In Vivo Analysis of Plant Intron Splicing Stamm Revision

Summary: Comments come in the following colors:

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Reagents added to the database (blue)

throughout the text: all the µ in µl (u in ul) got lost, the final version should be a pdf to avoid this problem, I did not mark this individually

should be moved after chapter 32: in vivo splicing reporter

Title: In vivo analysis of plant intron splicing.

Craig G. Simpson1, Michele Liney1, Diane Davidson1, Dominika Lewandowska1, Maria Kalyna3, Sean Chapman4, Andrea Barta3 and John WS Brown1,2

1Genetics Programme, Scottish Crop Research Institute, Dundee DD2 5DA, Scotland, UK; 2Division of Plant Sciences, University of Dundee at SCRI, Dundee DD2 5DA, Scotland, UK; 3Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna, Austria; 4Plant Pathology Programme, Crop Research Institute, Dundee DD2 5DA, Scotland

Address correspondence to: Craig G. Simpson, Genetics Programme, Scottish Crop Research Institute, Dundee DD2 5DA, Scotland, UK. E-mail: .

1. Abstract

The characterisation of plant intron signals required for efficient splicing has relied on in vivo splicing analyses due to the lack of a plant in vitro splicing extract. Different in vivo systems and a small number of particular introns have been utilised to determine the contribution that intron splicing signals make to efficient plant splicing and to allow comparisons both among the main plant families and to other organisms such as human and yeast. In addition, in vivo studies have addressed intron enhancement of expression, the roles of UA-rich sequences in plant introns and the functions of trans-acting factors. We describe protocols for analysing splicing behaviour using either plant protoplast or agroinfiltration systems.

Keywords: splicing reporter, protoplasts, transfection, transient splicing assays, agroinfiltration, RNA binding proteins

2. Theoretical background

2.1 Plant splicing analysis in vivo

Tremendous progress in our understanding of eukaryotic splicing has been made through the use of human nuclear or yeast whole cell extracts that are splicing competent (see chapter 26 for in vitro protocol and chapter 5 for a theoretical introduction). Such an in vitro splicing system has not been available for plants despite valiant numerous? attempts by different labs. Rem: people also tried mammalian tissue-specific splicing, which never worked, on can add a brain extract to hela extract and it sort of splices, do you do the same in plants?) In the absence of plant nuclear or whole cell extracts that support in vitro splicing, detailed analysis of plant splicing has progressed through the development of in vivo splicing analysis systems. Transcriptional assays in plants have commonly used transfection of plant protoplasts (plant cells stripped of their cell walls) as a rapid tool for promoter analysis. For analysing plant intron splicing, protoplast transfection of different intron constructs has been invaluable in defining intron splicing signals and features which determine the accuracy and efficiency of plant splicing (Goodall and Filipowicz, 1989, 1991; Simpson et al., 1996, 2000, 2002; Waigmann and Barta, 1992).

2.2 Splicing of plant and animal introns in reciprocal systems

Experiments to examine whether animal introns are spliced in plants and plant introns in animal splicing extracts gave variable results (Brown et al., 1986; Hartmuth and Barta, 1986; van Santen and Spritz, 1987). Naturally-occurring plant introns from a variety of plant species (wheat, oat, pea and soybean) and synthetic intron constructs have been accurately and efficiently spliced in HeLa cell in vitro splicing extracts, while other plant introns were spliced inefficiently or not at all in this system. The variation in splicing is likely to reflect the degree of similarity of the plant intron sequences to the requirements of the animal system (e.g. polypyrimidine tract sequence found near the 3’ splice site). On the other hand, with very few exceptions, transcripts containing animal introns have not been spliced when introduced into plant cells (Barta et al., 1986), again most likely reflecting the requirements of the plant splicing machinery for high UA content (>59%) in introns. In addition to plant/animal splicing differences, there are also differences in splicing efficiency between the two main branches of flowering plants (angiosperms): monocotyledonous (single seed leaf) and dicotyledonous (two seed leaves). Monocotyledons have a lower requirement for UA content of introns than dicotyledons and are therefore dicots are more restrictive in the introns which they can splice – for example, some monocotyledonous introns have been poorly spliced in dicotyledonous cells. Thus despite the many similarities in splicing signals and splicing factors found in plants and animals, there are clearly differences in splicing and in vivo splicing systems have been an essential development. We describe the construction of exemplar splicing reporters and protocols for two plant protoplast systems and a system based on agroinfiltration.

2.3 Plant splicing reporter constructs

A basic splicing reporter construct (pDH515) to study intron splicing was made by cloning an intronless zein storage protein gene behind the Cauliflower Mosaic Virus (CaMV) 35S RNA promoter and followed by the CaMV terminator sequence (Figure 1A). A restriction site was introduced into the zein gene to allow intron sequences (with short flanking exons) to be introduced. The advantage of using the zein coding region was that as a maize storage protein gene it was highly unlikely that a similar sequence would be present in the dicotyledonous cell systems used for protoplast production. Thus, primers to zein sequences flanking the inserted intron(s) could be used to specifically amplify pre-mRNAs and spliced mRNAs from the intron construct.

A sensitive splicing reporter for detailed studies of splicing signal sequences was based on a potato invertase mini-exon sequence. Mini-exons have requirements for strong or additional splicing signals to ensure that they are recognised and spliced into an mRNA. The GF invertase gene from potato (Acc #: AJ133765) consists of 6 exons and 5 introns. Exon 2 is a short 9 nt exon that is spliced by default into the final message (Simpson et el., 1996). Part of the GF invertase gene consisting of 50 nt of exon 1, intron 1 (219 nt), the 9nt mini-exon 2, intron 2 (108 nt) and 70nt of exon 3 was inserted into the unique BamHI site in the zein gene of expression vector pDH515 (Simpson et al., 1996, Simpson et al., 2000) (Figure 1B). This construct (inv1) has been used to generate a series of mutations in the splicing signals (Simpson et al., 2000, 2002) allowing it to be used to report on both splicing activation and repression.

Finally, the mini-exon system has also been used to generate a GFP-based splicing reporter to visualise changes in splicing behaviour. The first 9 nt of the 3’ end of exon 1, intron 1 (219 nt), the 9 nt mini-exon 2, intron 2 (108 nt) and 9nt of the 5’ end of exon 3 were fused to the 5’ end of the mGFP5 gene (Siemering et al., 1996). The exon 1 sequence was modified to include a translation initiation codon and the mini-exon 2 was modified to include an in-frame stop codon (Figure 1C). Skipping of the mini-exon would produce mGFP5 protein with an N-terminal extension of seven amino acids, while inclusion of the mini-exon would result in premature termination and expression of a five amino acid peptide or to degradation by the nonsense-mediated decay pathway. Can you give this construct a name?

Craig, John can you please insert the blue marked constructs into the reagent database:

http://csurs7.csr.uky.edu/sl_demo/login

user: superadmin

pw:golgi

the idea behind this is to collect reagents for Eurasnet, but also later make this reagents available for users (distributed by Dundee Cell products). Any user will be able to export data he wants to make available to a site there. However, at least internally, we should be able to see and exchange all the reagents.

2.4 Expression of trans-acting factors

The Arabidopsis genome encodes more than 200 proteins that contain recognised RNA binding domains. About half of these are highly conserved factors known to be involved in RNA processing events, in other eukaryotes, but the other half are plant-specific and most are of unknown function (Lorković, 2009). For example, plants contain many more SR protein genes than humans – some are orthologues of the human proteins while others are plant-specific (Kalyna and Barta, 2004). Similarly, Arabidopsis contains three genes with similarity to the human polypyrimidine tract binding protein (PTB), a negative regulator of splicing. One of the Arabidopsis PTB genes contains four RNA binding domains (RRM) and has the highest identity to human PTB while the remaining two are unique to plants. In vivo splicing reporter systems can be used to examine whether particular RNA-binding proteins influence splicing. This is achieved by over-expression of genes or cDNAs of RNA binding proteins from plant expression vectors usually driven by the CaMV 35S RNA promoter. These constructs are co-transfected or co-inoculated with splicing reporter constructs into plant cells. The inclusion of epitope tags allows monitoring of the expression of the RNA-binding protein or splicing factor from the plasmids.

3. Protocols

Transfection of plasmid DNA into plant protoplasts

Plant cells, unlike metazoan cells, have a large vacuole that makes up 80-90% of the cell volume and has an important role in cell shape maintenance (Oda et al., 2009). During protoplast preparation, the plant cell wall is removed and cells lose their shape and form spherical protoplasts that are susceptible to disruption by osmosis. Complex plant media that contain different salts and the plasmolysing agent mannitol are essential to maintain protoplasts and allow them to continue to function. We describe protocols for use of protoplasts from different sources of plant material: 1) tobacco leaves and 2) Arabidopsis cell cultures. When would you use tabacco vs Arabidopsis?

3.1 Protocol 1 Transfection of tobacco leaf protoplasts

The protocol described in detail below is based on that of Guerineau et al. (1988).

3.1.1 Solutions

To- (minus) = Solution To lacking sucrose, FeSO4 and Na2EDTA.

To+ (plus) = Solution To containing 0.02% Tween 20 or 80.

To- and To+ are made up from a number of different stock solutions:

Solution 1 10X

10.3 mM NH4NO3 8.25 g

9.4 mM KNO3 9.5 g

1.5 mM CaCl2.2H2O 2.2 g

0.75 mM MgSO4.7H2O 1.85 g

0.62 mM KH2PO4 0.85 g

Make up 200 ml

Solution 2 (for To+ only) 10X

100 μM FeSO4 0.278 g

100 μM Na2EDTA 0.372 g

Make up 200 ml

Solution 3 200X

16 μM H3BO3 200 mg

0.6 μM MnSO4 200 mg

3.5 μM ZnSO4.7H2O 20 mg

0.12 μM CuSO4.5H2O 6 mg

0.22 μM AlCl3 6 mg

0.13 μM NiCl2.6H2O 6 mg

0.06 μM KI 1 mg

Make up 200 ml

Solution 4 10X

555 μM myo-Inositol 1000 mg

3 μM Thiamine 10 mg

5 μM Pyridoxine 10 mg

8 μM Nicotinic acid (Niacin) 10 mg

2 μM Calcium Pantothenate 10 mg

0.04 μM Biotin 0.1 mg

(Make separate stock of biotin at 10 mg/ml dissolving first in 2 drops of 1 M NaOH. Add 100 μl)

Make up 10 ml

Calcium Nitrate Solution for 100 ml

0.275 M Ca(NO3)2 6.5 g

10 mM MES 10 ml 0.1 M MES pH 6

Autoclave

3.1.2 Preparation of tobacco leaf protoplasts

1. Select young, fully expanded leaves of tobacco (Nicotiana tabacum var. Xanthii) from plants grown in a controlled environment chamber at 20ºC in a 16h light/8h dark regime.

2. Avoid damaged or infested leaves; harvest 2 leaves for 4 transfection assays.

3. Sterilise leaves by soaking in 7% Domestos for 10 min at room temperature (RT).

4. Remove bleach and wash 4X in sterile water (~400 ml/wash).

5. Dry leaves by blotting gently with absorbent paper.

6. Peel the lower epidermis of the leaf off with a pair of fine forceps and place the leaves with exposed areas downwards onto 15 ml of enzyme solution in a 9 cm petri dish. Fill two dishes with peeled leaf material.