Translation regulation of Runx3.

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.