Phase separation of hnRNPA1 and TERRA regulates telomeric stability.

Telomeres are repetitive DNA sequences and associated protein complexes located at the end of chromatin. As a result of the DNA replication ending issue, telomeric DNA shortens during each cell cycle. The shelterin protein complex caps telomeric ends and forms a high-order protein-DNA structure to protect telomeric DNA. The stability of telomeres is critical for cellular function and is related to the progression of many human diseases. Telomeric repeat-containing RNA (TERRA) is a noncoding RNA transcribed from telomeric DNA regions. TERRA plays an essential role in regulating and maintaining the stability of telomeres. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA-binding proteins associated with complex and diverse biological processes. HnRNPA1 can recognize both TERRA and telomeric DNA. Previous research reported that hnRNPA1, TERRA, and POT1, a component of the shelterin complex, worked coordinately and displaced replication protein A from telomeric ssDNA after DNA replication, promoting telomere capping to preserve genomic integrity. However, the detailed molecular mechanism has remained unclear for over twenty years. Our study revealed the molecular structure through which the hnRNPA1 UP1 domain interacts with TERRA. Through structural analysis, we identified critical residues on the interacting surface between UP1 and TERRA. Furthermore, we proved that nucleic acids significantly increase the phase separation ability of hnRNPA1 and disrupting the UP1-TERRA interaction extraordinarily affects hnRNPA1 droplet formation both in vitro and in vivo. Taken together, these data revealed the molecular mechanism of the droplet formation of hnRNPA1 and TERRA and the possible function of the droplets for maintaining genomic stability.

PRMT1 and TDRD3 promote stress granule assembly by rebuilding the protein-RNA interaction network.

Stress granules (SGs) are membrane-less organelles (MLOs) or cytosolic compartments formed upon exposure to environmental cell stress-inducing stimuli. SGs are based on ribonucleoprotein complexes from a set of cytoplasmic proteins and mRNAs, blocked in translation due to stress cell-induced polysome disassembly. Post-translational modifications (PTMs) such as methylation, are involved in SG assembly, with the methylation writer PRMT1 and its reader TDRD3 colocalizing to SGs. However, the role of this writer-reader system in SG assembly remains unclear. Here, we found that PRMT1 methylates SG constituent RNA-binding proteins (RBPs) on their RGG motifs. Besides, we report that TDRD3, as a reader of asymmetric dimethylarginines, enhances RNA binding to recruit additional RNAs and RBPs, lowering the percolation threshold and promoting SG assembly. Our study enriches our understanding of the molecular mechanism of SG formation by elucidating the functions of PRMT1 and TDRD3. We anticipate that our study will provide a new perspective for comprehensively understanding the functions of PTMs in liquid-liquid phase separation driven condensate assembly.

Structure-Activity Relationship of Antibody-Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody-Phosphorodiamidate Morpholino Oligomer Conjugates for Drug Development.

Antibody-oligonucleotide conjugates (AOCs) are promising treatments for Duchenne muscular dystrophy (DMD). They work via induction of exon skipping and restoration of dystrophin protein in skeletal and heart muscles. The structure-activity relationships (SARs) of AOCs comprising antibody-phosphorodiamidate morpholino oligomers (PMOs) depend on several aspects of their component parts. We evaluate the SAR of antimouse transferrin receptor 1 antibody (αmTfR1)-PMO conjugates: cleavable and noncleavable linkers, linker location on the PMO, and the impact of drug-to-antibody ratios (DARs) on plasma pharmacokinetics (PK), oligonucleotide delivery to tissues, and exon skipping. AOCs containing a stable linker with a DAR9.7 were the most effective PMO delivery vehicles in preclinical studies. We demonstrate that αmTfR1-PMO conjugates induce dystrophin protein restoration in the skeletal and heart muscles of mdx mice. Our results show that αmTfR1-PMO conjugates are a potentially effective approach for the treatment of DMD.

Structural basis for TNIP1 binding to FIP200 during mitophagy.

TNIP1 has been increasingly recognized as a security check to finely adjust the rate of mitophagy by disrupting the recycling of the Unc-51-like kinase complex during autophagosome formation. Through tank-binding kinase 1-mediated phosphorylation of the TNIP1 FIP200 interacting region (FIR) motif, the binding affinity of TNIP1 for FIP200, a component of the Unc-51-like kinase complex, is enhanced, allowing TNIP1 to outcompete autophagy receptors. Consequently, FIP200 is released from the autophagosome, facilitating further autophagosome expansion. However, the molecular basis by which FIP200 utilizes its claw domain to distinguish the phosphorylation status of residues in the TNIP1 FIR motif for recognition is not well understood. Here, we elucidated multiple crystal structures of the complex formed by the FIP200 claw domain and various phosphorylated TNIP1 FIR peptides. Structural and isothermal titration calorimetry analyses identified the crucial residues in the FIP200 claw domain responsible for the specific recognition of phosphorylated TNIP1 FIR peptides. Additionally, utilizing structural comparison and molecular dynamics simulation data, we demonstrated that the C-terminal tail of TNIP1 peptide affected its binding to the FIP200 claw domain. Moreover, the phosphorylation of TNIP1 Ser123 enabled the peptide to effectively compete with the peptide p-CCPG1 (the FIR motif of the autophagy receptor CCPG1) for binding with the FIP200 claw domain. Overall, our work provides a comprehensive understanding of the specific recognition of phosphorylated TNIP1 by the FIP200 claw domain, marking an initial step toward fully understanding the molecular mechanism underlying the TNIP1-dependent inhibition of mitophagy.

Germ granule compartments coordinate specialized small RNA production.

Germ granules are biomolecular condensates present in most animal germ cells. One function of germ granules is to help maintain germ cell totipotency by organizing mRNA regulatory machinery, including small RNA-based gene regulatory pathways. The C. elegans germ granule is compartmentalized into multiple subcompartments whose biological functions are largely unknown. Here, we identify an uncharted subcompartment of the C. elegans germ granule, which we term the E granule. The E granule is nonrandomly positioned within the germ granule. We identify five proteins that localize to the E granule, including the RNA-dependent RNA polymerase (RdRP) EGO-1, the Dicer-related helicase DRH-3, the Tudor domain-containing protein EKL-1, and two intrinsically disordered proteins, EGC-1 and ELLI-1. Localization of EGO-1 to the E granule enables synthesis of a specialized class of 22G RNAs, which derive exclusively from 5' regions of a subset of germline-expressed mRNAs. Defects in E granule assembly elicit disordered production of endogenous siRNAs, which disturbs fertility and the RNAi response. Our results define a distinct subcompartment of the C. elegans germ granule and suggest that one function of germ granule compartmentalization is to facilitate the localized production of specialized classes of small regulatory RNAs.

Structural insights into the recognition of the A/T-rich motif in target gene promoters by the LMX1a homeobox domain.

LIM homeodomain transcription factor 1-alpha (LMX1a) is a neuronal lineage-specific transcription activator that plays an essential role during the development of midbrain dopaminergic (mDA) neurons. LMX1a induces the expression of multiple key genes, which ultimately determine the morphology, physiology, and functional identity of mDA neurons. This function of LMX1a is dependent on its homeobox domain. Here, we determined the structures of the LMX1a homeobox domain in complex with the promoter sequences of the Wnt family member 1 (WNT1) or paired like homeodomain 3 (Pitx3) gene, respectively. The complex structures revealed that the LMX1a homeobox domain employed its α3 helix and an N-terminal loop to achieve specific target recognition. The N-terminal loop (loop1) interacted with the minor groove of the double-stranded DNA (dsDNA), whereas the third α-helix (α3) was tightly packed into the major groove of the dsDNA. Structure-based mutations in the α3 helix of the homeobox domain significantly reduced the binding affinity of LMX1a to dsDNA. Moreover, we identified a nonsyndromic hearing loss (NSHL)-related mutation, R199, which yielded a more flexible loop and disturbed the recognition in the minor groove of dsDNA, consistent with the molecular dynamics (MD) simulations. Furthermore, overexpression of Lmx1a promoted the differentiation of SH-SY5Y cells and upregulated the transcription of WNT1 and PITX3 genes. Hence, our work provides a detailed elucidation of the specific recognition between the LMX1a homeobox domain and its specific dsDNA targets, which represents valuable information for future investigations of the functional pathways that are controlled by LMX1a during mDA neuron development.

Molecular mechanism of specific HLA-A mRNA recognition by the RNA-binding-protein hMEX3B to promote tumor immune escape.

Immunotherapy, including immune checkpoint inhibitors and adoptive cell transfer, has obtained great progress, but their efficiencies vary among patients due to the genetic and epigenetic differences. Human MEX3B (hMEX3B) protein is an RNA-binding protein that contains two KH domains at the N-terminus and a RING domain at its C-terminus, which has the activity of E3 ubiquitin ligase and is essential for RNA degradation. Current evidence suggests that hMEX3B is involved in many important biological processes, including tumor immune evasion and HLA-A regulation, but the sequence of substrate RNA recognized by hMEX3B and the functional molecular mechanisms are unclear. Here, we first screened the optimized hMEX3B binding sequence on the HLA-A mRNA and reported that the two tandem KH domains can bind with their substrate one hundred times more than the individual KH domains. We systematically investigated the binding characteristics between the two KH domains and their RNA substrates by nuclear magnetic resonance (NMR). Based on this information and the small-angle X-ray scattering (SAXS) data, we used molecular dynamics simulations to obtain structural models of KH domains in complex with their corresponding RNAs. By analyzing the models, we noticed that on the KH domains' variable loops, there were two pairs of threonines and arginines that can disrupt the recognition of the RNA completely, and this influence had also been verified both in vitro and in vivo. Finally, we presented a functional model of the hMEX3B protein, which indicated that hMEX3B regulated the degradation of its substrate mRNAs in many biological processes. Taken together, our research illustrated how the hMEX3B protein played a key role in translation inhibition during the immune response to tumor cells and provided an idea and a lead for the study of the molecular mechanism and function of other MEX3 family proteins.

Structural insights into the specific recognition of mitochondrial ribosome-binding factor hsRBFA and 12 S rRNA by methyltransferase METTL15.

Mitochondrial rRNA modifications are essential for mitoribosome assembly and its proper function. The m4C methyltransferase METTL15 maintains mitochondrial homeostasis by catalyzing m4C839 located in 12 S rRNA helix 44 (h44). This modification is essential to fine-tuning the ribosomal decoding center and increasing decoding fidelity according to studies of a conserved site in Escherichia coli. Here, we reported a series of crystal structures of human METTL15-hsRBFA-h44-SAM analog, METTL15-hsRBFA-SAM, METTL15-SAM and apo METTL15. The structures presented specific interactions of METTL15 with different substrates and revealed that hsRBFA recruits METTL15 to mitochondrial small subunit for further modification instead of 12 S rRNA. Finally, we found that METTL15 deficiency caused increased reactive oxygen species, decreased membrane potential and altered cellular metabolic state. Knocking down METTL15 caused an elevated lactate secretion and increased levels of histone H4K12-lactylation and H3K9-lactylation. METTL15 might be a suitable model to study the regulation between mitochondrial metabolism and histone lactylation.

A bivalent inhibitor against TDRD3 to suppress phase separation of methylated G3BP1.

The formation of membrane-less organelles is driven by multivalent weak interactions while mediation of such interactions by small molecules remains an unparalleled challenge. Here, we uncovered a bivalent inhibitor that blocked the recruitment of TDRD3 by the two methylated arginines of G3BP1. Relative to the monovalent inhibitor, this bivalent inhibitor demonstrated an enhanced binding affinity to TDRD3 and capability to suppress the phase separation of methylated G3BP1, TDRD3, and RNAs, and in turn inhibit the stress granule growth in cells. Our result paves a new path to mediate multivalent interactions involved in SG assembly for potential combinational chemotherapy by bivalent inhibitors.

ZNF524 directly interacts with telomeric DNA and supports telomere integrity.

Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing nonhomologous end joining (NHEJ) through its subunit TRF2. Here, we describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity, and reveal base-specific sequence recognition by cocrystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity.

ZBTB7A regulates primed-to-naïve transition of pluripotent stem cells via recognition of the PNT-associated sequence by zinc fingers 1-2 and recognition of γ-globin -200 gene element by zinc fingers 1-4.

ZBTB7A, a transcription factor containing a tandem array of four Cys2-His2 zinc fingers (ZFs), is vital for multiple physiological events through directional binding to different genomic loci. Our previously determined crystal structure of ZBTB7A in complex with a GCCCCTTCCCC sequence revealed that all four ZFs (ZF1-4) are involved in binding to γ-globin -200 gene element to repress fetal haemoglobin expression. Recently, it has been reported that ZBTB7A drives primed-to-naïve transition (PNT) of pluripotent stem cells through binding to a 12-bp consensus sequence ([AAGGACCCAGAT], referred to as PNT-associated sequence). Here, we report a crystal structure of ZBTB7A ZF1-3 in complex with the PNT-associated sequence. The structure shows that ZF1 and ZF2 primarily contribute to recognizing the GACCC core sequence mimicking the half part (GCCCC) of γ-globin -200 gene element via specific hydrogen bonding and van der Waals contacts. The mutations of key residues in ZF1-2 remarkably reduce their binding affinities for the PNT-associated sequence in vitro and cannot restore epiblast stem cells to the naïve pluripotent state in vivo. Collectively, our studies demonstrate that ZBTB7A mainly employs its ZF1-2 to recognize the PNT-associated sequence but recognizes γ-globin -200 gene element via ZF1-4, providing insights into the molecular mechanism for the diversity of ZBTB7A's genomic localization.

Structural insights into the recognition of telomeric variant repeat TTGGGG by broad-complex, tramtrack and bric-à-brac - zinc finger protein ZBTB10.

Multiple proteins bind to telomeric DNA and are important for the role of telomeres in genome stability. A recent study established a broad-complex, tramtrack and bric-à-brac - zinc finger (BTB-ZF) protein, ZBTB10 (zinc finger and BTB domain-containing protein 10), as a telomeric variant repeat-binding protein at telomeres that use an alternative method for lengthening telomeres). ZBTB10 specifically interacts with the double-stranded telomeric variant repeat sequence TTGGGG by employing its tandem C2H2 zinc fingers (ZF1-2). Here, we solved the crystal structure of human ZBTB10 ZF1-2 in complex with a double-stranded DNA duplex containing the sequence TTGGGG to assess the molecular details of this interaction. Combined with calorimetric analysis, we identified the vital residues in TTGGGG recognition and determined the specific recognition mechanisms that are different from those of TZAP (telomere zinc finger-associated protein), a recently defined telomeric DNA-binding protein. Following these studies, we further identified a single amino-acid mutant (Arg767Gln) of ZBTB10 ZF1-2 that shows a preference for the telomeric DNA repeat TTAGGG sequence. We solved the cocrystal structure, providing a structural basis for telomeric DNA recognition by C2H2 ZF proteins.

The recognition mode between hsRBFA and mitoribosome 12S rRNA during mitoribosomal biogenesis.

Eukaryotes contain two sets of genomes: the nuclear genome and the mitochondrial genome. The mitochondrial genome transcripts 13 mRNAs that encode 13 essential proteins for the oxidative phosphorylation complex, 2 rRNAs (12s rRNA and 16s rRNA), and 22 tRNAs. The proper assembly and maturation of the mitochondrial ribosome (mitoribosome) are critical for the translation of the 13 key proteins and the function of the mitochondrion. Human ribosome-binding factor A (hsRBFA) is a mitoribosome assembly factor that binds with helix 28, helix 44 and helix 45 of 12S rRNA and facilitates the transcriptional modification of 12S rRNA during the mitoribosomal biogenesis. Previous research mentioned that the malfunction of hsRBFA will induce the instability of mitoribosomes and affect the function of mitochondria, but the mechanisms underlying the interaction between hsRBFA and 12S rRNA and its influence on mitochondrial function are still unknown. In this study, we found that hsRBFA binds with double strain RNA (dsRNA) through its whole N-terminus (Nt) instead of the KH-like domain alone, which is different from the other homologous. Furthermore, we mapped the key residues that affected the RNA binding and maturation of mitoribosomes in vitro. Finally, we investigated how these residues affect mitochondrial functions in detail and systematically.

Phase separation of EB1 guides microtubule plus-end dynamics.

In eukaryotes, end-binding (EB) proteins serve as a hub for orchestrating microtubule dynamics and are essential for cellular dynamics and organelle movements. EB proteins modulate structural transitions at growing microtubule ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. However, the molecular mechanisms and physiochemical properties of the EB1 interaction network remain elusive. Here we show that EB1 formed molecular condensates through liquid-liquid phase separation (LLPS) to constitute the microtubule plus-end machinery. EB1 LLPS is driven by multivalent interactions among different segments, which are modulated by charged residues in the linker region. Phase-separated EB1 provided a compartment for enriching tubulin dimers and other plus-end tracking proteins. Real-time imaging of chromosome segregation in HeLa cells expressing LLPS-deficient EB1 mutants revealed the importance of EB1 LLPS dynamics in mitotic chromosome movements. These findings demonstrate that EB1 forms a distinct physical and biochemical membraneless-organelle via multivalent interactions that guide microtubule dynamics.

Two Binding Sites of SARS-CoV-2 Macrodomain 3 Probed by Oxaprozin and Meclomen.

Severe acute respiratory syndrome-coronavirus-1/2 (SARS-CoV-1/2) macrodomain 3 (Mac3) is critical for replication and transcription of the viral genome and is therefore a potential therapeutic target. Here, we solved the crystal structure of SARS-CoV-2 Mac3, which reveals a small-molecule binding pocket. Two low-molecular-weight drugs, oxaprozin and meclomen, induced different patterns of nuclear magnetic resonance (NMR) chemical shift perturbations (CSPs). Meclomen binds to site I of SARS-CoV-2 Mac3 with binding pose determined by NMR CSP and transferred paramagnetic relaxation enhancement, while oxaprozin binds to site II as revealed by the crystal structure. Interestingly, oxaprozin and meclomen both perturb residues in site I of SARS-CoV Mac3. Fluorescence polarization experiments further demonstrated that oxaprozin and meclomen inhibited the binding of DNA-G4s to SARS-CoV-2 Mac3. Our work identified two adjacent ligand-binding sites of SARS-CoV-2 Mac3 that shall facilitate structure-guided fragment linking of these compounds for more potent inhibitors.

Structural basis for Gemin5 decamer-mediated mRNA binding.

Gemin5 in the Survival Motor Neuron (SMN) complex serves as the RNA-binding protein to deliver small nuclear RNAs (snRNAs) to the small nuclear ribonucleoprotein Sm complex via its N-terminal WD40 domain. Additionally, the C-terminal region plays an important role in regulating RNA translation by directly binding to viral RNAs and cellular mRNAs. Here, we present the three-dimensional structure of the Gemin5 C-terminal region, which adopts a homodecamer architecture comprised of a dimer of pentamers. By structural analysis, mutagenesis, and RNA-binding assays, we find that the intact pentamer/decamer is critical for the Gemin5 C-terminal region to bind cognate RNA ligands and to regulate mRNA translation. The Gemin5 high-order architecture is assembled via pentamerization, allowing binding to RNA ligands in a coordinated manner. We propose a model depicting the regulatory role of Gemin5 in selective RNA binding and translation. Therefore, our work provides insights into the SMN complex-independent function of Gemin5.

Protocols for protein-DNA binding analysis of a zinc finger transcription factor bound to its cognate promoter.

Here, we describe protocols to interrogate the binding of the zinc fingers of the transcription factor ZBTB7A to the fetal γ-globin (HBG) promoter. We detail the steps for performing electrophoretic mobility shift assays (EMSAs), X-ray crystallography, and isothermal titration calorimetry (ITC) to explore this interaction. These techniques could readily be applied to the structural studies of other zinc finger transcription factors and cognate DNA sequences. For complete details on the use and execution of this protocol, please refer to Yang et al. (2021).

Structural insights reveal the specific recognition of meiRNA by the Mei2 protein.

In the fission yeast Schizosaccharomyces pombe, Mei2, an RNA-binding protein essential for entry into meiosis, regulates meiosis initiation. Mei2 binds to a specific non-coding RNA species, meiRNA, and accumulates at the sme2 gene locus, which encodes meiRNA. Previous research has shown that the Mei2 C-terminal RNA recognition motif (RRM3) physically interacts with the meiRNA 5' region in vitro and stimulates meiosis in vivo. However, the underlying mechanisms still remain elusive. We first employed an in vitro crosslinking and immunoprecipitation sequencing (CLIP-seq) assay and demonstrated a preference for U-rich motifs of meiRNA by Mei2 RRM3. We then solved the crystal structures of Mei2 RRM3 in the apo form and complex with an 8mer RNA fragment, derived from meiRNA, as detected by in vitro CLIP-seq. These results provide structural insights into the Mei2 RRM3-meiRNA complex and reveal that Mei2 RRM3 binds specifically to the UUC(U) sequence. Furthermore, a structure-based Mei2 mutation, Mei2F644A causes defective karyogamy, suggesting an essential role of the RNA-binding ability of Mei2 in regulating meiosis.