denote the organism of
origin, At=A. thaliana, Pp=P. patens, the last characters denote the gene name.
11
5.3 Gene expression analysis
It is known that expression levels and splicing patterns of transcripts encoding plant SR
proteins may vary in a developmental and tissue-specific manner (Golovkin and Reddy, 1998;
Lazar et al., 1995; Lopato et al., 1999; Lopato et al., 1996; Palusa et al., 2007). Therefore the
expression of the PpSR40, PpSR36 and PpSR39 genes were investigated in gametophyte and
protonema tissues. The expression of the PpSR40 and PpSR36 genes were upregulated in
protonema tissue whereas the expression of PpSR39 was similar in the two tissues (fig. 6a).
The expression of PpSR40 and PpSR36 was significantly greater in protonema than in
gametophyte (p= 0,019 and p= 0,025, respectively) post-hoc analysis also confirmed this
difference (fig. 6b). Furthermore, there was no measurable alternative splicing in the two
tissues.
Figure 7. Band density results of PCR products from the tissue-specific expression of the genes PpSR40,
PpSR36 and PpSR39 in gametophytes and protonema. Beta-tubulin 1 was used as endogenous control. (A) The
genes PpSR40 and PpSR36 show upregulation in protonemal tissue while the expression of PpSR39 is similar in
the two tissues. (B) ImageJ analysis of tissue-specific expression of the genes PpSR40, PpSR36 and PpSR39.
Gene expression data was normalized with beta-tubulin 1 as reference of endogenous overall transcription levels
(Data are presented as means ± SD fold-change to gametophyte, *: p=0.05, one-way ANOVA, N=2, Tukey and
Fisher method post-hoc analysis.
5.4 Overexpression of PpSR40 in P. patens
To further characterize the biological function of PpSR40 SR-protein genes, a cDNA-copy
was cloned into the pCMAK1 plasmid (fig. 1). pCMAK1 is a plasmid that is suitable for
overexpression in P. patens because it can be used for both bacterial transformation as well as
for transformation in P. patens. The plasmid’s expression cassette has a modified 35S
promoter called P7113 that is a strong constitutive promoter. A modified 35S promoter is
used since the 35S promoter is weak in P. patens (Horstmann et al., 2004; Saidi et al., 2009).
The target of the expression cassette is the genomic locus 213 (Schaefer and Zrÿd, 1997).
Following the transformation, 42 initial transformants were generated after selection on
selective medium containing 50 µg / ml zeocin (fig. 7a). After two weeks of non-selective
growth (fig 7b), the 42 transformants were further selected on to get stable transformants
(Ashton et al., 2000). After two weeks of selective growth six transformants showed
significantly greater growth (fig 7c). These are regarded as stable transformants, and have
thusly incorporated the expression cassette in the genome.
12
Figure 8. The post-transformational selection process. (A) The idea behind the selection process is that only
moss that have taken up the expression cassette will survive. (B) The surviving transformants are moved to nonselective medium for growth. This step will help in the generation of stable transformants. (C) Transformants
that have incorporated the cassette in the genome will show significant growth versus episomal transformants
(non-incorporated cassette).
There were six transformation lines which showed some characteristic phenotypes (Table 3).
Transformation lines one, two and three looked similar to wild-type phenotype colonies,
consisting of a foundation of protonema from which a considerable amount of them
differentiated into leafy gametophytes, giving the colonies a bushy appearance.
Transformation line four consisted primarily of protonema but of which few had
differentiated to gametophytes. The last two lines, five and six, consisted almost exclusively
of protonema, with none or very low amount of gametophytes. Furthermore, colony
composition seemed to have a connection with the water content in the colonies.
Transformation lines five and six had considerably higher amount of water than lines three
and four, which in turn, had higher water content than line one and two.
5.4.1 Gene expression analysis of pCMAK1:PpSR40 transformants
To determine the success of the overexpression of the pCMAK1:PpSR40 transformants, gene
expression analyses on the individual transformation lines were carried out. The results show
varied levels of overexpression in the different lines for PpSR40. Interestingly, the
overexpression of PpSR40 also affected the expression of PpSR36 and PpSR39 to some
extent. This was however greatly accentuated in line 2, in which the expression of PpSR36
and PpSR39 was almost as high as the overexpressed PpSR40.
Table 3. The observed phenotypes of the six transformant lines correlated with expression of PpSR40, PpSR36
and PpSR39. The scale for gametophyte distribution range from, low / none (+), some / few (++) and up to wildtype differentiation (+++). The amount of protonema is inversely correlated to the amount of gametophytes.
Water content scale from, normal (+), high (++) and very high (+++). The scale for SR-protein expression range
from up to 2-fold increase (+), 2 to 4-fold increase (++) and 6 to 8-fold increase (+++).
L1
L2
L3
L4
L5
L6
Gametophyte
+++
+++
+++
++
+
+
Protonema
+
+
+
++
+++
+++
Water
+
+
++
++
+++
+++
PpSR40
+++
+++
+++
+
+++
++
13
PpSR36
+
+++
+
++
-
PpSR39
++
+++
++
++
++
+
Figure 9. Band density results of PCR products of the genes PpSR40, PpSR36 and
PpSR39 from the pCMAK1:PpSR40 transformants. Beta-tubulin 1 was used as
endogenous control. The six different transformant lines show varied overexpression
of PpSR40. Furthermore, the overexpression of PpSR40 did also affect the
expression of PpSR36 and PpSR39 to some extent. Gene expression data was
normalized with beta-tubulin 1 as reference of endogenous overall transcription
levels (Data are presented as means ± SD fold-change to control).
6. Discussion
6.1 Protein identification and characterization
Three novel SR-proteins of the SR-subfamily were identified in Physcomitrella patens. They
are highly similar in their intron-exon structure. This is not strange as phylogenetic analysis
suggest they are the result of two gene duplication and subsequent sequence divergence. This
is in agreement with similar events in A. thaliana in which three rounds of genome
duplications has likely occurred (Simillion et al., 2002), in which 12 SR-proteins are located
on the duplicated regions (Kalyna and Barta, 2004). It has previously been shown that SRprotein genes after gene duplication and further diversification are under positive selection
(Escobar et al., 2006). To measure positive selection, the number of synonymous substitutions
per synonymous site (dS) and the number of nonsynonymous substitutions per
nonsynonymous site (dN) is estimated (Escobar et al., 2006). This means that there has been
selection on mutations that alters the amino acid sequence and thus diversifying the SRprotein gene family. The SR-proteins recognize and bind exonic splicing enhancers (ESEs)
(Maniatis and Tasic, 2002), diversification of the sequence binding region in the SR-proteins
could possibly increase the number of substrates of the SR-protein gene family. This suggests
that it is beneficial for the organism to have more SR-proteins and thus increasing protein
diversity. But mutations can also occur in regulatory sequences that control spatial and
temporal expression (Kalyna and Barta, 2004). The A. thaliana SR-proteins atSRp30 and
atSRp34/SR1 are both expressed during early root formation, whereas atSRp34/SR1 in later
stages is only expressed in the growing rot tip (Lopato et al., 1999). This highlights the
potential functional overlap, or perhaps redundancy, and later on highly specialized function
of different SR-protein gene families.
14
6.2 SR-protein transcripts in P. patens
SR-proteins are essential for AS (Dauksaite and Akusjärvi, 2004) and are themselves known
to be subject to AS (Isshiki et al., 2006; Kalyna et al., 2003; Lopato et al., 1999; Palusa et al.,
2007). Therefore AS was investigated in PpSR40, PpSR36 and PpSR39 transcripts by
analyzing ESTs. In the ESTs examined, no alternative transcripts that affected the coding
sequences and thus affecting the protein products were found. However, the analysis revealed
different transcriptional starts and subtle variations in the 5’ UTR but also an alternative 5’
UTR exon in the PpSR40 transcripts. The UTRs, primarily the 3’ UTR, is thought to contain
various regulatory sequences and is believed to affect the stability of the transcript
(Mazumder et al., 2003). Due to the low number of ESTs found and no full-length cDNA, not
all transcripts may be available and also dependent on sequencing methods (5’ or 3’) different
regions of the transcripts are underrepresented. The ESTs are all from standard conditions but
from different tissues, like gametophytes, protonema and protoplasts. Even though not seen in
this EST-analysis SR-proteins are known to be alternatively spliced (Isshiki et al., 2006;
Lopato et al., 1999; Tanabe et al., 2007). If alternative splicing introduces a PTC, the
transcript will translate a truncated protein. Proteins containing PTCs are often not translated
they are rather the target of NMD (Belgrader et al., 1994; Maquat, 2004). AS and NMD might
play an important role in post-transcriptional gene regulation through RUST (Lewis et al.,
2003), a process which is highly conserved in mammalian SR-proteins (Lareau et al., 2007)
and has been suggested to be important for the autoregulation of SR-protein expression (Ni et
al., 2007).
In P. patens no signs of AS of SR-proteins were seen in control conditions in different
tissues. These first results indicate that regulation of SR-proteins through NMD in tissues
does not occur. This does not however rule out that AS of SR-proteins in P. patens is used in
developmental stages and tissues.
6.3 Gene expression analysis
Gene expression analysis of SR-proteins in A. thaliana have shown that SR-proteins are
differentially expressed in tissues and developmental stages (Golovkin and Reddy, 1998;
Kalyna et al., 2003; Lazar et al., 1995; Lopato et al., 1999; Lopato et al., 1996; Palusa et al.,
2007). While in Rice, Oryza sativa, SR-proteins are not markedly differentially expressed in
tissues (Isshiki et al., 2006).
Gene expression analysis of the SR-proteins PpSR40, PpSR36 and PpSR39 showed tissuespecific differential expression. The genes PpSR40 and PpSR36 showed significant
differential expression between protonema and gametophyte tissue, whereas PpSR39 did not.
This is consistent with the notion that in certain developmental stages or in different tissues
there could be a functional overlap of SR-proteins.
In A. thaliana and P. patens there is differential expression of SR-proteins between tissues
but O. sativa have no marked difference in tissue-specific expression. P. patens, representing
early diverging land plants show, as A. thaliana, variation of SR-protein concentration
between tissues and developmental stages. This poses a question whether this tissue-specific
gene expression differential is an ancestral mechanism which has been lost in O. sativa or
independently evolved in A. thaliana and P. patens. To test this hypothesis, further
experimentation on more plant SR-proteins would need to be carried out.
6.4 Overexpression of PpSR40 in P. patens
6.4.1 Transformation of P. patens
The generation of stable transformants in P. patens has been proven to be time consuming.
Even though P. patens readily take up transformation cassettes and with great efficiency by
15
homologous recombination can incorporate supplied linearized DNA into the genome, a
strenuous post-transformational procedure is required. It is known that intact bacterial
plasmids are replicated in P. patens cells without integration into the genome (Ashton et al.,
2000). All moss transformants, whether they have incorporated the transformation cassette or
exist extrachromosomally, will be able to survive and grow on selection (Hohe et al., 2004).
This results in a surplus of transformants that are not stable. The non-stable transformants
have the same morphology and able to be differentiated from the stable transformants that
successfully incorporated the transformation cassette in the genome. When subjecting the
moss transformants with two rounds of selection, in which between they are grown without
selection, stable transformants that incorporate the transformation cassette show significant
growth. The second round of selection ensures that extrachromosomally replicating plasmid
are lost (Hohe et al., 2004).
In this overexpression experiment 42 initial transformants were able to grow on the first
round of selection, after a second round of selection six moss colonies show significant
growth and were regarded as stable transformants. This shows that the overexpression of
PpSR40 is not inherently toxic. The six transformant lines exhibit differences in their
morphology. Lines one, two and three differentiates similarly as wild-type moss, whereas line
four colonies consist primarily as protonema with a few gametophytes and lines five and six
colonies consist of undifferentiated protonema.
It is however unclear what the reasons for the distinct phenotypes are. Moss that undergoes
transformation will sometimes acquire more chromosomes in the plant nucleus, a
phenomenon called polyploidy (Schween et al., 2005). P. patens protoplasts are haploid but
can after transformation become diploid or tetraploid. The ploidy of moss affect the
differentiation and gametophyte morphology (Schween et al., 2005).
Transformant lines one, two and three show wild-type differentiation which indicates
haploid or diploid moss (Schween et al., 2005). Haploid moss can in most cases be
distinguished from diploid moss by studying the morphology of gametophytes (Schween et
al., 2005). However, transformant lines four, five and six have few, to none gametophytes
thus displaying the characteristics of tetraploidy (Schween et al., 2005). But to be certain of
the ploidy of the moss lines, flowcytometric analysis needs to be employed.
6.4.2 Gene expression analysis of P. patens transformants
When the relative concentration of SR-proteins is altered the splice-sites of target genes may
be alternatively determined (Lazar and Goodman, 2000; Smith and Valcárcel, 2000). The six
P. patens transformant lines show varied levels of overexpression of PpSR40, ranging from
2.5- to 7.5-fold relative wild-type. Increased gene expression was not limited to PpSR40, but
also increased expression of PpSR36/39 in most lines. In moss transformant line two, all three
SR-protein genes were increased about 7-fold relative wild-type. How the ploidy of the moss
affect gene expression and specifically expression of SR-protein is not clear. But it is known
that SR-protein concentration can be altered when subjected to stress (Palusa et al., 2007;
Tanabe et al., 2007). The overexpression of a specific SR-protein could be stressful for the
plant. A. thaliana plants overexpressing atRSZ33 have reduced viability (Kalyna et al., 2003),
suggesting that control of the expression levels of SR-proteins is vital. It has been proposed
that AS and RUST and subsequent NMD play and role in SR-protein level regulation. In the
six PpSR40 overexpression lines which all had elevated PpSR40 levels, no significant
alteration of splicing patterns were seen. It is known that SR-proteins determined splice-site
usage in a concentration dependent manner. These first results suggest that PpSR40, PpSR36
and PpSR39 are not substrates for PpSR40. Furthermore, the results show that the SR-protein
levels in these overexpression lines are not regulation through RUST.
16
Overexpression studies of SR-proteins in other plants, A. thaliana and O. sativa, show
alteration of splicing patterns of both the overexpressed protein, but also other SR-proteins
(Isshiki et al., 2006; Kalyna et al., 2003; Lopato et al., 1999). Alteration of these splicing
patterns results in a decrease of the expression of full-length transcript and thereby regulation
SR-protein expression (Isshiki et al., 2006; Kalyna et al., 2003; Lopato et al., 1999). Why is
this not seen in moss? The SR-protein regulation in A. thaliana and O. sativa might be more
refined than in the early diverging moss P. patens. Both A. thaliana and O. sativa is more
morphological complex and have more complex biological processes. In complex life forms,
homeostasis is of utmost importance and SR-proteins play pivotal roles in gene expression.
6.5 Conclusion
The three identified novel SR-proteins of the SR-subfamily in P. patens show high similarity
in intron-exon structure and in protein domain distribution. The SR-protein gene family is
likely the result of gene duplication with subsequent sequence divergence, also seen in the
flowering plant A. thaliana. The proteins PpSR40 and PpSR36 show differential tissuespecific expression, while PpSR39 does not. Tissue-specific expression of SR-proteins is seen
in A. thaliana but not in O. sativa. Overexpression of PpSR40 does not alter splicing pattern
of PpSR40, PpSR36 and PpSR39 in contrast to overexpression of SR-proteins in other plants.
P. patens show most of the SR-protein characteristics as seen in later diverging plants,
suggesting that the SR-protein regulation mechanisms was already established in the common
land plant ancestor and that was further refined in flowering plants. However, more research
on plant SR-proteins would need to be carried out on more early diverging land plants e.g.
Chlamydomonas Reinhardtii or Selaginella moellendorffii.
7. Acknowledgements
I would like to thank my supervisor Dr. Johan Edqvist for all his help and guidance. I would
like to thank Monika Edstam for all the assistance in the lab. I would also like to thank Jessica
Olsen for all help in the lab, ideas and rewarding discussions.
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