The nucleotidyl transferase URT1-directed mRNA surveillance is critical for embryo and seedling development in Arabidopsis.

In Arabidopsis, the nucleotidyl transferase UTP: RNA URIDYLYLTRANSFERASE (URT1) has been identified as a key mediator of mRNA uridylation and metabolic regulation. While its biochemical function in RNA modification has been established, the physiological relevance of URT1 across different developmental stages remains poorly understood. This study revealed that URT1 exhibits genetic interactions similar to those of EXORIBONUCLEASE 4 (XRN4) during developmental transitions, particularly at the globular embryo stage and during seedling establishment. Simultaneous dysfunction of URT1 and the SUPER-KILLER (SKI) complex resulted in pronounced embryonic lethality during the globular-to-heart stage transition, with residual mortality persisting through seedling development. Small RNA deep sequencing (sRNA-seq) analyses identified certain members of the HEAT SHOCK PROTEIN 90/70 (HSP90/70) families as primary targets of URT1 and SKI2 during embryogenesis. Our findings demonstrate that URT1 cooperates with the SKI complex to suppress the production of RDR6-dependent aberrant small interfering RNAs (siRNAs) from HSP90/70 loci during embryonic development. In addition, transcriptome profiling demonstrated that URT1 selectively targets a distinct subset of XRN4 substrate mRNAs in seedlings, demonstrating a possible role for URT1 in the 5'-3' cytosolic mRNA decay pathway. Notably, through characterization of the prime editor-generated catalytically inactive urt1 allele, we confirmed that the catalytic uridylation activity of URT1 is critical for plant development. Collectively, our findings establish URT1 as an important developmental regulator that orchestrates 5'-3' cytosolic mRNA decay through uridylation activity, highlighting its essential role in maintaining RNA homeostasis during embryogenesis and seedling establishment.