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

University of Melbourne

Bio21 Institute

Ph.D. Program

Decapping Decapitating

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

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

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)

Glu-53 Glu-57 Nudix box

Part I Bioinformatics Analysis

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

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

Intron Number

What are intron phases ?

Intron Phases

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

Nudix box Motif 4

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

Publication…….

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

Decapping Decapitating

mRNA Decapping Enzyme

cDNA coding for mRNA decapping enzyme 1000 bp 1100 bp M

His-tag recombinant protein

GST-tag recombinant protein

Protein folding and Inclusion Bodies

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

Degradation of GST-AtDCP2

Disorder in AtDCP2 protein

Proteases and Protease inhibitors

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)

PDZ domains

PDZ binding domain

Mutation of the PDZ binding domain

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

Comparison of stability of AtDcp2

Radiolabeled cap assay – Assaying Decapping

Mutational Analysis

Wildtype and 10 Mutant proteins

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 +/- 0.128 - E154A 0.011 +/- 0.002 350 E158A 0.024 +/- 0.013 160

A Deletion Mutant – Kh like motif

The KH-like motif

Positive charged amino acids in BoxB

Publication

Thank You