In Vivo Analysis of Plant Intron Splicing

Summary:

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

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, DundeeDD2 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:

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 110X

10.3 mM NH4NO38.25 g

9.4 mM KNO39.5 g

1.5 mM CaCl2.2H2O2.2 g

0.75 mM MgSO4.7H2O1.85 g

0.62 mM KH2PO40.85 g

Make up 200 ml

Solution 2 (for To+ only) 10X

100 μM FeSO40.278 g

100 μM Na2EDTA0.372 g

Make up 200 ml

Solution 3200X

16 μM H3BO3200 mg

0.6 μM MnSO4200 mg

3.5 μM ZnSO4.7H2O20 mg

0.12 μM CuSO4.5H2O6 mg

0.22 μM AlCl36 mg

0.13 μM NiCl2.6H2O6 mg

0.06 μM KI1 mg

Make up 200 ml

Solution 410X

555 μM myo-Inositol1000 mg

3 μM Thiamine10 mg

5 μM Pyridoxine10 mg

8 μM Nicotinic acid (Niacin)10 mg

2 μM Calcium Pantothenate10 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 Solutionfor 100 ml

0.275 M Ca(NO3)26.5 g

10 mM MES10 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.

7. Cover in aluminium foil and incubate overnight at 25ºC.

8. Released protoplasts fall to the bottom of the petri dish; pipette protoplasts onto a sterile 100 µm sieve (use sterile disposable pipettes).

9. Transfer filtered protoplasts into 2x sterile 10 ml plastic round bottom tubes (round bottom tubes used throughout the protocol) and centrifuged at low speed (32xg) for 5 min at RT.

10. Remove supernatant and resuspend protoplasts in 10 ml To- and divide into 2x10 ml tubes.

11. Prepare 4x10 ml tubes with 2.5 ml 16% sucrose and gently layer 5 ml of protoplasts onto the sucrose solution.

12. Centrifuge samples at 130xg for 5 min at RT.

13. Protoplasts band at the sucrose/protoplast media interface and are collected with a glass pipette and transferred to two new 10 ml tubes and suspended in 10 ml of To-.

14. The number of protoplasts is determined by haemocytometer.

15. Centrifuge protoplast suspension at 32xg for 5 min at RT and the supernatant removed.

16. Resuspend protoplasts in To- to a concentration of approximately 1x106 protoplasts/ml. (This should give a dark green colour in a volume of around 2 ml).

3.1.3 PEG-mediated transfection of tobacco protoplasts

1. Precipitate up to 30 μg plasmid DNA with 1/16 volume 5M NaCl and 2.5 volumes of ethanol. For co-transfection of two plasmids (e.g. splicing reporter and protein factor), 30 µg of each plasmid (60 µg total) are precipitated.

2. Resuspend plasmid DNA pellet in 20 μl water.

3. Aliquot 200 μl protoplasts (approximately 200,000 protoplasts) per assay into a 10 ml round bottom tubes (each experiment usually consisted of 8 assays).

4. Add plasmid DNA and mix gently.

5. Add 200 μl PEG solution drop-wise, mixing gently while adding and leave to stand for 20 min.

(Note: There is enough time to do 8 assays comfortably in a 20 min period – time each assay accurately).

6. Add 4x 200 μl Calcium Nitrate solution very slowly and mix carefully.

7. Finally, add 4 ml Calcium Nitrate solution (giving a final volume of 5 ml) and leave to stand for 20 min.

(Note: 4 assays can be done in a 20 min period so a second set of assays can be done while the first is left to stand).

8. Centrifuge assays at 32xg for 3 min at RT.

9. Remove supernatant and resuspend protoplasts in 5 ml To+ by gentle shaking.

10. Pour the protoplasts gently into a small petri dish (3 cm diameter) and seal with Nescofilm.

11. Incubate at 25ºC for 24 hrs under light.

12. Collect protoplasts by centrifugation at 32xg for 5 min at RT.

13. Remove supernatant and transfer protoplasts to microfuge tube with a glass pipette and centrifuge at 110xgfor 1 min.

14. Remove as much supernatant as possible and flash freeze protoplasts in liquid nitrogen.

15. Store at -80ºC until RNA extraction.

3.2 Protocol 2 Transfection of Arabidopsis cell suspension protoplasts

3.2.1 Solutions

Growth medium for Arabidopsis cell suspension for 1 liter

Murashige and Skoog medium including Gamborg B5 vitamins (Duchefa)4.4 g

87 mM Sucrose30 g

4.5 µM 2,4-D (10 mg/ml) 100 µl

pH 5.8 with KOH

Filter sterilise.

Store at 4ºC

Make separate stock of 2.4-Dichlorophenoxyacetic acid (2.4-D) at 10 mg/ml dissolving in dimethyl sulfoxide. Store aliquotes of 100 µl at -20ºC.

B5-0.28 M Sucrose Solutionfor 1 liter

Gamborg B5 medium including vitamins (Duchefa)3.18 g

0.28 M Sucrose95.76 g

pH 5.5 with KOH

Filter sterilise

Store at 4ºC

B5-0.34 M Glucose Mannitol (GM) Solutionfor 1 liter

Gamborg B5 medium including vitamins (Duchefa) 3.18 g

0.17 M Glucose30.5 g

0.17 M Mannitol 30.5 g

4.5 µM 2.4-D (10 mg/ml) 100 µl

pH to 5.5 with KOH

Filter sterilize

Store at 4ºC

Protoplasting Enzyme Solutionfor 100 ml

1% Cellulase (Duchefa)1 g

0.2% Macerozyme (Duchefa)0.2 g

Make up to 100 ml with B5-0.34 M GM Solution

Stir slowly for 30 min. Filter through Whatmann paper.

Filter sterilise

Store aliquots of 25 ml at -20ºC

PEG Solutionfor 10 ml

30% PEG 60003 g

0.1 M Ca(NO3)2.4H2O0.24 g

0.45 M Mannitol0.82 g

pH 9.0 with KOH (adjust pH carefully, may take several hours)

Filter sterilise

Store at -20ºC

Calcium Nitrate Solutionfor 100 ml

0.275 M Ca(NO3)2. 4H2O 6.5 g

Filter sterilize

Store at room temperature

3.2.2 Preparation of protoplasts from Arabidopsis cell cultures

1. Grow Arabidopsis cell suspension cultures in 40 ml of the growth medium in 250 ml flasks at 23°C in the dark with shaking at 200 rpm (Model G25 Incubator Shaker - New Brunswick Scientific Co. Inc). Dilute the cells every 7 days into 3 parts of growth medium.

2. Transfer 40 ml of a 5 days post-subculture Arabidopsis cell suspension into a sterile 50 ml centrifuge tube and spin at 1500 rpm (Heraeus Megafuge 1.OR) for 5 min at RT.

3. Remove supernatant, add 25 ml of enzyme solution and fill up to 50 ml (total volume) with B5-0.34 M GM solution.

4. Resuspend the cells and transfer 25 ml to each of two large petri dishes (15 cm diameter).

5. Incubate the plates in an incubator shaker at 50 rpm for 3-4 h at 25°C. Check the cells every hour during incubation.

6. Filter the cells through a sterile 100 µm sieve, collecting the filtrate into 50 ml centrifuge tubes.

7. Spin at 1500 rpm for 5 min and discard the supernatant.

8. Resuspend the pellets in 25 ml of B5-0.28 M Sucrose solution and transfer each into two 14 ml round-bottom tubes.

9. Spin at 800 rpm for 7 min. The protoplasts should float on the top of the solution - if not, increase the centrifugation time.

10. Collect the floating protoplasts into a new 14 ml tube, add B5-0.28 M Sucrose solution to fill the tubes.

11. Spin at 1000 rpm for 6 min.

12. Collect all protoplasts into one 14 ml tube, count the protoplast density using a haemocytometer.

13. Resuspend protoplasts in B5-0.28 M Sucrose solution to a density of 1 x 106 protoplasts/ml.

3.2.3 PEG-mediated transfection of Arabidopsis protoplasts

1. Put 5 µl (up to a maximum of 15 µg) of plasmid DNA in 15 µl of water per assay in a microfuge tube.

2. Add 2 x 105 protoplasts in 50 µl per tube, mix protoplasts and DNA gently.

3. Immediately add 150 µl of PEG solution. Mix well.

4. Incubate 15 min at RT in the dark.

5. Wash PEG solution by adding Calcium Nitrate solution in two steps of 0.5 ml.

6. Mix by inverting.

7. Spin 7 min 800 rpm.

8. Remove supernatant.

9. Resuspend in 0.5 ml of B5-0.34 M GM.

10. Incubate protoplasts in tubes laid on the side in the dark at 25°C for 12-24 h.

11. Collect protoplasts by centrifugation for 5 min at 1500 rpm.

12. Remove all supernatant and freeze protoplasts in liquid nitrogen.

13. Store at -80°C until RNA extraction.

3.3 Protocol 3 – Agrobacterium mediated infiltration of Nicotiana benthamiana leaves

Infiltration of Agrobacterium containing expression plasmids into leaves of N.benthamiana provides a second rapid method of expression of splicing reporters and trans-acting factors for splicing analysis. The Agrobacterium-mediated infiltration method is widely used in RNA silencing assays and the method described here is adapted from Voinnet et al. (2003). The splicing reporter is based on the invertase mini-exon construct fused to GFP (Figure 1C) such that splicing can be detected on the basis of fluorescence and by RT-PCR of RNA extracted from the infiltrated region. For expression of proteins, coding sequences are PCR amplified with gene-specific primers that also introduce a 5’ AscI site and a 3’ NotI site. Amplification products were cloned in to pGEM-T Easy (Invitrogen) and then recovered by digestion with AscI and NotI prior to cloning into pGRAB, a derivative of the binarynot clear to me what binary means in this context pGreen II 0229 (Hellens et al., 2000). The plasmid pGRAB contains the 35S promoter and terminator cassette from a derivative of pRTL2 (Carrington and Freed, 1990) with a modified multiple cloning site, containing AscI and NotI sites, inserted between the TEV leader sequence and 35S terminator sequence.