Biology subjects

Loerch, S.

Publications and source records attributed to Loerch, S..

9 recordsLinked to original sources

bioRxiv2026

SF3B1 Phosphorylation Prompts U2AF2 Dissociation for Widespread Control of pre-mRNA Splicing

Reversible pre-mRNA splicing factor phosphorylation is a well-documented feature of spliceosome assembly, activation, and disassembly, yet its functional and mechanistic roles are still emerging. SF3B1, a core spliceosome subunit, is extensively phosphorylated before the first catalytic step of pre-mRNA splicing. Intriguingly, most SF3B1 phosphorylation sites surround its binding sites for U2AF2, an early-stage pre-mRNA splicing factor. Here, we discovered that SF3B1 phosphorylation significantly decreases its association with U2AF2. We determined two crystal structures revealing electrostatic repulsion between an acidic U2AF2 -helix and the negatively-charged phosphoryl group of SF3B1. Variants with amino acid substitutions that prevent or mimic SF3B1 phosphorylation perturbed thousands of splice sites, primarily those marked by uridine-rich splice site signals recognized by U2AF2. Collectively, our findings demonstrate a widespread but previously unrecognized role for SF3B1 phosphorylation as a gateway for U2AF2 dissociation so that pre-mRNA splicing can proceed.

biochemistry↗

bioRxiv2025

Separation of halide oxidation and substrate halogenation chemistries rationalizes site-selective vanadium dependent haloperoxidase catalysis

Vanadium-dependent haloperoxidases (VHPOs) catalyze the halogenation of organic molecules under mild aqueous conditions. Selective bacterial VHPOs exhibit exquisite regio- and enantiocontrol, however the precise mechanisms dictating selectivity have remained elusive. We have solved the single-particle cryo-electron microscopy (cryo-EM) structure of a selective bromoperoxidase from Enhygromyxa salina (esVHPO). Mutagenesis demonstrates that halide oxidation and substrate halogenation occur in two distinct pockets, with halide transfer mediated by critical lysine residue K329. Isolation of a stable intermediate following bromide oxidation (BrOx) enables single turnover catalysis in the presence of organic substrate; subsequent application of a chemoselective fluorescent probe provides support for an intermediate bromamine involved in selectivity. Cryo-EM of the BrOx state reveals a camera shutter mechanism that compacts the halide entry tunnel and vanadate pocket, minimizing the premature dissociation of hypohalous acid. These findings collectively unveil a multilayered halogen trapping and transfer mechanism and provide a rationale for selective VHPO catalysis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/684477v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@6154b9org.highwire.dtl.DTLVardef@169fdorg.highwire.dtl.DTLVardef@d56940org.highwire.dtl.DTLVardef@f11028_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

In situ single particle classification reveals distinct 60S maturation intermediates in cells

Electron cryo-microscopy (cryo-EM) can generate high-resolution views of cells with faithful preservation of molecular structure. In situ cryo-EM, therefore, has enormous potential to reveal the atomic details of biological processes in their native context. However, in practice, the utility of in situ cryo-EM is limited by the difficulty of reliably locating and confidently identifying molecular targets (particles) and their conformational states in the crowded cellular environment. We recently showed that 2DTM, a fine-grained template-based search applied to cryo-EM micrographs, can localize particles in two-dimensional views of cells with high precision. Here we demonstrate that the signal-to-noise ratio (SNR) observed with 2DTM can be used to differentiate related complexes in focused ion beam (FIB)-milled cell sections. We apply this method in two contexts to locate and classify related intermediate states of 60S ribosome biogenesis in the Saccharomyces cerevisiae cell nucleus. In the first, we separate the nuclear pre-60S population from the cytoplasmic mature 60S population, using the subcellular localization to validate assignment. In the second, we show that relative 2DTM SNRs can be used to separate mixed populations of nuclear pre-60S that are not visually separable. We use a maximum likelihood approach to define the probability of each particle belonging to each class, thereby establishing a statistic to describe the confidence of our classification. Without the need to generate 3D reconstructions, 2DTM can be applied even when only a few target particles exist in a cell.

biophysics↗

bioRxiv2022

A UHM - ULM interface contributes to U2AF2 and SF3B1 association for pre-mRNA splicing

In the early stages of spliceosome assembly, the 3 splice site is recognized by sequential complexes of U2AF2 with SF1 followed by the SF3B1 subunit of the U2 small nuclear ribonucleoprotein particle. The U2AF2 - SF1 interface comprises a U2AF homology motif (UHM) of U2AF2 and a well-characterized U2AF ligand motif (ULM)/coiled coil region of SF1. However, the structure of the U2AF2 - SF3B1 interface and its importance for pre-mRNA splicing is unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8 [A] resolution. The trajectory of the SF3B1 ULM across the U2AF2 UHM surface differed from prior UHM/ULM structures. This distinctive structure is expected to modulate the orientations of the fulllength proteins. Using isothermal titration calorimetry, we established similar binding affinities of a minimal U2AF2 UHM - SF3B1 ULM complex and a nearly full-length U2AF2 protein binding the N-terminal SF3B1 region, with or without an auxiliary SF3B6 subunit. We showed that key residues at the U2AF2 UHM - SF3B1 ULM interface are required for high affinity association and co-immunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2 - SF3B1 interface altered splicing of representative human transcripts. Further analysis of these transcripts and genome-wide data sets indicated that the subset of splice sites co-regulated by U2AF2 and SF3B1 are largely distinct from those co-regulated by U2AF2 and SF1. Altogether, these findings support distinct structural and functional roles for the sequential SF1 and SF3B1 complexes with U2AF2 during the pre-mRNA splicing process.

biochemistry↗

bioRxiv2021

Functionally distinct roles for eEF2K in the control of ribosome availability and p-body abundance in sensory neurons

Processing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies specifically in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splitting in vitro. Collectively, the data suggest that eEF2 phosphorylation defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons.

molecular biology↗

bioRxiv2021

Small-molecule ligands can inhibit -1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases since 2000 have high-lighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates -1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored a group of 6 small-molecule ligands, evaluating their activity against the frameshift signals from a panel of representative bat CoVs--the most likely source of future zoonoses--as well as SARS-CoV-2 and MERS-CoV. We found that whereas some ligands had notable activity against only a few of the frameshift signals, the serine protease inhibitor nafamostat suppressed -1 PRF significantly in several of them, while having limited to no effect on -1 PRF caused by frameshift signals from other viruses used as negative controls. These results suggest it is possible to find small-molecule ligands that inhibit -1 PRF specifically in a broad spectrum of CoVs, establishing the frameshift signal as a viable target for developing pan-coronaviral therapeutics.

biochemistry↗

bioRxiv2020

Intercellular Arc signaling regulates vasodilation

Injury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in DRG neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of privileged translation in vitro and in vivo. Inflammatory cues promote local translation of Arc in the skin. Arc deficient mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles, we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found that the excessive thermal responses and vasodilation observed in Arc defective mice are rescued by injection of Arc-containing extracellular vesicles into the skin. Our findings suggest that activity-dependent production of Arc in afferent fibers regulates neurogenic inflammation through intercellular signaling. HIGHLIGHTSO_LIRibosome profiling identifies Arc as a target of activity-dependent translation C_LIO_LIArc is present in the DRG, spinal cord, and skin C_LIO_LIInduced Arc biosynthesis in skin requires the presence of afferent fibers C_LIO_LIArc-deficient mice have exaggerated inflammation that is rescued by Arc-containing extracellular vesicles C_LI

neuroscience↗

bioRxiv2020

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Forster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.

biochemistry↗

bioRxiv2018

Cryo-EM structure of an amyloid fibril from systemic amyloidosis

Systemic AA amyloidosis is a worldwide occurring disease of humans and animals that arises from the misfolding of serum amyloid A protein. To provide insights into the molecular basis of this disease we used electron cryo-microscopy and determined the structure of an ex vivo amyloid fibril purified from AA amyloidotic mice at 3.0 [A] resolution. The fibril consists of C-terminally truncated serum amyloid A protein arranged into a compactly folded all-{beta} conformation. The structure identifies the protein N-terminus as central for the assembly of this fibril and provides a mechanism for its prion-like replication. Our data further explain how amino acid substitutions within the tightly packed fibril core can lead to amyloid resistance in vivo.

biophysics↗