Disrupting Roquin-1 interaction with Regnase-1 induces autoimmunity and enhances antitumor responses.

Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies.

Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation.

Cytoplasmic FUS aggregates are a pathological hallmark in a subset of patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). A key step that is disrupted in these patients is nuclear import of FUS mediated by the import receptor Transportin/Karyopherin-ß2. In ALS-FUS patients, this is caused by mutations in the nuclear localization signal (NLS) of FUS that weaken Transportin binding. In FTD-FUS patients, Transportin is aggregated, and post-translational arginine methylation, which regulates the FUS-Transportin interaction, is lost. Here, we show that Transportin and arginine methylation have a crucial function beyond nuclear import-namely to suppress RGG/RG-driven phase separation and stress granule association of FUS. ALS-associated FUS-NLS mutations weaken the chaperone activity of Transportin and loss of FUS arginine methylation, as seen in FTD-FUS, promote phase separation, and stress granule partitioning of FUS. Our findings reveal two regulatory mechanisms of liquid-phase homeostasis that are disrupted in FUS-associated neurodegeneration.

A naturally occurring 22-amino acid fragment of human hemoglobin A inhibits autophagy and HIV-1.

Autophagy is an evolutionarily ancient catabolic pathway and has recently emerged as an integral part of the innate immune system. While the core machinery of autophagy is well defined, the physiological regulation of autophagy is less understood. Here, we identify a C-terminal fragment of human hemoglobin A (HBA1, amino acids 111-132) in human bone marrow as a fast-acting non-inflammatory inhibitor of autophagy initiation. It is proteolytically released from full-length HBA1 by cathepsin E, trypsin or pepsin. Biochemical characterization revealed that HBA1(111-132) has an in vitro stability of 52 min in human plasma and adopts a flexible monomeric conformation in solution. Structure-activity relationship studies revealed that the C-terminal 13 amino acids of HBA1(120-132) are sufficient to inhibit autophagy, two charged amino acids (D127, K128) mediate solubility, and two serines (S125, S132) are required for function. Successful viruses like human immunodeficiency virus 1 (HIV-1) evolved strategies to subvert autophagy for virion production. Our results show that HBA1(120-132) reduced virus yields of lab-adapted and primary HIV-1. Summarizing, our data identifies naturally occurring HBA1(111-132) as a physiological, non-inflammatory antagonist of autophagy. Optimized derivatives of HBA1(111-132) may offer perspectives to restrict autophagy-dependent viruses.

Human TRMT2A methylates tRNA and contributes to translation fidelity.

5-Methyluridine (m5U) is one of the most abundant RNA modifications found in cytosolic tRNA. tRNA methyltransferase 2 homolog A (hTRMT2A) is the dedicated mammalian enzyme for m5U formation at tRNA position 54. However, its RNA binding specificity and functional role in the cell are not well understood. Here we dissected structural and sequence requirements for binding and methylation of its RNA targets. Specificity of tRNA modification by hTRMT2A is achieved by a combination of modest binding preference and presence of a uridine in position 54 of tRNAs. Mutational analysis together with cross-linking experiments identified a large hTRMT2A-tRNA binding surface. Furthermore, complementing hTRMT2A interactome studies revealed that hTRMT2A interacts with proteins involved in RNA biogenesis. Finally, we addressed the question of the importance of hTRMT2A function by showing that its knockdown reduces translation fidelity. These findings extend the role of hTRMT2A beyond tRNA modification towards a role in translation.

Depletion of the RNA-binding protein PURA triggers changes in posttranscriptional gene regulation and loss of P-bodies.

The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs are not affected. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders.

Trnp1 organizes diverse nuclear membrane-less compartments in neural stem cells.

TMF1-regulated nuclear protein 1 (Trnp1) has been shown to exert potent roles in neural development affecting neural stem cell self-renewal and brain folding, but its molecular function in the nucleus is still unknown. Here, we show that Trnp1 is a low complexity protein with the capacity to phase separate. Trnp1 interacts with factors located in several nuclear membrane-less organelles, the nucleolus, nuclear speckles, and condensed chromatin. Importantly, Trnp1 co-regulates the architecture and function of these nuclear compartments in vitro and in the developing brain in vivo. Deletion of a highly conserved region in the N-terminal intrinsic disordered region abolishes the capacity of Trnp1 to regulate nucleoli and heterochromatin size, proliferation, and M-phase length; decreases the capacity to phase separate; and abrogates most of Trnp1 protein interactions. Thus, we identified Trnp1 as a novel regulator of several nuclear membrane-less compartments, a function important to maintain cells in a self-renewing proliferative state.

Methylated HNRNPK acts on RPS19 to regulate ALOX15 synthesis in erythropoiesis.

Post-transcriptional control is essential to safeguard structural and metabolic changes in enucleated reticulocytes during their terminal maturation to functional erythrocytes. The timely synthesis of arachidonate 15-lipoxygenase (ALOX15), which initiates mitochondria degradation at the final stage of reticulocyte maturation is regulated by the multifunctional protein HNRNPK. It constitutes a silencing complex at the ALOX15 mRNA 3' untranslated region that inhibits translation initiation at the AUG by impeding the joining of ribosomal 60S subunits to 40S subunits. To elucidate how HNRNPK interferes with 80S ribosome assembly, three independent screens were applied. They consistently demonstrated a differential interaction of HNRNPK with RPS19, which is localized at the head of the 40S subunit and extends into its functional center. During induced erythroid maturation of K562 cells, decreasing arginine dimethylation of HNRNPK is linked to a reduced interaction with RPS19 in vitro and in vivo. Dimethylation of residues R256, R258 and R268 in HNRNPK affects its interaction with RPS19. In noninduced K562 cells, RPS19 depletion results in the induction of ALOX15 synthesis and mitochondria degradation. Interestingly, residue W52 in RPS19, which is frequently mutated in Diamond-Blackfan Anemia (DBA), participates in specific HNRNPK binding and is an integral part of a putative aromatic cage.

Structural basis for the recognition of transiently structured AU-rich elements by Roquin.

Adenylate/uridylate-rich elements (AREs) are the most common cis-regulatory elements in the 3'-untranslated region (UTR) of mRNAs, where they fine-tune turnover by mediating mRNA decay. They increase plasticity and efficacy of mRNA regulation and are recognized by several ARE-specific RNA-binding proteins (RBPs). Typically, AREs are short linear motifs with a high content of complementary A and U nucleotides and often occur in multiple copies. Although thermodynamically rather unstable, the high AU-content might enable transient secondary structure formation and modify mRNA regulation by RBPs. We have recently suggested that the immunoregulatory RBP Roquin recognizes folded AREs as constitutive decay elements (CDEs), resulting in shape-specific ARE-mediated mRNA degradation. However, the structural evidence for a CDE-like recognition of AREs by Roquin is still lacking. We here present structures of CDE-like folded AREs, both in their free and protein-bound form. Moreover, the AREs in the UCP3 3'-UTR are additionally bound by the canonical ARE-binding protein AUF1 in their linear form, adopting an alternative binding-interface compared to the recognition of their CDE structure by Roquin. Strikingly, our findings thus suggest that AREs can be recognized in multiple ways, allowing control over mRNA regulation by adapting distinct conformational states, thus providing differential accessibility to regulatory RBPs.

Challenging a Preconception: Optoacoustic Spectrum Differs from the Optical Absorption Spectrum of Proteins and Dyes for Molecular Imaging.

Optoacoustic (photoacoustic) imaging has seen marked advances in detection and data analysis, but there is less progress in understanding the photophysics of common optoacoustic contrast agents. This gap blocks the development of novel agents and the accurate analysis and interpretation of multispectral optoacoustic images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. Herein, we employ MLS to analyze contrast agents (methylene blue, rhodamine 800, Alexa Fluor 750, IRDye 800CW, and indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720, and smURFP). We found that the optical absorption spectrum does not correlate with the optoacoustic spectrum for the majority of the analytes. We determined that for dyes, the transition underlying an aggregation state has more optoacoustic signal generation efficiency than the monomer transition. For proteins we found a favored optoacoustic relaxation that stems from the neutral or zwitterionic chromophores and unreported photoswitching behavior of tdTomato and HcRed. We then crystalized HcRed in its photoswitch optoacoustic state, confirming structurally the change in isomerization with respect to HcReds' fluorescence state. Finally, on the example of the widely used label tdTomato and the dye indocyanine green, we show the importance of correct photophysical (e.g., spectral and kinetic) information as a prerequisite for spectral-unmixing for in vivo imaging.

Ferritin-Displayed GLP-1 with Improved Pharmacological Activities and Pharmacokinetics.

Glucagon-like peptide-1 (GLP-1) is an incretin (a type of metabolic hormone that stimulates a decrease in blood glucose levels), holding great potential for the treatment of type 2 diabetes mellitus (T2DM). However, its extremely short half-life of 1-2 min hampers any direct clinical application. Here, we describe the application of the heavy chain of human ferritin (HFt) nanocage as a carrier to improve the pharmacological properties of GLP-1. The GLP-HFt was designed by genetic fusion of GLP-1 to the N-terminus of HFt and was expressed in inclusion bodies in E. coli. The refolding process was developed to obtain a soluble GLP-HFt protein. The biophysical properties determined by size-exclusion chromatography (SEC), dynamic light scattering (DLS), circular dichroism (CD), transmission electron microscopy (TEM), and X-ray crystallography verified that the GLP-HFt successfully formed a 24-mer nanocage with GLP-1 displayed on the external surface of HFt. The in vivo pharmacodynamic results demonstrated that the GLP-HFt nanocage retained the bioactivity of natural GLP-1, significantly reduced the blood glucose levels for at least 24 h in a dose-dependent manner, and inhibited food intake for at least 8-10 h. The half-life of the GLP-HFt nanocage was approximately 52 h in mice after subcutaneous injection. The prolonged half-life and sustained control of blood glucose levels indicate that the GLP-HFt nanocage can be further developed for the treatment of T2DM. Meanwhile, the HFt nanocage proves its great potential as a universal carrier that improves the pharmacodynamic and pharmacokinetic properties of a wide range of therapeutic peptides and proteins.

The large family of PC4-like domains - similar folds and functions throughout all kingdoms of life.

RNA- and DNA-binding domains are essential building blocks for specific regulation of gene expression. While a number of canonical nucleic acid binding domains share sequence and structural conservation, others are less obviously linked by evolutionary traits. In this review, we describe a protein fold of about 150 aa in length, bearing a conserved ß-ß-ß-ß-α-linker-ß-ß-ß-ß-α topology and similar nucleic acid binding properties but no apparent sequence conservation. The same overall fold can also be achieved by dimerization of two proteins, each bearing a ß-ß-ß-ß-α topology. These proteins include but are not limited to the transcription factors PC4 and P24 from humans and plants, respectively, the human RNA-transport factor Pur-α (also termed PURA), as well as the ssDNA-binding SP_0782 protein from Streptococcus pneumonia and the bacteriophage coat proteins PP7 and MS2. Besides their common overall topology, these proteins share common nucleic acids binding surfaces and thus functional similarity. We conclude that these PC4-like domains include proteins from all kingdoms of life and are much more abundant than previously known.

A high-content screen for small-molecule regulators of epithelial cell-adhesion molecule (EpCAM) cleavage yields a robust inhibitor.

Epithelial cell-adhesion molecule (EpCAM) is a transmembrane protein that regulates cell cycle progression and differentiation and is overexpressed in many carcinomas. The EpCAM-induced mitogenic cascade is activated via regulated intramembrane proteolysis (RIP) of EpCAM by ADAM and γ-secretases, generating the signaling-active intracellular domain EpICD. Because of its expression pattern and molecular function, EpCAM is a valuable target in prognostic and therapeutic approaches for various carcinomas. So far, several immunotherapeutic strategies have targeted the extracellular domain of EpCAM. However, targeting the intracellular signaling cascade of EpCAM holds promise for specifically interfering with EpCAM's proliferation-stimulating signaling cascade. Here, using a yellow fluorescence protein-tagged version of the C-terminal fragment of EpCAM, we established a high-content screening (HCS) of a small-molecule compound library (n = 27,280) and characterized validated hits that target EpCAM signaling. In total, 128 potential inhibitors were initially identified, of which one compound with robust inhibitory effects on RIP of EpCAM was analyzed in greater detail. In summary, our study demonstrates that the development of an HCS for small-molecule inhibitors of the EpCAM signaling pathway is feasible. We propose that this approach may also be useful for identifying chemical compounds targeting other disorders involving membrane cleavage-dependent signaling pathways.

Crystal structure of a biliverdin-bound phycobiliprotein: Interdependence of oligomerization and chromophorylation.

Small, ultra-red fluorescence protein (smURFP) introduces the non-native biliverdin (BV) chromophore to phycobiliproteins (PBPs), allowing them to be used as transgenic labels for in vivo mammalian imaging. Presently, no structural information exists for PBPs bound to the non-native BV chromophore, which limits the further development of smURFP and related proteins as imaging labels or indicators. Here we describe the first crystal structure of a PBP bound to BV. The structures of smURFP-Y56R with BV and smURFP-Y56F without BV reveal unique oligomerization interfaces different from those in wild-type PBPs bound to native chromophores. Our structures suggest that the oligomerization interface affects the BV binding site, creating a link between oligomerization and chromophorylation that we confirmed through site-directed mutagenesis and that may help guide efforts to improve the notorious chromophorylation of smURFP and other PBPs engineered to bind BV.

mRNA transport meets membrane traffic.

Active transport and local translation of mRNAs ensure the appropriate spatial organization of proteins within cells. Recent work has shown that this process is intricately connected to membrane trafficking. Here, we focus on new findings obtained in fungal model systems. Important highlights are that RNA-binding proteins recognize cargo mRNA synergistically and that mRNAs are co-transported with membranous compartments such as the endoplasmic reticulum (ER) and endosomes. We further discuss a novel concept of endosome-coupled translation that loads shuttling endosomes with septin cargo, a process important for correct septin filamentation. Interestingly, evidence is accumulating that RNA and membrane trafficking are also tightly interwoven in higher eukaryotes, suggesting that this phenomenon is a common theme and not an exception restricted to fungi.

Functional dissection of the paired domain of Pax6 reveals molecular mechanisms of coordinating neurogenesis and proliferation.

To achieve adequate organ development and size, cell proliferation and differentiation have to be tightly regulated and coordinated. The transcription factor Pax6 regulates patterning, neurogenesis and proliferation in forebrain development. The molecular basis of this regulation is not well understood. As the bipartite DNA-binding paired domain of Pax6 regulates forebrain development, we examined mice with point mutations in its individual DNA-binding subdomains PAI (Pax6(Leca4), N50K) and RED (Pax6(Leca2), R128C). This revealed distinct roles in regulating proliferation in the developing cerebral cortex, with the PAI and RED subdomain mutations reducing and increasing, respectively, the number of mitoses. Conversely, neurogenesis was affected only by the PAI subdomain mutation, phenocopying the neurogenic defects observed in full Pax6 mutants. Genome-wide expression profiling identified molecularly discrete signatures of Pax6(Leca4) and Pax6(Leca2) mutations. Comparison to Pax6 targets identified by chromatin immunoprecipitation led to the identification and functional characterization of distinct DNA motifs in the promoters of target genes dysregulated in the Pax6(Leca2) or Pax6(Leca4) mutants, further supporting the distinct regulatory functions of the DNA-binding subdomains. Thus, Pax6 achieves its key roles in the developing forebrain by utilizing particular subdomains to coordinate patterning, neurogenesis and proliferation simultaneously.

Role of Loc1p in assembly and reorganization of nuclear ASH1 messenger ribonucleoprotein particles in yeast.

Directional transport of mRNA is a universal feature in eukaryotes, requiring the assembly of motor-dependent RNA-transport particles. The cytoplasmic transport of mRNAs is preceded by the nuclear assembly of pre-messenger ribonucleoprotein particles (mRNPs). In budding yeast, the asymmetric synthesis of HO 1 (ASH1) pre-mRNP originates already cotranscriptionally and passes through the nucleolus before its nuclear export. The nucleolar localization of ASH1 mRNA protein 1 (Loc1p) is required for efficient ASH1 mRNA localization. Immunoprecipitation experiments have revealed that Loc1p forms cocomplexes with other components of the ASH1 transport complex. However, it remains unclear how Loc1p is recruited into this mRNP and why Loc1p is important for ASH1 mRNA localization. Here we demonstrate that Loc1p undergoes a direct and specific interaction with the ASH1 mRNA-binding Swi5p-dependent HO expression protein 2 (She2p). This cocomplex shows higher affinity and specificity for RNA bearing localization elements than the individual proteins. It also stabilizes the otherwise transient binding of She2p to ASH1 mRNA, suggesting that cooperative mRNA binding of Loc1p with She2p is the required nuclear function of Loc1p for ASH1 mRNA localization. After nuclear export, myosin-bound She3p joins the ASH1 mRNP to form a highly specific cocomplex with She2p and ASH1 mRNA. Because Loc1p is found only in the nucleus, it must be removed from the complex directly before or after export. In vitro and in vivo experiments indicate that the synergistic interaction of She2p and She3p displaces Loc1p from the ASH1 complex, allowing free Loc1p to rapidly reenter the nucle(ol)us. Together these findings suggest an ordered process of nuclear assembly and reorganization for the maturation of localizing ASH1 mRNPs.

Structural features of Argonaute-GW182 protein interactions.

MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to target mRNAs, leading to gene silencing. However, Ago proteins are not the actual mediators of gene silencing but interact with a member of the GW182 protein family (also known as GW proteins), which coordinates all downstream steps in gene silencing. GW proteins contain an N-terminal Ago-binding domain that is characterized by multiple GW repeats and a C-terminal silencing domain with several globular domains. Within the Ago-binding domain, Trp residues mediate the direct interaction with the Ago protein. Here, we have characterized the interaction of Ago proteins with GW proteins in molecular detail. Using biochemical and NMR experiments, we show that only a subset of Trp residues engage in Ago interactions. The Trp residues are located in intrinsically disordered regions, where flanking residues mediate additional weak interactions, that might explain the importance of specific tryptophans. Using cross-linking followed by mass spectrometry, we map the GW protein interactions with Ago2, which allows for structural modeling of Ago-GW182 interaction. Our data further indicate that the Ago-GW protein interaction might be a two-step process involving the sequential binding of two tryptophans separated by a spacer with a minimal length of 10 aa.

ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions.

Asymmetric ASH1 mRNA transport during mitosis of budding yeast constitutes one of the best-studied examples of mRNA localization. Recently, 2 studies used in vitro motility assays to prove that motile ASH1 mRNA-transport complexes can be reconstituted entirely from recombinant factors. Both studies, however, differed in their conclusions on whether cargo RNA itself is required for particle assembly and thus activation of directional transport. Here we provide direct evidence that stable complexes do assemble in absence of RNA at physiologic conditions and even at ionic strengths above cellular levels. These results directly confirm the previous notion that the ASH1 transport machinery is not activated by the cargo RNA itself, but rather through protein-protein interactions.

Production of pure and functional RNA for in vitro reconstitution experiments.

Reconstitution of protein complexes has been a valuable tool to test molecular functions and to interpret in vivo observations. In recent years, a large number of RNA-protein complexes has been identified to regulate gene expression and to be important for a range of cellular functions. In contrast to protein complexes, in vitro analyses of RNA-protein complexes are hampered by the fact that recombinant expression and purification of RNA molecules is more difficult and less well established than for proteins. Here we review the current state of technology available for in vitro experiments with RNAs. We outline the possibilities to produce and purify large amounts of homogenous RNA and to perform the required quality controls. RNA-specific problems such as degradation, 5' and 3' end heterogeneity, co-existence of different folding states, and prerequisites for reconstituting RNAs with recombinantly expressed proteins are discussed. Additionally a number of techniques for the characterization of direct and indirect RNA-protein interactions are explained.

A cytoplasmic complex mediates specific mRNA recognition and localization in yeast.

In eukaryotes, hundreds of mRNAs are localized by specialized transport complexes. For localization, transcripts are recognized by RNA-binding proteins and incorporated into motor-containing messenger ribonucleoprotein particles (mRNPs). To date, the molecular assembly of such mRNPs is not well understood and most details on cargo specificity remain unresolved. We used ASH1-mRNA transport in yeast to provide a first assessment of where and how localizing mRNAs are specifically recognized and incorporated into mRNPs. By using in vitro-interaction and reconstitution assays, we found that none of the implicated mRNA-binding proteins showed highly specific cargo binding. Instead, we identified the cytoplasmic myosin adapter She3p as additional RNA-binding protein. We further found that only the complex of the RNA-binding proteins She2p and She3p achieves synergistic cargo binding, with an at least 60-fold higher affinity for localizing mRNAs when compared to control RNA. Mutational studies identified a C-terminal RNA-binding fragment of She3p to be important for synergistic RNA binding with She2p. The observed cargo specificity of the ternary complex is considerably higher than previously reported for localizing mRNAs. It suggests that RNA binding for mRNP localization generally exhibits higher selectivity than inferred from previous in vitro data. This conclusion is fully consistent with a large body of in vivo evidence from different organisms. Since the ternary yeast complex only assembles in the cytoplasm, specific mRNA recognition might be limited to the very last steps of mRNP assembly. Remarkably, the mRNA itself triggers the assembly of mature, motor-containing complexes. Our reconstitution of a major portion of the mRNA-transport complex offers new and unexpected insights into the molecular assembly of specific, localization-competent mRNPs and provides an important step forward in our mechanistic understanding of mRNA localization in general.