Biology subjects

Marzluff, W. F.

Publications and source records attributed to Marzluff, W. F..

8 recordsLinked to original sources

bioRxiv2026

Differential control of both cell cycle-regulated and quantitative histone mRNA expression by Drosophila Mute

Coupling histone gene expression to S phase of the cell cycle is essential for genome duplication and stability. Activation of Cyclin E/Cdk2 at the G1-S transition stimulates high-level expression of histone genes during S phase, but how histone genes are turned off at the end of S phase is not understood. Here we demonstrate that the essential Drosophila gene mute functions to repress inappropriate histone mRNA accumulation outside of S phase by counteracting Cyclin E/Cdk2-dependent phosphorylation of Mxc, which activates histone gene expression. Additionally, Mute plays contrasting roles in histone gene expression during S phase by promoting high levels of H1, H2a and H2b expression but not H3 and H4. Although Mute is present only at replication-dependent histone genes, its loss leads to 801 differentially regulated genes, primarily those involved in muscle related processes in late-stage embryos. Thus, disruptions of histone gene expression control alters the transcriptome resulting in developmental defects.

cell biology↗

bioRxiv2025

Molecular mechanisms of recruitment, function and regulation of UPF1 in histone mRNA decay

Animal replication-dependent histone mRNAs end in a conserved stem loop (SL) instead of the canonical poly(A) tail present in all other eukaryotic mRNAs. Degradation of the histone SL at the end of the S-phase is initiated by the stem-loop binding protein SLBP and its interplay with the RNA helicase UPF1 and the exoribonuclease 3hExo. We report direct interactions between SLBP and UPF1 and show that the unstructured SLBP N-terminus wraps around the UPF1 helicase core, contacting it at multiple sites. Although binding of SLBP to UPF1 impedes unwinding activity, it is critical for efficient histone mRNA decay in cells, as unwinding of the SL facilitates degradation by 3hExo. Here we show that the UPF1-activator, UPF2, binds 3hExo, and that UPF2-mediated activation of UPF1 overrides the inhibitory effect of SLBP. Our results highlight the intricate network of UPF1-centric protein-protein and protein/RNA interactions that fine-tunes its unwinding activity and orchestrates timely and efficient degradation of histone mRNA.

biochemistry↗

bioRxiv2025

Composition and RNA binding specificity of metazoan RNase MRP

Ribonuclease (RNase) MRP is a conserved RNA-based enzyme that is essential for maturation of ribosomal RNA (rRNA) in eukaryotes. However, the composition and RNA substrate specificity of this multisubunit ribonucleoprotein complex in higher eukaryotes remain a mystery. Here, we identify NEPRO and C18ORF21 as constitutive subunits of metazoan RNase MRP. Both proteins are specific to RNase MRP and are the only ones distinguishing this enzyme from the closely related RNase P, which selectively cleaves transfer RNA-like substrates. We find that NEPRO and C18ORF21 each form a complex with all other subunits of RNase MRP, stabilize its catalytic RNA, and are required for rRNA maturation and cell proliferation. We harness our discovery to identify a full suite of in vivo RNA targets of each enzyme, including positions of potential cleavage sites at nucleotide resolution. These findings resolve the general composition of metazoan RNase MRP, illuminate its RNA binding specificity, and provide valuable assets for functional exploration of this essential eukaryotic enzyme.

molecular biology↗

bioRxiv2025

SUMO2 Promotes Histone Pre-mRNA Processing by Stabilizing Histone Locus Body Interactions and Facilitating U7 snRNP Assembly

Histone mRNAs are the only non-polyadenylated mRNAs in eukaryotic cells and require specialized processing in the histone locus body (HLB), a nuclear body where essential processing factors, including the U7 snRNP, are concentrated. Recent studies have revealed that misregulation of histone pre-mRNA processing can lead to polyadenylation of histone mRNAs and disruption of histone protein homeostasis. Despite links to human disease, the factors contributing to polyadenylation of histone mRNAs and the mechanisms underlying HLB assembly and U7 snRNP biogenesis remain unclear. Here, we report novel functions of the small ubiquitin-related modifier 2 (SUMO2) in promoting histone pre-mRNA processing. Using a SUMO2 knockout osteosarcoma cell line, we identified a defect in 3 end cleavage and a global increase in histone mRNA polyadenylation. Subsequent analysis of HLBs revealed increased dynamics and reduced levels of the U7 snRNP complex. By over-expressing U7 snRNP-specific components, Lsm11 and U7 snRNA, we rescued U7 snRNP levels and processing defects in SUMO2 knockout cells. Through analysis of Lsm11, we identified a SUMO-interacting motif in its N-terminus required for efficient formation of U7 snRNP. Collectively, we demonstrate that SUMO2 promotes histone pre-mRNA 3 end processing by stabilizing HLB interactions and facilitating U7 snRNP assembly.

molecular biology↗

bioRxiv2024

Cell cycle-regulated transcriptional pausing of Drosophila replication-dependent histone genes

Coordinated expression of replication-dependent (RD) histones genes occurs within the Histone Locus Body (HLB) during S phase, but the molecular steps in transcription that are cell cycle regulated are unknown. We report that Drosophila RNA Pol II promotes HLB formation and is enriched in the HLB outside of S phase, including G1-arrested cells that do not transcribe RD histone genes. In contrast, the transcription elongation factor Spt6 is enriched in HLBs only during S phase. Proliferating cells in the wing and eye primordium express full-length histone mRNAs during S phase but express only short nascent transcripts in cells in G1 or G2 consistent with these transcripts being paused and then terminated. Full-length transcripts are produced when Cyclin E/Cdk2 is activated as cells enter S phase. Thus, activation of transcription elongation by Cyclin E/Cdk2 and not recruitment of RNA pol II to the HLB is the critical step that links histone gene expression to cell cycle progression in Drosophila.

cell biology↗

bioRxiv2020

A region of Drosophila SLBP distinct from the histone pre-mRNA binding and processing domains is essential for deposition of histone mRNA in the oocyte

During Drosophila oogenesis, large amounts of histone mRNA and proteins are deposited in the developing oocyte. These are sufficient for the first 14 embryonic cell cycles and provide the developing embryo with sufficient histone proteins until the zygotic histone genes are activated. The maternally deposited histone mRNA is synthesized in stage 10b of oogenesis after completion of endoreduplication of the nurse cells. Histone mRNAs are the only cellular mRNAs that are not polyadenylated, ending instead in a conserved stemloop instead of a polyA tail. The Stem-loop binding protein (SLBP) binds the 3 end of histone mRNA and is essential for both the biosynthesis and translation of histone mRNA. We report that a 10 aa region in SLBP, which is not required for processing in vitro, is essential for transcription of histone mRNA in the stage 10b oocyte. In stage 10b the Histone Locus Bodies (HLBs) produce histone mRNAs in the absence of phosphorylation of Mxc, normally required for histone gene expression in S-phase cells. Mutants expressing this SLBP develop normally, produce small amounts of polyadenylated histone mRNA throughout development, but little histone mRNA in stage 10b resulting in death of the embryos in the first hr of development.

developmental biology↗

bioRxiv2020

Drosophila Histone Locus Body assembly and function involves multiple interactions

The histone locus body (HLB) assembles at replication-dependent (RD) histone loci and concentrates factors required for RD histone mRNA biosynthesis. The D. melanogaster genome has a single locus comprised of [~]100 copies of a tandemly arrayed repeat unit containing one copy of each of the 5 RD histone genes. To determine sequence elements required for D. melanogaster HLB formation and histone gene expression, we used transgenic gene arrays containing 12 copies of the histone repeat unit that functionally complement loss of the [~]200 endogenous RD histone genes. A 12x histone gene array in which all H3-H4 promoters were replaced with H2a-H2b promoters does not form an HLB or express high levels of RD histone mRNA in the presence of the endogenous histone genes. In contrast, this same transgenic array is active in HLB assembly and RD histone gene expression in the absence of the endogenous RD histone genes and rescues the lethality caused by homozygous deletion of the RD histone locus. The HLB formed in the absence of endogenous RD histone genes on the mutant 12x array contains all known factors present in the wild type HLB including CLAMP, which normally binds to GAGA repeats in the H3-H4 promoter. These data suggest that multiple protein-protein and/or protein-DNA interactions contribute to HLB formation, and that the large number of endogenous RD histone gene copies sequester available factor(s) from attenuated transgenic arrays, thereby preventing HLB formation and gene expression.

cell biology↗

bioRxiv2020

Single-Cell Analysis of the 3D Topologies of Genomic Loci Using Genome Architecture Mapping

Although each cell within an organism contains a nearly identical genome sequence, the three-dimensional (3D) packing of the genome varies among individual cells, influencing cell-type-specific gene expression. Genome Architecture Mapping (GAM) is the first genome-wide experimental method for capturing 3D proximities between any number of genomic loci without ligation. GAM overcomes several limitations of 3C-based methods by sequencing DNA from a large collection of thin sections sliced from individual nuclei. The GAM technique measures locus co-segregation, extracts radial positions, infers chromatin compaction, requires small numbers of cells, does not depend on ligation, and provides rich single-cell information. However, previous analyses of GAM data focused exclusively on population averages, neglecting the variation in 3D topology among individual cells. We present the first single-cell analysis of GAM data, demonstrating that the slices from individual cells reveal intercellular heterogeneity in chromosome conformation. By simultaneously clustering both slices and genomic loci, we identify topological variation among single cells, including differential compaction of cell cycle genes. We also develop a geometric model of the nucleus, allowing prediction of the 3D positions of each slice. Using GAM data from mouse embryonic stem cells, we make new discoveries about the structure of the major mammalian histone gene locus, which is incorporated into the Histone Locus Body (HLB), including structural fluctuations and putative causal molecular mechanisms. Our methods are packaged as SluiceBox, a toolkit for mining GAM data. Our approach represents a new method of investigating variation in 3D genome topology among individual cells across space and time.

bioinformatics↗