The maternal-to-zygotic transition (MZT) is a conserved step in animal development, where control is passed from the maternal to the zygotic genome. Although the MZT is typically considered from its impact on the transcriptome, we previously found that three maternally deposited Drosophila RNA binding proteins (ME31B, Trailer Hitch [TRAL], and Cup) are also cleared during the MZT by unknown mechanisms. Here, we show that these proteins are degraded by the ubiquitin-proteasome system. Marie Kondo, an E2 conjugating enzyme, and the E3 CTLH ligase are required for the destruction of ME31B, TRAL, and Cup. Structure modeling of the Drosophila CTLH complex suggests that substrate recognition is different than orthologous complexes. Despite occurring hours earlier, egg activation mediates clearance of these proteins through the Pan Gu kinase, which stimulates translation of Kondo mRNA. Clearance of the maternal protein dowry thus appears to be a coordinated, but as-yet underappreciated, aspect of the MZT.

Giardia lamblia is a human pathogen of worldwide importance with limited treatment options. Its unusual molecular biology presents targets for new therapies and the opportunity to explore the fundamental features of important biological mechanisms. We determined the structure of the G. lamblia 80S ribosome by cryoelectron microscopy, revealing how it combines eukaryotic and bacterial features. The structure reveals regions that are rapidly evolving, including depletion of A and U bases from its rRNA. Specific features of the G. lamblia ribosome suggest it is less prone to stall on problematic peptide sequences, and that the organism uses altered ribosome quality control pathways compared to other eukaryotes. Examination of translation initiation factor binding sites suggests these interactions are conserved despite a divergent initiation mechanism. This work defines key new questions regarding ribosome-centric biological pathways in G. lamblia and motivates new experiments to explore potential targetable mechanisms.

During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3' UTR of an mRNA, thus determining much of its post-transcriptional fate. Here, using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding Giardia lamblia cleavage sites for over 2600 genes. We find that G. lamblia uses a AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how G. lamblia lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes that show evidence of alternative polyadenylation. These results suggest that despite pared down cleavage and polyadenylation machinery, 3' end formation still appears to be an important regulatory step for gene expression in G. lamblia.

During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3′ UTR of an mRNA, thus determining much of its post-transcriptional fate. Using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding Giardia lamblia cleavage sites for over 2600 genes. We find that G. lamblia uses an AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how G. lamblia lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes with evidence of alternative polyadenylation. These results suggest that despite pared-down cleavage and polyadenylation machinery, 3′ end formation still appears to be an important regulatory step for gene expression in G. lamblia.

HIGHLIGHTS• Degradation of ME31B requires the PNG kinase, but not fertilization • The ubiquitin-proteasome system degrades ME31B via CTLH E3 ligase and the UBC-E2H/Kondo ubiquitin-conjugating enzyme• The association of ME31B with the CTLH complex does not require PNG activity• PNG kinase mediates the translational upregulation of Kondo at egg activation 3 SUMMARYThe maternal-to-zygotic transition (MZT) is a conserved step in animal development, where control is passed from the maternal genome to the zygotic one. Although the MZT is typically considered from its impact on the transcriptome, we previously found that three maternally deposited Drosophila RNA binding proteins (ME31B, Trailer Hitch[TRAL], and Cup) are also cleared during the MZT by unknown mechanisms. Here, weshow that these proteins are degraded by the ubiquitin-proteasome system. Kondo, an E2 conjugating enzyme, and the E3 CTLH ligase are required for the destruction of ME31B, TRAL, and Cup. Importantly, despite occurring hours earlier, egg activation establishes the timer for clearance of these proteins by activating the Pan Gu kinase, which in turn stimulates translation of Kondo mRNA. In other words, egg activation triggers a series of regulatory events that culminate in the degradation of maternally deposited RNA binding proteins several hours later. Clearance of the maternal protein dowry thus appears to be a coordinated, but as-yet underappreciated, aspect of the MZT. 4