SPARP-seq reveals poly(A) tail shortening as a hallmark of the active human translatome

The poly(A) tail at the 3’ end of mRNAs plays a critical role in the synthesis of proteins, but its dynamic relationship with the translation process has been unclear. Here, using a novel single-molecule approach SPARP-seq that integrates poly(A) assessment with ribosome profiling, we demonstrate that poly(A) tail shortening is a universal hallmark of active translation in human cells. We find that most mRNAs begin translation on single ribosome (monosome) with long tails that progressively shorten as additional ribosomes are recruited. In contrast, highly expressed housekeeping genes are translated efficiently on monosomes and possess intrinsically short poly(A) tails. Furthermore, we find that translationally repressed transcripts, such as those retaining introns, are trapped on monosomes with long tails. Pharmacological inhibition of translation induces global poly(A) tail lengthening, confirming that deadenylation is coupled to ongoing translation. These findings reveal a dynamic system where the poly(A) tail integrates mRNA status with translational fate, defining the monosome as a central hub for post-transcriptional control.