Reproductive Ageing: Declining translational capacity as a potential driver for oocyte meiotic instability.

In Brief: This point of view article focuses on the potential contribution of defects in protein synthesis (translation) to the incidence of oocyte meiotic failure. We discuss the potential cause of diminished oocyte translation during aging and the impact of these deficits on the function of the meiotic spindle. Abstract: Errors during female meiosis lead to embryonic aneuploidy and miscarriage and occur with increasing frequency during aging. The underlying molecular changes that drive female meiotic instability remain a subject of debate. Developing oocytes undergo a tremendous increase in cytoplasmic volume over several months of follicle development and rely on long-lived mRNAs and ribosomes accumulated during this growth phase for subsequent meiotic maturation. In this point of view article, we discuss how the unique reliance on stores of long-lived mRNAs and ribosomes may represent an Achilles' heel for oocyte function and how alterations that reduce the translational capacity of oocytes could be a factor significantly contributing to female infertility. Understanding these mechanisms could lead to new therapeutic strategies to improve fertility outcomes.

End-to-end pipeline for differential analysis of pausing in ribosome profiling data.

Ribosome profiling is a powerful technique which maps the distribution of ribosomes along mRNAs to analyze translation genome-wide. Ribosome density can be affected by multiple factors, such as changes to translation initiation or elongation rates. We describe the application of a metric for identifying genes rate-limited by these rates by analyzing the relative distribution of ribosome footprints along transcripts. This protocol also details two sample analyses comparing gene translation efficiencies and the distribution of ribosome densities on downloadable datasets. For complete details on the use and execution of this protocol, please refer to Flanagan et al. (2022).

FMRP-dependent production of large dosage-sensitive proteins is highly conserved.

Mutations in FMR1 are the most common heritable cause of autism spectrum disorder. FMR1 encodes an RNA-binding protein, FMRP, which binds to long, autism-relevant transcripts and is essential for normal neuronal and ovarian development. In contrast to the prevailing model that FMRP acts to block translation elongation, we previously found that FMRP activates the translation initiation of large proteins in Drosophila oocytes. We now provide evidence that FMRP-dependent translation is conserved and occurs in the mammalian brain. Our comparisons of the mammalian cortex and Drosophila oocyte ribosome profiling data show that translation of FMRP-bound mRNAs decreases to a similar magnitude in FMRP-deficient tissues from both species. The steady-state levels of several FMRP targets were reduced in the Fmr1 KO mouse cortex, including a ∼50% reduction of Auts2, a gene implicated in an autosomal dominant autism spectrum disorder. To distinguish between effects on elongation and initiation, we used a novel metric to detect the rate-limiting ribosome stalling. We found no evidence that FMRP target protein production is governed by translation elongation rates. FMRP translational activation of large proteins may be critical for normal human development, as more than 20 FMRP targets including Auts2 are dosage sensitive and are associated with neurodevelopmental disorders caused by haploinsufficiency.

Prolonged ovarian storage of mature Drosophila oocytes dramatically increases meiotic spindle instability.

Human oocytes frequently generate aneuploid embryos that subsequently miscarry. In contrast, Drosophila oocytes from outbred laboratory stocks develop fully regardless of maternal age. Since mature Drosophila oocytes are not extensively stored in the ovary under laboratory conditions like they are in the wild, we developed a system to investigate how storage affects oocyte quality. The developmental capacity of stored mature Drosophila oocytes decays in a precise manner over 14 days at 25°C. These oocytes are transcriptionally inactive and persist using ongoing translation of stored mRNAs. Ribosome profiling revealed a progressive 2.3-fold decline in average translational efficiency during storage that correlates with oocyte functional decay. Although normal bipolar meiotic spindles predominate during the first week, oocytes stored for longer periods increasingly show tripolar, monopolar and other spindle defects, and give rise to embryos that fail to develop due to aneuploidy. Thus, meiotic chromosome segregation in mature Drosophila oocytes is uniquely sensitive to prolonged storage. Our work suggests the chromosome instability of human embryos could be mitigated by reducing the period of time mature human oocytes are stored in the ovary prior to ovulation.

Fragile X mental retardation 1 gene enhances the translation of large autism-related proteins.

Mutations in the fragile X mental retardation 1 gene (FMR1) cause the most common inherited human autism spectrum disorder. FMR1 influences messenger RNA (mRNA) translation, but identifying functional targets has been difficult. We analyzed quiescent Drosophila oocytes, which, like neural synapses, depend heavily on translating stored mRNA. Ribosome profiling revealed that FMR1 enhances rather than represses the translation of mRNAs that overlap previously identified FMR1 targets, and acts preferentially on large proteins. Human homologs of at least 20 targets are associated with dominant intellectual disability, and 30 others with recessive neurodevelopmental dysfunction. Stored oocytes lacking FMR1 usually generate embryos with severe neural defects, unlike stored wild-type oocytes, which suggests that translation of multiple large proteins by stored mRNAs is defective in fragile X syndrome and possibly other autism spectrum disorders.

Making the cut: intramembrane cleavage by a rhomboid protease promotes ERAD.

Endoplasmic reticulum­associated degradation (ERAD) is a cellular protein quality-control process that disposes of proteasomal substrates from the early secretory pathway. Recent work shows that the endoplasmic reticulum­resident rhomboid protease RHBDL4 facilitates ERAD by recognizing and cleaving integral membrane substrates. The work indicates that intramembrane proteolysis may have a general role in the extraction of misfolded membrane proteins from the endoplasmic reticulum.

Unassembled CD147 is an endogenous endoplasmic reticulum-associated degradation substrate.

Degradation of folding- or assembly-defective proteins by the endoplasmic reticulum-associated degradation (ERAD) ubiquitin ligase, Hrd1, is facilitated by a process that involves recognition of demannosylated N-glycans by the lectin OS-9/XTP3-B via the adaptor protein SEL1L. Most of our knowledge of the machinery that commits proteins to this fate in metazoans comes from studies of overexpressed mutant proteins in heterologous cells. In this study, we used mass spectrometry to identify core-glycoslyated CD147 (CD147(CG)) as an endogenous substrate of the ERAD system that accumulates in a complex with OS-9 following SEL1L depletion. CD147 is an obligatory assembly factor for monocarboxylate transporters. The majority of newly synthesized endogenous CD147(CG) was degraded by the proteasome in a Hrd1-dependent manner. CD147(CG) turnover was blocked by kifunensine, and interaction of OS-9 and XTP3-B with CD147(CG) was inhibited by mutations to conserved residues in their lectin domains. These data establish unassembled CD147(CG) as an endogenous, constitutive ERAD substrate of the OS-9/SEL1L/Hrd1 pathway.

Derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant α-1 antitrypsin from the endoplasmic reticulum.

The degradation of misfolded secretory proteins is ultimately mediated by the ubiquitin-proteasome system in the cytoplasm, therefore endoplasmic reticulum-associated degradation (ERAD) substrates must be dislocated across the ER membrane through a process driven by the AAA ATPase p97/VCP. Derlins recruit p97/VCP and have been proposed to be part of the dislocation machinery. Here we report that Derlins are inactive members of the rhomboid family of intramembrane proteases and bind p97/VCP through C-terminal SHP boxes. Human Derlin-1 harboring mutations within the rhomboid domain stabilized mutant α-1 antitrypsin (NHK) at the cytosolic face of the ER membrane without disrupting the p97/VCP interaction. We propose that substrate interaction and p97/VCP recruitment are separate functions that are essential for dislocation and can be assigned respectively to the rhomboid domain and the C terminus of Derlin-1. These data suggest that intramembrane proteolysis and protein dislocation share unexpected mechanistic features.

Defining human ERAD networks through an integrative mapping strategy.

Proteins that fail to correctly fold or assemble into oligomeric complexes in the endoplasmic reticulum (ER) are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). Although many individual components of the ERAD system have been identified, how these proteins are organized into a functional network that coordinates recognition, ubiquitylation and dislocation of substrates across the ER membrane is not well understood. We have investigated the functional organization of the mammalian ERAD system using a systems-level strategy that integrates proteomics, functional genomics and the transcriptional response to ER stress. This analysis supports an adaptive organization for the mammalian ERAD machinery and reveals a number of metazoan-specific genes not previously linked to ERAD.

A two-hybrid assay to study protein interactions within the secretory pathway.

Interactions of transcriptional activators are difficult to study using transcription-based two-hybrid assays due to potent activation resulting in false positives. Here we report the development of the Golgi two-hybrid (G2H), a method that interrogates protein interactions within the Golgi, where transcriptional activators can be assayed with negligible background. The G2H relies on cell surface glycosylation to report extracellularly on protein-protein interactions occurring within the secretory pathway. In the G2H, protein pairs are fused to modular domains of the reporter glycosyltransferase, Och1p, and proper cell wall formation due to Och1p activity is observed only when a pair of proteins interacts. Cells containing interacting protein pairs are identified by selectable phenotypes associated with Och1p activity and proper cell wall formation: cells that have interacting proteins grow under selective conditions and display weak wheat germ agglutinin (WGA) binding by flow cytometry, whereas cells that lack interacting proteins display stunted growth and strong WGA binding. Using this assay, we detected the interaction between transcription factor MyoD and its binding partner Id2. Interfering mutations along the MyoD:Id2 interaction interface ablated signal in the G2H assay. Furthermore, we used the G2H to detect interactions of the activation domain of Gal4p with a variety of binding partners. Finally, selective conditions were used to enrich for cells encoding interacting partners. The G2H detects protein-protein interactions that cannot be identified via traditional two-hybrid methods and should be broadly useful for probing previously inaccessible subsets of the interactome, including transcriptional activators and proteins that traffic through the secretory pathway.