Antisense RNAs constitute a significant part of the human genome, yet their functions in key physiological processes remain poorly understood due to complexity and the lack of suitable models. Here, we report the intricate regulatory co-ordination between antisense RNA and its encoded protein involved in safeguarding the essential physiological processes during interphase and mitosis, using antisense RNA ARHGEF17-AS1 and its encoded protein, MSEP as a model system. Mechanistically, ARHGEF17-AS1 orchestrates actin-cytoskeleton organization in interphase and mitotic spindle pole integrity via the RNF10-RPS3-Twinfilin-Dynein pathway, involving monoubiquitination dependent functional switch of RPS3 during interphase to mitosis. While MSEP ensures spindle integrity through the TPX2-Aurora A pathway. Thus, our investigations provide molecular insights into how ARHGEF17-AS1 and its protein product MSEP maintain critical cell cycle stage-specific processes through unique and coordinated actions. These findings establish a compelling model for investigating the collaborative actions between antisense RNA and its encoded protein in cellular physiology.