Evaluating Malat1 -derived 3’end sequences for functional transgene expression in human cells using synthetic mRNA delivery

The poly(A) tail is a key structural element found in almost all mammalian messenger RNAs, required for stabilizing the transcript and enhancing translation. Here, we evaluate poly(A) replacement strategies based on sequence elements derived from the long non-coding RNA Malat1. Malat1 undergoes RNase P processing to generate a 3′ end protected by a conserved triple helix. Importantly, this triple helix structure was shown to enhance translation when placed downstream of an open reading frame. We compared Malat1-derived 3′ end motifs with canonical poly(A) tails incorporated into a synthetic reporter RNA encoding a green fluorescent protein. Our results corroborate the central role of the poly(A) tail for efficient expression, showing that the tested Malat1-derived motifs can generally support translation of the reporter mRNA, although they consistently underperform compared to canonical poly(A) tails. Future work should optimize triplex sequence and folding to enhance translation capacity, test additional triplex structures, and explore small molecule to regulate stability and translation of triplex-containing RNAs for applications in synthetic biology and RNA-based therapeutics.