1. A Biological Guillotine
Heads and tails of messenger RNA decay in plants

3. University of Melbourne

4. Bio21 Institute

5. Ph.D. Program

6. Decapping
Decapitating

7. Nudix Hydrolases
A diverse superfamily of pyrophosphatases found in all three domains – bacteria, eukarya and archaea
0 in Mycoplasma pneumonia to 33 in Frankia sp. EAN1pec
Substrates - nucleoside diphosphate linked to another moiety X, hence the name Nudix
Nudix motif – 23 amino acids, the catalytic site of this enzyme superfamily

8. Type of substrate Example NDP-X format
Nucleoside triphosphates
8-oxo-dGTP/dGTP
NDP-P
Dinucleoside polyphosphates
Ap4A
NDP-P(n)A
Nucleotide sugars
ADP-ribose
NDP-sugar
mRNA
Capped (7-methyl guanosine) RNA
NDP-P-RNA
Nicotinamide dinucleotides
NADH
NDP-ribose-nicotinamide
Coenzyme A compounds
Coenzyme A
NDP(P)-pantothenate-β-mercaptoethylamine

9. Enzyme Organism Publication
MutT enzyme
E. coli
(Abeygunawardana et al., 1995)
GDP-mannose mannosyl hydrolase
(Gabelli et al., 2004)
ADP-ribose pyrophosphatase
(Gabelli et al., 2001)
Coenzyme A pyrophosphatase
Deinococcus radiodurans
(Kang et al., 2003)
DR1025
D. radiodurans
(Ranatunga et al., 2004)
DR0079
(Buchko et al., 2004)
MTH1
Homo sapiens
(Mishima et al., 2004)
H. sapiens
(Zha et al., 2006)
(Shen et al., 2003)
Ap4A hydrolase
(Swarbrick et al., 2005)
Lupinus angustifolius
(Swarbrick et al., 2000)
Caenorhabditis elegans
(Bailey et al., 2002)
Mycobacterium tuberculosis
mRNA decapping enzyme
Schizosaccharomyces pombe
(She et al., 2006)
U8 snoRNA decapping enzyme
X. laevis
(Scarsdale et al., 2006)
Unknown Nudix enzyme
Pyrobaculum aerophilum
(Wang et al., 2002)

10. Glu-53
Glu-57
Nudix box

11. Part I Bioinformatics Analysis

12. Bioinformatics Analysis
(1) The HMMs of the seed and full alignments for the superfamily of Nudix hydrolases from the PFAM database were used to scan the proteome of A. thaliana (E-value < 0.01); The total non-redundant sequences from both the seed and full alignment based searches were considered for identification purposes
93 sequences
(2) HMM model for Nudix hydrolases derived from the two-element fingerprint for Nudix hydrolase in the PROSITE database was used to scan the proteome of A. thaliana (E-value < 0.01)
151 sequences
(3) The combined non-redundant matches from the dual searches were further scrutinized with the conserved sequence ‘REXXEE’ to narrow the search to exact Nudix hydrolases
– 26 sequences
Final manual analysis established the presence of 25 Nudix hydrolases encoded by the Arabidopsis genome
PFAM Database
Prosite Database
Common
Sequences

14. Size of encoded protein
Gene ID
Size of encoded protein
Family
At1g30110
175
ApnA hydrolase
At3g10620
216
At5g06340
227
At1g12880
203
DIPP
At1g14860
176
At1g73540
198
At1g18300
207
At2g01670
182
At3g12600
180
At3g26690
202
At1g28960
285
Coenzyme A pyrophosphatase
At2g33980
302
At5g45940
222
At2g04430
283
GFTNE
At2g04450
At4g12720
282
At5g47240
At5g47650
278
At1g68760
147
Dihydroneopterin triphosphate hydrolase
At1g79690
772
Isopentenyl diphosphate isomerase
At2g42070
280
Unknown
At4g11980
309
ADP-ribose pyrophosphatase
At5g13570
373
mRNA decapping enzyme
At5g20070
438
NADH hydrolase
At4g25440
668

15. Intron Number

16. What are intron phases ?

17. Intron Phases

20. Motif 4 – A Unique Motif
A single motif (motif 4) was present in all members of the GFTNE family and was also found in both a member of the ApnA hydrolase family (At1g30110) and the only member of the NADH hydrolase family (At5g20070)
Always found C-terminal to the Nudix box

22. Nudix box
Motif 4

23. Motif 4 A case of an exon shuffling event during evolution
A likely substrate binding domain
Flexible region within distal helix
Nudix box
Motif 4

25. Publication…….

26. Characterization of the mRNA decapping enzyme in Arabidopsis thaliana
Part II
Characterization of the mRNA decapping enzyme in Arabidopsis thaliana

28. Decapping
Decapitating

29. mRNA Decapping Enzyme

30. cDNA coding for mRNA decapping enzyme
1000 bp
1100 bp M

31. His-tag recombinant protein

32. GST-tag recombinant protein

34. Protein folding and Inclusion Bodies

35. Expression Conditions
Vector
Fusion
Temp
Induction of
expression
Solubility
Yields of purified protein
Primary problems
encountered
pDESTTM15
GST
(N-terminus)
37ºC
Yes
Insoluble -
Insolubility
23ºC
15ºC
Soluble
Low
Low yields / degradation
pDESTTM17
His
pBADTM49
Thioredoxin (N-terminus) No
Lack of induction
pGEX-6P-3
High
Degradation

36. Degradation of GST-AtDCP2

37. Disorder in AtDCP2 protein

38. Proteases and Protease inhibitors

39. Proteases in the litterature – ones which contain a PDZ domain
Localisation
Publication
DegS
Periplasm
(Walsh et al., 2003)
DegP
DegQ
Tsp
(Beebe et al., 2000)
ClpA
Cytoplasm
Levchenko et al .(1997)
ClpB
Levchenko et al. (1997)
ClpX
ClpY
Lee et al .(2003)

40. PDZ domains

41. PDZ binding domain

43. Mutation of the PDZ binding domain

44. Another strategy – His tag at the C-terminal end (With modified PDZ-binding domain)

45. Comparison of stability of AtDcp2

46. Radiolabeled cap assay – Assaying Decapping

48. Mutational Analysis

49. Wildtype and 10 Mutant proteins

52. Fold reduction in decapping activity
Nudix Box Mutations
Protein identity
Decapping activity /- SEM
( % Decapping / min )
Fold reduction in decapping activity
GST-AtDcp2-PD-His10
3.85 +/ -
E154A /
350
E158A /
160

53. A Deletion Mutant – Kh like motif

55. The KH-like motif

57. Positive charged amino acids in BoxB

61. Publication

62. Thank You