Chromatoid body integrates piRNA, SMG6 and m⁶A pathways to control mRNAs in the male germline

Abstract

Spermatogenesis requires tightly controlled transcriptome regulation, supported by the PIWI-interacting RNA (piRNA) and nonsense-mediated decay (NMD) pathways, both concentrated in the chromatoid body (CB) of haploid spermatids. We previously showed that the NMD endonuclease SMG6 interacts with the piRNA-binding protein PIWIL1, and that loss of either factor results in overlapping mRNA dysregulation, suggesting functional cooperation. Here, we demonstrate that SMG6 and PIWIL1 assemble with shared RNA-regulatory proteins and bind common mRNA targets in the testis. Functional experiments in GC-2spd cells revealed cooperative regulation of selected transcripts, including Ccny and Taf1d, and established that SMG6 is required for piRNA-guided degradation of these targets, implicating its endonuclease activity in the piRNA pathway. Target mRNAs were mA-methylated, and this modification shaped their expression levels, SMG6 binding, and CB localization. Moreover, intact target mRNAs accumulated in CBs isolated from Smg6-cKO testes, indicating defective CB-associated decay. Together, these findings uncover a key role for the CB in mRNA regulation through coordinated action of the NMD and piRNA pathways.