The Giardia lamblia ribosome structure reveals divergence in several biological pathways and the mode of emetine function.

Giardia lamblia is a deeply branching protist and a human pathogen. Its unusual biology presents the opportunity to explore conserved and fundamental molecular mechanisms. We determined the structure of the G. lamblia 80S ribosome bound to tRNA, mRNA, and the antibiotic emetine by cryo-electron microscopy, to an overall resolution of 2.49 Å. The structure reveals rapidly evolving protein and nucleotide regions, differences in the peptide exit tunnel, and likely altered ribosome quality control pathways. Examination of translation initiation factor binding sites suggests these interactions are conserved despite a divergent initiation mechanism. Highlighting the potential of G. lamblia to resolve conserved biological principles; our structure reveals the interactions of the translation inhibitor emetine with the ribosome and mRNA, thus providing insight into the mechanism of action for this widely used antibiotic. Our work defines key questions in G. lamblia and motivates future experiments to explore the diversity of eukaryotic gene regulation.

Precise gene models using long-read sequencing reveal a unique poly(A) signal in Giardia 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.

The E2 Marie Kondo and the CTLH E3 ligase clear deposited RNA binding proteins during the maternal-to-zygotic transition.

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 Kdo mRNA. Clearance of the maternal protein dowry thus appears to be a coordinated, but as-yet underappreciated, aspect of the MZT.

Bestselling author and organizing consultant Marie Kondo has helped people around the world declutter their homes by getting rid of physical items that do not bring them joy. Keeping the crowded environment inside a living cell organized also requires work and involves removing molecules that are no longer needed. A fertilized egg cell, for example, contains molecules from the mother that regulate the initial stages as it develops into an embryo. Later on, the embryo takes control of its own development by destroying these inherited molecules and switches to making its own instead. This process is called the maternal-to-zygotic transition. The molecules passed from the mother to the egg cell include proteins and messenger RNAs (molecules that include the coded instructions to make new proteins). Previous research has begun to reveal how the embryo destroys the mRNAs it inherits from its mother and how it starts to make its own. Yet almost nothing is known about how an embryo gets rid of its mother's proteins. To address this question, Zavortink, Rutt, Dzitoyeva et al. used an approach known as an RNA interference screen to identify factors required to destroy three maternal proteins in fruit fly embryos. The experiments helped identify one enzyme that worked together with another larger enzyme complex to destroy the maternal proteins. This enzyme belongs to a class of enzymes known as ubiquitin-conjugating enzymes (or E2 enzymes) and it was given the name "Kdo", short for "Marie Kondo". Further experiments showed that the mRNAs that code for the Kdo enzyme were present in unfertilized eggs, but in a repressed state that prevented the eggs from making the enzyme. Once an egg started to develop into an embryo, these mRNAs became active and the embryo started to make Kdo enzymes. This led to the three maternal proteins being destroyed during the maternal-to-zygotic transition. These findings reveal a new pathway that regulates the destruction of maternal proteins as the embryo develops. The next challenge will be identifying other maternal proteins that do not "spark joy" and understanding the role their destruction plays in the earliest events of embryonic development.