Alternative translation initiation generates the N-terminal truncated form of RUNX1 that retains hematopoietic activity.

Transcription factor RUNX1 plays a crucial role in hematopoiesis, and its activity is tightly regulated at both transcriptional and post-translational levels. However, translational control of RUNX1 expression has not been fully understood. In this study, we demonstrated that RUNX1b mRNA is translated from two alternative initiation sites (Met-1 and Met-25), giving full-length RUNX1b and a shorter protein lacking the first 24 amino acids (RUNX1ΔN24). Presence/absence of strong Kozak consensus sequences around Met-1 determines which initiation site is mainly used in RUNX1b cDNA. Selective disruption of either Met-1 or Met-25 abrogates expression of the corresponding protein, while facilitating production of another protein. The RUNX1b cDNA containing 65bp natural promoter sequences mainly produces full-length RUNX1b in human cord blood cells, but disruption of Met-1 in this cDNA also induced translation from Met-25. Consistent with these data, disruption of endogenous RUNX1b around Met-1 using CRISPR/Cas9 induced selective expression of RUNX1ΔΝ24 in several leukemia cell lines. RUNX1ΔN24 protein is more stable than full-length RUNX1b and retains hematopoietic activity. We also found that FLAG-tagged full-length RUNX1 showed altered activity, indicating the influence of N-terminal FLAG-tag on RUNX1 function. The alternative translation initiation of RUNX1b may participate in fine-tuning of RUNX1 activity.