Purification of TAP-tagged XRN2 and XRN3

Slide 1: 

Purification of TAP-tagged Arabidopsis thaliana’s proteins XRN2 and XRN3 for isolation of protein complexes Supervisor: Joanna Kufel, PhD Monika Bazyl, MSc

Slide 2: 

Mechanisms of normal mRNA degradation: the deadenylation dependent pathway (Garneau et. al., 2007)

Slide 3: 

Aligned members of the XRN family (Kastenmayer et. al., 2000)

Slide 4: 

Transformation of wild type (Col0) Arabidopsis thaliana plants with AtXRN2-TAP and AtXRN3-TAP constructs and transformation of insertion mutant xrn2-2 with AtXRN2-TAP construct RESULTS (Rubio et. al., 2005) two copies of the protein A IgG binding domain (2xIgG-BD) an eight amino acid sequence corresponding to the 3C protease cleavage site (3C) six histidine stretch (6xHis) nine repeats of the myc epitope (9xmyc)

Slide 5: 

Selection of homozygotic lines on MS medium with gentamicin PCR analysis on DNA isolated from lines: xrn2-2/XRN2-TAPa (line 1 and 6), Col0/XRN3-TAPa (line 2,4,7,9) Col0/ XRN2-TAPa (line 3 and 8) and Col0 (line 5 and 10). In reactions from lines 1-5 the primers complemantar to the sequence of XRN3 and TAP tag have been used and in lines 6-10 to the sequence of XRN2 and TAP tag.

Slide 6: 

RT-PCR analysis of Arabidopsis lines Products of PCR made on cDNA obtained from Col0/XRN3-TAP (line 1-4), lines 1’-4’- negative controls of RT-PCR; used primers are complemantar to the 3’ end of XRN3 gene and TAP tag Products of PCR made on cDNA obtained from Col0/XRN2-TAP (line 1 and 2) and xrn2-2/XRN2-TAP (line 3), lines 1’-3’ – negative controls; used primers are complemantar to the 3’ end of XRN2 gene and TAP tag A B

Slide 7: 

Western blot analysis Western blot analysis with anty-Myc antibodies of extracts from Col0, Col0/XRN3-TAPa, Col0/XRN2-TAPa and xrn2-2/XRN2-TAPa;

Slide 8: 

Transformation of Arabidopsis thalina insertion mutants xrn2-1 and xrn2-3 with AtXRN2-TAP construct Western blot analysis with anty-Myc antibodies of extracts from Col0, Col0/XRN3-TAPa, xrn2-1/XRN2-TAPa and xrn2-3/XRN2-TAPa

Slide 9: 

(Rubio et. al., 2005)

Slide 10: 

Western blot of different fractions obtained during the XRN3-TAP purification: 1- total protein extract 2- unbound protein after IgG bead incubation 3- eluate from IgG beads using 3C protease 4- discarded Ni-NTA column flow through fraction 5- final purified protein Col0/XRN3 Col0

Slide 11: 

Final purified protein extract from Col0/XRN3 XRN3 (5'-3' exoribonuclease 3) [Arabidopsis thaliana]   cell wall-plasma membrane linker protein homolog [Arabidopsis thaliana]   GAST1 protein homolog   protease inhibitor/seed storage/lipid transfer protein (LTP) family protein [Arabidopsis thaliana] ribosomal protein L12 Elongation factor Tu (EF-Tu) 30S ribosomal protein S3 acid phosphatase class B family protein SKS4 (SKU5 Similar 4); copper ion binding oxidoreductase Final purified protein extract from Col0 (control) cell wall-plasma membrane linker protein homolog [Arabidopsis thaliana]   GAST1 protein homolog   protease inhibitor/seed storage/lipid transfer protein (LTP) family protein [Arabidopsis thaliana] acid phosphatase class B family protein leucine-rich repeat family protein jacalin lectin family protein cell wall-plasma membrane linker protein homolog protease inhibitor/seed storage/lipid transfer protein (LTP) family protein beta-glucosidase ribosomal protein L16 Peroxisomal (S)-2-hydroxy-acid oxidase ribosomal protein S8 Proteins identification by mass spectrometry

Slide 12: 

One step purification Western blot of different fractions obtained during the XRN3-TAP purification on IgG beads: 1,1’- total extracts from Col0/XRN3 and Col0 2,2’- unbound protein after IgG bead incubation 3,3’- eluate from IgG beads using 3C protease Western blot of different fractions obtained during the XRN3-TAP purification on NiNTA column: 1,1’- total extracts from Col0/XRN3 and Col0 2,2’- discarded Ni-NTA column flow through fraction 3,3’- eluate from NiNTA column

Slide 13: 

Done: transgenic plants overexpressing XRN3-TAP protein setting up conditions for tandem affinity purification Problems: impossible overexpression of XRN2-TAP protein low efficiency of purification To do: pull-down experiments isolation of nuclei

Slide 14: 

Literature Kastenmayer JP, Green PJ. (2000). Novel features of the XRN-family in Arabidopsis: evidence that AtXRN4, one of several orthologs of nuclear Xrn2p/Rat1p, functions in the cytoplasm. Proc Natl Acad Sci U S A. 97:13985-90. Rubio V, Shen Y, Saijo Y, Liu Y, Gusmaroli G, Dinesh-Kumar SP, Deng XW. (2005). An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation. Plant J. 41:767-78. Puig O, Caspary F, Rigaut G, Rutz B, Bouveret E, Bragado-Nilsson E, Wilm M, Seraphin B. (2001). The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods. 24:218-29. Johnson AW. (1997). Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively. Mol Cell Biol. 17:6122-30. Garneau NL, Wilusz J, Wilusz CJ. (2007). The highways and byways of mRNA decay. Nat Rev Mol Cell Biol. 8:113-26.