Biology subjects

Ameres, S. L.

Publications and source records attributed to Ameres, S. L..

12 recordsLinked to original sources

bioRxiv2026

A pseudohelicase-centered complex couples assembly-dependent RNA cleavage to poly(UG)ylation

Robust RNA interference in Caenorhabditis elegans requires Argonaute targeted RNAs to be cleaved by RDE 8 and poly(UG)ylated by MUT 2, generating templates for RNA dependent RNA polymerases that synthesize secondary small RNAs. RDE 8 and MUT 2 are physically coupled within the cleavage and poly(UG)ylation (CPUG) complex, together with the pseudonuclease NYN 1 and MUT 15, but how their activities are controlled and coordinated is unknown. Structural and biochemical analyses reveal that the RDE 8/NYN 1 nuclease module is autoinhibited, restricting cleavage outside CPUG. We identify MUT 15, the CPUG scaffold linking this nuclease module to MUT 2, as a pseudohelicase. MUT 15 remodels the nuclease module, licensing RDE 8 activity upon complex assembly. Reconstituted CPUG synthesizes long poly(UG) tails, with the substrate 3' end determining tailing efficiency and register. Within CPUG, RNA engagement by MUT 2 limits RDE 8 access, while poly(UG) tails themselves are not cleaved by RDE 8, preventing re cleavage. Together, CPUG couples assembly dependent RNA cleavage to downstream processing, imposing safety and directionality.

biochemistry↗

bioRxiv2026

Kinetic logic of uridylation-mediated RNA decay

3'-terminal uridylation marks structured non-coding RNAs for cytoplasmic decay, yet how uridylation is quantitatively coupled to exonucleolytic degradation remains unclear. Here, we dissect the kinetic logic of uridylation-mediated RNA surveillance in Drosophila melanogaster. Using biochemical reconstitution together with high-throughput enzymology and quantitative modeling, we show that the terminal uridylyl transferase Tailor generates discrete oligo(U) intermediates through product-dependent kinetic tuning, while co-substrate promiscuity suppresses sustained processivity under physiological nucleotide conditions. Massively parallel binding and decay assays further reveal how the 3'-to-5' exoribonuclease Dis3l2 selectively degrades Tailor-primed RNAs by integrating 3'-proximal uridine content and defined 3'-end accessibility--features encoded by short, kinetically tuned oligo(U) intermediates centered on four nucleotides--to enable productive threading of RNA substrates along an extended RNA-binding path to the catalytic site. Together, our findings establish a quantitative framework in which uridylation encodes decay competence through transient RNA 3'-end states that are matched to the mechanistic requirements for decay. HighlightsO_LIThe TUTase Tailor kinetically tunes uridylation to generate short, discrete oligo(U) intermediates C_LIO_LIMixed nucleotide availability suppresses sustained processive uridylation C_LIO_LIDis3l2 decodes 3'-proximal uridine content and end accessibility to commit RNAs to decay C_LIO_LIShort oligo(U) tails encode RNA decay competence through transient RNA 3'-end states C_LI

biochemistry↗

bioRxiv2025

Dual regulation of the unfolded protein response by IGF2BP3 during ER stress

Misfolded protein accumulation in the endoplasmic reticulum (ER) perturbs cellular homeostasis, causing pathological ER stress. The Unfolded Protein Response (UPR) is a highly conserved signaling cascade that restores ER homeostasis by countering ER protein overload. Transcriptional response is paramount for UPR signaling and negating ER stress. While multiple UPR-linked mRNAs are post-transcriptionally regulated, the mechanisms mediating this regulation are unclear. Here, we demonstrate that the highly conserved RNA-binding protein IGF2BP3 interacts with transcripts encoding a subset of UPR effectors. During ER stress, IGF2BP3 destabilized many of these target transcripts, including UPR targets. In contrast, IGF2BP3 stabilized mRNAs encoding transcriptional regulators and thereby upregulated expression of UPR target genes. This dual regulation allows IGF2BP3 to differentially upregulate stress response genes while tuning down the expression of other transcripts during ER stress, relieving protein folding load during this critical response. Our data reveal that posttranscriptional mechanisms control transcription, thus forming gene regulatory networks that robustly tune the UPR.

cell biology↗

bioRxiv2025

Hierarchical assembly and functional resilience of the mammalian RNA exosome

Most eukaryotic proteins assemble into multisubunit complexes that coordinate essential cellular functions, yet the principles governing their assembly and proteostatic control remain largely undefined. Here, we systematically dissect the cellular assembly and functional organization of the RNA exosome, an essential ribonucleolytic complex, using an inducible dual-guide CRISPR/Cas9 system in mouse embryonic stem cells. We reveal a sequential assembly pathway where Exosc2, Exosc4, and Exosc7 initiate complex formation, facilitating the incorporation of barrel and cap subunits in a defined hierarchy. Unlike other structural subunits, the terminally incorporated cap subunit Exosc1 is dispensable for cell viability, revealing a modular, functionally resilient architecture. We demonstrate that orphan subunits are selectively degraded via the ubiquitin-proteasome system, enforcing stringent quality control over RNA exosome biogenesis. These findings establish a framework for decoding the assembly logic of essential macromolecular machines and uncover previously unrecognized plasticity in the composition and function of the RNA exosome.

molecular biology↗

bioRxiv2024

ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA

Type II interferon (IFN{gamma}) signaling is essential for innate immunity and critical for effective immunological checkpoint blockade in cancer immunotherapy. Genetic screen identification of post-transcriptional regulators of this pathway has been challenging since such factors are often essential for cell viability. Here, we utilize our inducible CRISPR/Cas9 approach to screen for key post-transcriptional regulators of IFN{gamma} signaling, and in this way identify ERH and the ERH-associated splicing and RNA export factors MAGOH, SRSF1, and ALYREF. Loss of these factors impairs post-transcriptional mRNA maturation of JAK2, a crucial kinase for IFN{gamma} signaling, resulting in abrogated JAK2 protein levels and diminished IFN{gamma} signaling. Further analysis highlights a critical role for ERH in preventing intron retention in AU-rich regions in specific transcripts, such as JAK2. This regulation is markedly different from previously described retention of GC-rich introns. Overall, these findings reveal that post-transcriptional JAK2 processing is a critical rate-limiting step for the IFN{gamma}-driven innate immune response.

cell biology↗

bioRxiv2024

Proteome-scale tagging and functional screening in mammalian cells by ORFtag

Determining protein function in a systematic manner is a key goal of modern biology, but remains challenging with current approaches. Here, we present ORFtag, a versatile, cost-effective and highly efficient method for the massively-parallel tagging and functional interrogation of proteins at proteome scale. Using mouse embryonic stem cells, we showcase ORFtags utility through screens for transcriptional activators, repressors and post-transcriptional regulators. Each screen finds known and novel regulators, including long ORFs not accessible to other methods, revealing that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions frequently function as transactivators.

systems biology↗

bioRxiv2023

Mime-seq 2.0: a method to sequence microRNAs from specific mouse cell types

Many microRNAs (miRNAs) are expressed with high spatiotemporal specificity during organismal development, with some being limited to rare cell types, often embedded in complex tissues. Yet most miRNA profiling efforts remain at the tissue and organ levels. To overcome challenges in accessing the microRNomes from tissue-embedded cells, we had previously developed mime-seq (miRNome by methylation dependent sequencing), a technique in which cell-specific miRNA methylation in C. elegans and Drosophila enabled chemo-selective sequencing without the need for cell sorting or biochemical purification. Here, we present mime-seq 2.0 for profiling miRNAs from specific mouse cell types. We engineered a chimeric RNA methyltransferase that is tethered to Argonaute and efficiently methylates miRNAs at their 3-terminal 2OH in mouse and human cell lines. We also generated a transgenic mouse for conditional expression of this methyltransferase, which can be used to direct methylation of miRNAs in a cell-type of choice. We validated the use of this mouse by profiling miRNAs from B cells and bone marrow plasma cells.

molecular biology↗

bioRxiv2023

Mutual homeostasis of charged proteins

Protein dosage is regulated to maintain cellular homeostasis and health. The dosage of proteins containing disordered low complexity domains (LCDs) must be particularly well-controlled to prevent aberrant disease, yet no mechanism to maintain homeostasis has been identified1, 2. Here we report a mutual homeostatic mechanism that controls the concentration of such proteins, termed interstasis, in which proteins with similar LCDs co-regulate their combined dosage through collective negative feedback. We focused on the mechanism that exploits the fundamental multivalency of GA-rich RNA regions that encode charged LCDs, including those with arginine-enriched mixed charge domains (R-MCDs). Modest variations in the abundance of an R-MCD protein change the properties of nuclear speckles, a protein-RNA condensate, selectively trapping multivalent GA-rich mRNAs to promote their nuclear retention. This interstasis depends on conserved codon biases, shared by amniotes, which enhance the multivalency of GA-rich regions encoding charged LCDs. The threshold of interstasis is modulated by CLK kinases, which affect the nuclear speckle localisation of proteins such as TRA2B, key binder of GA-rich RNAs. Notably, many classes of LCDs are encoded by RNA regions containing multivalency-enhancing codon biases, each preferentially bound by specific proteins, suggesting that interstasis might co-regulate many classes of functionally related LCD-containing proteins through dose-sensitivity of various types of protein-RNA condensates.

molecular biology↗

bioRxiv2022

SLAMseq resolves the kinetics of maternal and zygotic gene expression in early zebrafish embryogenesis

The maternal-to-zygotic transition (MZT) is a key developmental process in metazoan embryos that involves the activation of zygotic transcription (ZGA) and degradation of maternal transcripts. We employed metabolic mRNA sequencing (SLAMseq) to deconvolute the compound embryonic transcriptome in zebrafish. While mitochondrial zygotic transcripts prevailed prior to MZT, we uncover the spurious transcription of hundreds of short and intron-poor nuclear genes as early as the 2-cell stage. Upon ZGA, most zygotic transcripts originate from thousands of maternal-zygotic (MZ) genes that are transcribed at rates comparable to those of hundreds of purely zygotic genes and replenish maternal mRNAs at distinct timescales. Rapid replacement of MZ transcripts involves transcript decay features unrelated to major maternal degradation pathways and promotes de novo synthesis of the core gene expression machinery by increasing poly(A)-tail length and translation efficiency. SLAMseq hence provides unprecedented insights into the timescales, molecular features and regulation of MZT during zebrafish embryogenesis.

developmental biology↗

bioRxiv2020

NMD is required for timely cell fate transitions by fine-tuning gene expression and controlling translation

Cell fate transitions depend on balanced rewiring of transcription and translation programmes to mediate ordered developmental progression. Here we identify a feedback loop between nonsense-mediated mRNA decay (NMD) and translation initiation. We show that NMD controls the translation initiation factor Eif4a2 and its premature termination codon encoding isoform (Eif4a2PTC). NMD deficiency leads to translation of a specific truncated Eif4a2 protein, which elicits increased translation rates and is causative for significant delays in mouse embryonic stem cell (ESC) differentiation. Our results show identical mRNA targets for Smg5, Smg6 and Smg7, but illustrate a clear hierarchy between KOs in amplitude of target deregulation and differentiation phenotype (Smg5 > Smg6 > Smg7). This hierarchy highlights heterodimer independent functions for Smg5 and Smg7. Together, our findings expose an intricate link between mRNA stability and translation initiation control, that must be maintained for normal dynamics of cell state transitions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2020

Sister-chromatid-sensitive Hi-C reveals the conformation of replicated human chromosomes

The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C)1-3 has revealed a complex genomic landscape of internal chromosome structures in vertebrate cells4-11 yet how sister chromatids topologically interact in replicated chromosomes has remained elusive due to their identical sequences. Here, we present sister-chromatid-sensitive Hi-C (scsHi-C) based on nascent DNA labeling with 4-thio-thymidine. Genome-wide conformation maps of human chromosomes revealed that sister chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired, characterized by facultative heterochromatin, as well as insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister chromatid topologies and our scsHi-C technology will make it possible to dissect how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.

cell biology↗

bioRxiv2017

Thiol-linked alkylation for the metabolic sequencing of RNA

Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry-based epitranscriptomics-sequencing technology that uncovers 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. When applied to mouse embryonic stem cells, SLAM-seq provides global and transcript-specific insights into pluripotency-associated gene expression. We validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner.\n\nOne Sentence SummaryChemical nucleotide-analog derivatization provides global insights into transcriptional and post-transcriptional gene regulation

molecular biology↗