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Translation regulation of Runx3.

Abstract Runx3 protein products that are translated from the distal (P1)- and proximal (P2)-promoter transcripts appear on Western blots as a 47-46kDa doublet corresponding to full-length proteins bearing the P1- and P2-N-termini respectively. An additional 44kDa protein band, the origin and nature of which was unclear, is also detected. Transfection of full-length Runx3 cDNA bearing the P2 N-terminus (P2-cDNA) into HEK293 cells resulted in expression of both 46 and 44kDa proteins. Sequence analysis of the P2-cDNA revealed an in-frame ATG 90bp downstream (+90ATG) of the proximal +1ATG. Insertion of an N-terminal HA-tag into P2-cDNA immediately downstream of the +1ATG produced HA-tagged 46kDa and untagged 44kDa proteins, consistent with the possibility that the latter was translated through initiation at the internal +90ATG site. Deleting or blocking the activity of the +1ATG, the natural cap-dependent translation initiation site in P2-cDNA, abrogated production of the 46kDa Runx3 protein while facilitating production of the 44kDa product. These findings supported the notion that Runx3 44kDa protein resulted from internal translation initiation at the +90ATG. Northern blot and RT-PCR analyses performed on RNA from P2-cDNA transfected cells showed a single transcript and product respectively, of the expected size, ruling out the possibility that the 44kDa protein was translated from transcripts originating at a cryptic promoter or produced by alternative splicing. Taken together, the data indicate that the 44kDa protein results from translation initiation at the internal ATG and that Runx3, like its family members Runx1 and Runx2, contains a mechanism for internal mRNA translation initiation.
PMID
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Authors

Mayor MeshTerms
Keywords
Journal Title blood cells, molecules & diseases
Publication Year Start




PMID- 20554226
OWN - NLM
STAT- MEDLINE
DCOM- 20101207
LR  - 20100803
IS  - 1096-0961 (Electronic)
IS  - 1079-9796 (Linking)
VI  - 45
IP  - 2
DP  - 2010 Aug 15
TI  - Translation regulation of Runx3.
PG  - 112-6
LID - 10.1016/j.bcmd.2010.04.001 [doi]
AB  - Runx3 protein products that are translated from the distal (P1)- and proximal
      (P2)-promoter transcripts appear on Western blots as a 47-46kDa doublet
      corresponding to full-length proteins bearing the P1- and P2-N-termini
      respectively. An additional 44kDa protein band, the origin and nature of which
      was unclear, is also detected. Transfection of full-length Runx3 cDNA bearing the
      P2 N-terminus (P2-cDNA) into HEK293 cells resulted in expression of both 46 and
      44kDa proteins. Sequence analysis of the P2-cDNA revealed an in-frame ATG 90bp
      downstream (+90ATG) of the proximal +1ATG. Insertion of an N-terminal HA-tag into
      P2-cDNA immediately downstream of the +1ATG produced HA-tagged 46kDa and untagged
      44kDa proteins, consistent with the possibility that the latter was translated
      through initiation at the internal +90ATG site. Deleting or blocking the activity
      of the +1ATG, the natural cap-dependent translation initiation site in P2-cDNA,
      abrogated production of the 46kDa Runx3 protein while facilitating production of 
      the 44kDa product. These findings supported the notion that Runx3 44kDa protein
      resulted from internal translation initiation at the +90ATG. Northern blot and
      RT-PCR analyses performed on RNA from P2-cDNA transfected cells showed a single
      transcript and product respectively, of the expected size, ruling out the
      possibility that the 44kDa protein was translated from transcripts originating at
      a cryptic promoter or produced by alternative splicing. Taken together, the data 
      indicate that the 44kDa protein results from translation initiation at the
      internal ATG and that Runx3, like its family members Runx1 and Runx2, contains a 
      mechanism for internal mRNA translation initiation.
CI  - 2010 Elsevier Inc. All rights reserved.
FAU - Bone, Karen Rae
AU  - Bone KR
AD  - Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot,
      Israel.
FAU - Gruper, Yaron
AU  - Gruper Y
FAU - Goldenberg, Dalia
AU  - Goldenberg D
FAU - Levanon, Ditsa
AU  - Levanon D
FAU - Groner, Yoram
AU  - Groner Y
LA  - eng
GR  - Medical Research Council/United Kingdom
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20100602
PL  - United States
TA  - Blood Cells Mol Dis
JT  - Blood cells, molecules & diseases
JID - 9509932
RN  - 0 (Codon, Initiator)
RN  - 0 (Core Binding Factor Alpha 3 Subunit)
RN  - 0 (Protein Isoforms)
RN  - 0 (Runx3 protein, mouse)
SB  - IM
MH  - Animals
MH  - Blotting, Western
MH  - Cell Line
MH  - Codon, Initiator
MH  - Core Binding Factor Alpha 3 Subunit/*genetics
MH  - Dogs
MH  - Mice
MH  - Peptide Chain Initiation, Translational/*genetics
MH  - Protein Biosynthesis/*genetics
MH  - Protein Isoforms/*biosynthesis
MH  - Sequence Analysis, DNA
EDAT- 2010/06/18 06:00
MHDA- 2010/12/14 06:00
CRDT- 2010/06/18 06:00
PHST- 2010/04/05 00:00 [received]
PHST- 2010/04/06 00:00 [accepted]
PHST- 2010/06/18 06:00 [entrez]
PHST- 2010/06/18 06:00 [pubmed]
PHST- 2010/12/14 06:00 [medline]
AID - S1079-9796(10)00115-4 [pii]
AID - 10.1016/j.bcmd.2010.04.001 [doi]
PST - ppublish
SO  - Blood Cells Mol Dis. 2010 Aug 15;45(2):112-6. doi: 10.1016/j.bcmd.2010.04.001.
      Epub 2010 Jun 2.