We believe the putative At3G48190 protein is likely to be an artifactual chimera of two
separate proteins, a PWWP-RRM protein and an ATM kinase protein, for we know of no direct
data demonstrating the PWWP-RRM sequences are physically joined to the AtATM homologous
region, and there is significant evidence against At3G48190 being a single, composite protein in
other plants:
1) In plant species where EST sequences encoding proteins homologous to the N-terminal
portion of AtATM 1-836 AA are known, translated C-terminal regions are highly
homologous and contain stop codons
>Brassica oleracea
QKPKNPPISVESMKQNLLMMTAMLDKSGDHLSRETKAKLKSDISALLEKISSMPSSSSSSS*
>Raphanus sativus
RELNEKPTSVDSVKQNLLMITAMLEKSGDRLSRETKAKLKSEVTELLEKVSSMPCSSSSSSSA*
>Quercus petraea
ANPGEAPPLDSIRQNLQMMTSMLEKSGDNLSPEMRAKLESEIKGLLHKVTSMAGSSST*
> Manihot esculenta
SMPAEAPPIDFIRQNLEMMTSMLEKSGDNLSLEMRAKLETEIKGLLKKVTSMXXSSSS*
> Theobroma cacao
PHLGDAPPIDFIRQNLEMMTSMLERSGDNLSPEMKAKLESEIKGLLKKVSSLPNSSSS*
>Populus trichocarpa
PKLAEAPPIDFIRQNLEMMTSMLEKSGDNLSPEMRAKLEIEIKGLLKKVSSLPSSSS*
> Euphorbia esula
PAPAPAPPIDLIRQNLEMMTSMLEKSGDNLSPEMRAKLETEIKGLLKKVSSLPGSSSSS*
>Carica papaya
CHPGEVPPIDFIKQNLEAMTSMLEKSGDNLSPEMRVKLESEIKGLLEKISSMPSSSSS*
>Gossypium hirsutum
PRLGDAPPIDVIKKNLEMMTSMLEKSGDNLSPEMKAKLESEIKGLLKKVSHCPGSSS*
> Glycine max
PRLGDAPPIDVIKKNLEMMTSMLEKSGDNLSPEMKAKLESEIKGLLKKVSHCPGSSS*
> Solanum tuberosum
PSNGEGPDLVVIKQNLESMTTMLEKAGDNISPEMKAKLESEVKGFLEKVSNMVGSSSS*
> Solanum lycopersicum
GAVPSNGEGPDLVVIKQNLESMTTMLEKAGDNISPEIKAKLESEVKGFLEKVSTMVGSSSS*
> Theobroma cacao
SSKKSLASQKSADQASQNSADQASQLNFFRHKLEMLTSMLEKSDEKMSSEIKSKVHSEIKGLLEKVNTMVKSSS*
>Nicotiana tabacum
PSNGEGPDLVVIKQNLEAMTSMLEKAGDNISPEMRAKLENEVKGFLKKVSSMVGSSSS*
>Capsella rubella
PKPNSIPTSVESMRQNLLMMTAMLENSGDSLSRETKAKLKSEITGLLEKVSSMPSSSSS*
Translation of EST sequences containing C-termini of PWWP-RRM proteins from different species. (Note
that all have in frame stop codon).
2) The deduced C-terminus of Arabidopsis PWWP-RRM N-terminal portion of At3G48190 ends
with a stop codon and is highly homologous to proteins from other species.
1 MKLQNPDKKT LREGFSQESS VVALDSGVLA MSGLKCDGKF PVKDVLMEEG
51 GDKVRKIQVS GGNISLVVDF SGARTSSNNF FESNASCVNE NLVKGNGYRE
101 DETQEFLVGN LVWVMTKYKK WWPGEVVDFK ADAKESFMVR SIGQSHLVSW
151 FASSKLKPFK ESFEQVLNQR NDNGFFDALQ KAMSLLSNSL KLDMTCSCIA
201 DGNGIVSAQN ITTRKNKPLI LREFSVDRLE PKEFVTQLKN IAKCVLNAGV
251 LESTVMQSQL SAFYTLFGHK QIPMAQLHEN EGRKSFTAKM SDSKFIGSPS
301 ICAGNSRKRF RKEWFRKFVS EVDNVSARDD LVNVPPSDLI SKLKLLAVGY
351 NCSEETENIG LFEWFFSKFR ISVYHDENAY KMQLANMAGF KDLMLATNAN
401 RGTVQKTLKS KKIGKSKMEP LNGVSVADTE QKTFELQISK KSNIESLNGV
451 SVADTEQKTF ELQILEKSNI ESLNGVSTPN IDHEASKSNN SGKTKINHII
501 GHSNFPSSVA KVQLAKDFQD KLLVQAPDRK AMTADTLSRP AAILVPDLNS
551 GGNALGTAEF DHMQRPETLI QHNVCPQEEK TPRSTILNFQ VTAHQGVSGT
601 QFVSSQPTSY KHFTSADLFT YSGKKKRGRK RKNAEELPIV AHASATTGIP
651 DLNGTNTEPT LVLPQVEPTQ RRRRRKKEES PNGLTRGITI LFLKFSSQVS
701 MPSRDDLTST FSAFGPLDSS ETHVSEEFSG AQVAFVSSAD AIEAVKSLEK
751 ANPFGETLVN FRLQQKLITV QRNIAPRMPV ISHVSPVPKP NNIPTSMDAM
801 RQNLLMMTAM LEKSGDSLSR ETKAKLKSEI TGLLEKVSSI PSSSSS*
The predicted C-terminus sequence is shown, with the sequence generated by extending reading frame
beyond putative AtATM 2/3 exon splice site is shown in red. The sequence used to search the plant EST
database is italicized.
Comparison of predicted C- terminal sequence from Arabidopsis At3G48190 PWWP-RRM protein region
AA 788-836 (annotated as N-terminal portion of AtATM) with C-terminal sequences ending with stop
codon generated from ESTs from different plant species.
Plant EST sequences were searched with AT3G48190 protein fragment (AA 788-836). No EST sequences
encompassing this region in Arabidopsis are represented in the Arabidopsis EST database. EST clones
from other species (including closely related species Brassica oleracea, Raphanus sativus and Capsella
rubella) have in frame stop codons supporting that the N –terminal portion of the Arabidopsis AtATM
protein is most likely a separate PWWP-RRM protein. (Note that the evidence of PWWP- RRM region of
AtATM mRNA sequence was generated by RT-PCR (that could potentially generate aberrant product)
and has no other independent confirmation in ESTs).
3) The deduced Arabidopsis ATM N-terminal sequence is highly homologous to the
annotated N-terminal ATM regions from other species lacking PWWP-RRM
extension
4)
In most species PWWP-RRM protein and ATM protein are located on separate
chromosomes
While in the Brassicacea a putative PWWP-RRM protein is located in close proximity (1.6 kb) to the
chromosomal region encoding the ATM catalytic domain, in other species homologs of At3G48190 are
located on separate chromosomes. For example, the Theobroma cacao gene encoding 1076 AA PWWPRRM protein homologous to PWWP-RRM N-terminal segment of AtATM region 50-856 AA is located on
chromosome 10, while the gene encoding a 3039 AA protein homologous to AtATM region 820-3856 AA
containing ATM catalytic domain is located on chromosome 3. (In Medicago truncatula, Solanum
lycopersicum, Phaseolus vulgaris, Citrus sinensis etc gene arrangement is similar to Theobroma cacao).