Biology subjects

Mello, C. C.

Publications and source records attributed to Mello, C. C..

8 recordsLinked to original sources

bioRxiv2026

El Ni&ntildeo, capture success, and wing fluctuating asymmetry in Drosophila sturtevanti Duda, 1927

Fluctuating asymmetry (FA) is widely used as a proxy for developmental instability, although reduced rather than increased FA, has occasionally been reported under particularly challenging conditions, possibly because environmental filtering removes less developmentally stable individuals. Here, we investigated temporal variation in wing shape asymmetry in males of Drosophila sturtevanti collected in 2022, 2024, and 2026 at the same sampling site, an urban Atlantic Forest fragment. Sampling in 2022 and 2026 were completed during single field expeditions in early April, whereas in 2024 repeated collections were required until mid-May because of markedly low capture success. We therefore examined whether this difference was associated with unusual environmental conditions and whether FA was higher in 2024, as expected under greater developmental perturbation. Daily temperature and relative humidity were examined from early March onward, encompassing approximately one month before adult emergence. The 2024 sampling period was warmer and drier than the corresponding periods in 2022 and 2026 and coincided with the final phase of the strong 2023-2024 El Ni&ntildeo event. Contrary to our prediction, FA was significantly lower in 2024. The magnitude of directional asymmetry remained similar among years, although the spatial pattern of the DA vector differed between 2024 and 2026. The coincidence of reduced capture success and reduced FA is consistent with the hypothesis that adverse conditions may impose demographic filtering, such that individuals expressing greater developmental instability are less likely to contribute to the sampled adult population.

evolutionary biology↗

bioRxiv2026

Systematic Identification of Germ Granule Proteins Reveals Specialized Roles in RNAi and Small RNA Inheritance

Biomolecular condensates, such as germ granules, organize RNAi pathways critical for fertility and genome regulation. Yet, the protein composition and functional contributions of these condensates remain poorly defined. Here, we applied TurboID proximity labeling to the Caenorhabditis elegans germ granule protein SIMR-1, integrating mass spectrometry with genetic screening, CRISPR-based tagging, and small RNA sequencing. This systematic approach identified several previously uncharacterized germ granule proteins that contribute to fertility, germline immortality, exogenous RNAi, and transgenerational inheritance. Small RNA sequencing of 21 mutants revealed broad and class-specific defects in siRNA and miRNA biogenesis, with distinct factors associated with defects in WAGO-class 22G-RNAs, CSR-class 22G-RNAs, or histone-directed small RNAs. Among these, we identified PINT-1, a highly disordered protein that directly interacts with and is recruited to germ granules by the PIWI Argonaute PRG-1. PINT-1 is required for piRNA-dependent and -independent secondary siRNA biogenesis and germline development. Comparative genomics revealed that PINT-1 has co-evolved with PRG-1 across clade V nematodes, with a conserved structured N-terminus and a rapidly diverging repeat-rich intrinsically disordered region. Together, our findings expand the germ granule proteome and reveal how distinct condensate components contribute to specialized functions within the small RNA pathways, while highlighting an evolutionarily co-adapted PIWI interactor critical for siRNA biogenesis.

genetics↗

bioRxiv2022

A family of C. elegans VASA homologs control Argonaute pathway specificity and promote transgenerational silencing

Germline Argonautes direct transcriptome surveillance within peri-nuclear membraneless organelles called nuage. In C. elegans, a family of Vasa-related Germ Line Helicase (GLH) proteins localize in, and promote the formation of nuage called P granules. Previous studies have implicated GLH proteins in inherited silencing but direct roles in amplification of small RNAs, or in target mRNA or Argonatue binding have not been identified. Here we show that GLH proteins compete with each other to control Argonaute pathway specificity, bind directly to Argonaute-target mRNAs and act to promote the amplification of small RNAs required for transgenerational inheritance. We show that the ATPase cycle of GLH-1 regulates its direct binding to the Argonaute WAGO-1 which engages amplified small RNAs. Our findings support a dynamic and direct role for GLH proteins in inherited silencing beyond their role as structural components of nuage.

molecular biology↗

bioRxiv2020

The RNA phosphatase PIR-1 regulates endogenous small RNA pathways in C. elegans

Eukaryotic cells regulate 5 triphosphorylated (ppp-) RNAs to promote cellular functions and prevent recognition by antiviral RNA sensors. For example, RNA capping enzymes possess triphosphatase domains that remove the {gamma} phosphates of ppp-RNAs during RNA capping. Members of the closely related PIR1 family of RNA polyphosphatases remove both the {beta} and {gamma} phosphates from ppp-RNAs. Here we show that C. elegans PIR-1 dephosphorylates ppp-RNAs made by cellular RdRPs and is required for the maturation of 26G-RNAs, Dicer-dependent small RNAs that regulate thousands of genes during spermatogenesis and embryogenesis. PIR-1 also regulates the CSR-1 22G-RNA pathway and has critical functions in both somatic and germline development. Our findings suggest that PIR-1 modulates both Dicer-dependent and - independent Argonaute pathways, and provide insight into how cells and viruses use a conserved RNA phosphatase to regulate and respond to ppp-RNA species.

molecular biology↗

bioRxiv2020

Melting dsDNA donor molecules potentiates precision genome editing in C. elegans

CRISPR genome editing has revolutionized genetics in many organisms. In the nematode Caenorhabditis elegans one injection into each of the two gonad arms of an adult hermaphrodite exposes hundreds of meiotic germ cells to editing mixtures, permitting the recovery of multiple indels or small precision edits from each successfully injected animal. Unfortunately, particularly for long insertions, editing efficiencies can vary widely, necessitating multiple injections, and often requiring co-selection strategies. Here we show that melting double stranded DNA (dsDNA) donor molecules prior to injection increases the frequency of precise homology-directed repair (HDR) by several fold for longer edits. We describe troubleshooting strategies that enable consistently high editing efficiencies resulting, for example, in up to 100 independent GFP knock-ins from a single injected animal. These efficiencies make C. elegans by far the easiest metazoan to genome edit, removing barriers to the use and adoption of this facile system as a model for understanding animal biology.

genetics↗

bioRxiv2019

GCNA interacts with Spartan and Topoisomerase II to regulate genome stability.

GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan/DVC-1 and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. We show that GCNA and Topoisomerase 2 (Top2) physically interact and colocalize on condensed chromosomes during mitosis, when Spartan is not present. We show that C. elegans gcna-1 mutants are sensitive to Top2 poison and accumulate mutations consistent with low fidelity repair of DNA damage, leading to loss of fitness and fertility over generations. We also demonstrate that mouse GCNA interacts with TOP2, and Gcna-mutant mice exhibit abnormalities consistent with the inability to process DPCs, including chromatin condensation and crossover defects. Together, our findings provide evidence that GCNA maintains genomic integrity by processing Top2 DPCs in the germline and early embryo, where the genome is challenged with an increased DPC burden.

developmental biology↗

bioRxiv2018

5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome Editing

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5'-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

molecular biology↗

bioRxiv2018

Identification of piRNA binding sites reveals the Argonaute regulatory landscape of the C. elegans germline

piRNAs (Piwi-interacting small RNAs) engage Piwi Argonautes to silence transposons and promote fertility in animal germlines. Genetic and computational studies have suggested that C. elegans piRNAs tolerate mismatched pairing and in principle could target every transcript. Here we employ in vivo cross-linking to identify transcriptome-wide interactions between piRNAs and target RNAs. We show that piRNAs engage all germline mRNAs and that piRNA binding follows microRNA-like pairing rules. Targeting correlates better with binding energy than with piRNA abundance, suggesting that piRNA concentration does not limit targeting. In mRNAs silenced by piRNAs, secondary small RNAs accumulate at the center and ends of piRNA binding sites. In germline-expressed mRNAs, however, targeting by the CSR-1 Argonaute correlates with reduced piRNA binding density and suppression of piRNA-associated secondary small RNAs. Our findings reveal physiologically important and nuanced regulation of individual piRNA targets and provide evidence for a comprehensive post transcriptional regulatory step in germline gene expression.

genetics↗