Heteromeric RNP Assembly at LINEs Controls Lineage-Specific RNA Processing.

Long mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs repress splicing and 3' end processing within and around LINEs. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are located far from exons. These RBPs insulate the LINEs and the surrounding intronic regions from RNA processing. Upon evolutionary divergence, changes in RNA motifs within LINEs lead to gradual loss of their insulation. Hence, older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for the assembly of repressive RBPs and also contribute to the evolution of new, lineage-specific transcripts in mammals. VIDEO ABSTRACT.

Asymmetric friction of nonmotor MAPs can lead to their directional motion in active microtubule networks.

Diverse cellular processes require microtubules to be organized into distinct structures, such as asters or bundles. Within these dynamic motifs, microtubule-associated proteins (MAPs) are frequently under load, but how force modulates these proteins' function is poorly understood. Here, we combine optical trapping with TIRF-based microscopy to measure the force dependence of microtubule interaction for three nonmotor MAPs (NuMA, PRC1, and EB1) required for cell division. We find that frictional forces increase nonlinearly with MAP velocity across microtubules and depend on filament polarity, with NuMA's friction being lower when moving toward minus ends, EB1's lower toward plus ends, and PRC1's exhibiting no directional preference. Mathematical models predict, and experiments confirm, that MAPs with asymmetric friction can move directionally within actively moving microtubule pairs they crosslink. Our findings reveal how nonmotor MAPs can generate frictional resistance in dynamic cytoskeletal networks via micromechanical adaptations whose anisotropy may be optimized for MAP localization and function within cellular structures.

Higher-Order Assembly of BRCC36-KIAA0157 Is Required for DUB Activity and Biological Function.

BRCC36 is a Zn(2+)-dependent deubiquitinating enzyme (DUB) that hydrolyzes lysine-63-linked ubiquitin chains as part of distinct macromolecular complexes that participate in either interferon signaling or DNA-damage recognition. The MPN(+) domain protein BRCC36 associates with pseudo DUB MPN(-) proteins KIAA0157 or Abraxas, which are essential for BRCC36 enzymatic activity. To understand the basis for BRCC36 regulation, we have solved the structure of an active BRCC36-KIAA0157 heterodimer and an inactive BRCC36 homodimer. Structural and functional characterizations show how BRCC36 is switched to an active conformation by contacts with KIAA0157. Higher-order association of BRCC36 and KIAA0157 into a dimer of heterodimers (super dimers) was required for DUB activity and interaction with targeting proteins SHMT2 and RAP80. These data provide an explanation of how an inactive pseudo DUB allosterically activates a cognate DUB partner and implicates super dimerization as a new regulatory mechanism underlying BRCC36 DUB activity, subcellular localization, and biological function.

SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.

Molecular mechanism underlying selective inhibition of mRNA nuclear export by herpesvirus protein ORF10.

Viruses employ multiple strategies to inhibit host mRNA nuclear export. Distinct to the generally nonselective inhibition mechanisms, ORF10 from gammaherpesviruses inhibits mRNA export in a transcript-selective manner by interacting with Rae1 (RNA export 1) and Nup98 (nucleoporin 98). We now report the structure of ORF10 from MHV-68 (murine gammaherpesvirus 68) bound to the Rae1-Nup98 heterodimer, thereby revealing detailed intermolecular interactions. Structural and functional assays highlight that two highly conserved residues of ORF10, L60 and M413, play critical roles in both complex assembly and mRNA export inhibition. Interestingly, although ORF10 occupies the RNA-binding groove of Rae1-Nup98, the ORF10-Rae1-Nup98 ternary complex still maintains a comparable RNA-binding ability due to the ORF10-RNA direct interaction. Moreover, mutations on the RNA-binding surface of ORF10 disrupt its function of mRNA export inhibition. Our work demonstrates the molecular mechanism of ORF10-mediated selective inhibition and provides insights into the functions of Rae1-Nup98 in regulating host mRNA export.

RNF8 mediates NONO degradation following UV-induced DNA damage to properly terminate ATR-CHK1 checkpoint signaling.

RNF8 plays a critical role in DNA damage response (DDR) to initiate ubiquitination-dependent signaling. To better characterize the role of RNF8 in UV-induced DDR, we searched for novel substrates of RNF8 and identified NONO as one intriguing substrate. We found that: (i) RNF8 ubiquitinates NONO and (ii) UV radiation triggers NONO ubiquitination and its subsequent degradation. Depletion of RNF8 inhibited UV-induced degradation of NONO, suggesting that RNF8 targets NONO for degradation in response to UV damage. In addition, we found that 3 NONO lysine residues (positions 279, 290 and 295) are important for conferring its instability in UV-DDR. Depletion of RNF8 or expression of NONO with lysine to arginine substitutions at positions 279, 290 and 295 prolonged CHK1 phosphorylation over an extended period of time. Furthermore, expression of the stable mutant, but not wild-type NONO, induced a prolonged S phase following UV exposure. Stable cell lines expressing the stable NONO mutant showed increased UV sensitivity in a clonogenic survival assay. Since RNF8 recruitment to the UV-damaged sites is dependent on ATR, we propose that RNF8-mediated NONO degradation and subsequent inhibition of NONO-dependent chromatin loading of TOPBP1, a key activator of ATR, function as a negative feedback loop critical for turning off ATR-CHK1 checkpoint signaling in UV-DDR.

In situ 3D visualization of biomineralization matrix proteins.

Calcium biominerals occur in all major animal phyla, and through biomolecular control, exhibit such diverse structures as exoskeletons, shells, bones, teeth and earstones (otoliths). Determining the three-dimensional expression of key biomineral proteins, however, has proven challenging as typical protein identification methods either lose spatial resolution during dissolution of the mineral phase or are costly and limited to two-dimensional expression of high abundance proteins. Here we present a modification of the CLARITY and ACT-PRESTO protocols to visualize and confirm, for the first time, the timing of expression and function of two key regulators of biomineralization.

Neuron-enriched phosphatase and actin regulator 3 (Phactr3)/ nuclear scaffold-associated PP1-inhibiting protein (Scapinin) regulates dendritic morphology via its protein phosphatase 1-binding domain.

Phosphatase and actin regulator 3/nuclear scaffold-associated protein phosphatase 1-inhibiting protein (Phactr3/Scapinin) is an actin- and protein phosphatase 1 (PP1)-binding protein known to negatively regulate axon elongation. In this study, we examined the expression pattern of Phactr3/Scapinin in several tissues and investigated the effect of Phactr3/Scapinin on dendritic morphology of cortical neurons. Results showed that Phactr3/Scapinin expression was up-regulated in the developing brain and enriched in neurons and in the postsynaptic density fraction, but not in astrocytes. Overexpression of wild type or mutant Phactr3/Scapinin, which lacked actin-binding activity, resulted in increased dendritic complexity and percentage of spines with a mushroom or stubby shape, as well as a decrease in spine density. However, overexpression of mutant Phactr3/Scapinin that lacked PP1-binding activity did not. Taken together, these findings suggest that Phactr3/Scapinin expression is neuronal and might contribute to synaptic formation via distinct actin- and PP1-binding domains involved in dendritic and axonal morphology, respectively.

Grafting a short chameleon sequence from αB crystallin into a ß-sheet scaffold protein.

Many protein and peptide sequences are self-assembled into ß-sheet-rich fibrous structures called amyloids. Their atomic details provide insights into fundamental knowledge related to amyloid diseases. To study the detailed structure of the amyloid, we have developed a model system that mimics the self-assembling process of the amyloid within a water-soluble protein, termed peptide self-assembly mimic (PSAM). PSAM enables capturing of a peptide sequence within a water-soluble protein, thus making structural and energetics-related studies possible. In this work, we extend our PSAM approach to a naturally occurring chameleon sequence from αB crystallin. We chose "Val-Leu-Gly-Asp-Val (VLGDV)", a five amino-acid sequence, which forms a ß-turn in the native structure and a ß-barrel in the amyloid oligomer cylindrin, as a grafting sequence to the PSAM scaffold. The crystal structure revealed that the sequence grafting induced ß-sheet bending at the grafted site. We further investigated the role of the central glycine residue and found that its role in the ß-sheet bending is dependent on the neighboring residues. The ability of PSAM to observe the structural alterations induced by the grafted sequence provides an opportunity to evaluate the structural impact of a sequence from the peptide self-assembly.

LGN/mInsc and LGN/NuMA complex structures suggest distinct functions in asymmetric cell division for the Par3/mInsc/LGN and Gαi/LGN/NuMA pathways.

Asymmetric cell division requires the establishment of cortical cell polarity and the orientation of the mitotic spindle along the axis of cell polarity. Evidence from invertebrates demonstrates that the Par3/Par6/aPKC and NuMA/LGN/Gαi complexes, which are thought to be physically linked by the adaptor protein mInscuteable (mInsc), play indispensable roles in this process. However, the molecular basis for the binding of LGN to NuMA and mInsc is poorly understood. The high-resolution structures of the LGN/NuMA and LGN/mInsc complexes presented here provide mechanistic insights into the distinct and highly specific interactions of the LGN TPRs with mInsc and NuMA. Structural comparisons, together with biochemical and cell biology studies, demonstrate that the interactions of NuMA and mInsc with LGN are mutually exclusive, with mInsc binding preferentially. Our results suggest that the Par3/mInsc/LGN and NuMA/LGN/Gαi complexes play sequential and partially overlapping roles in asymmetric cell division.

SFPQ•NONO and XLF function separately and together to promote DNA double-strand break repair via canonical nonhomologous end joining.

A complex of two related mammalian proteins, SFPQ and NONO, promotes DNA double-strand break repair via the canonical nonhomologous end joining (c-NHEJ) pathway. However, its mechanism of action is not fully understood. Here we describe an improved SFPQ•NONO-dependent in vitro end joining assay. We use this system to demonstrate that the SFPQ•NONO complex substitutes in vitro for the core c-NHEJ factor, XLF. Results are consistent with a model where SFPQ•NONO promotes sequence-independent pairing of DNA substrates, albeit in a way that differs in detail from XLF. Although SFPQ•NONO and XLF function redundantly in vitro, shRNA-mediated knockdown experiments indicate that NONO and XLF are both required for efficient end joining and radioresistance in cell-based assays. In addition, knockdown of NONO sensitizes cells to the interstrand crosslinking agent, cisplatin, whereas knockdown of XLF does not, and indeed suppresses the effect of NONO deficiency. These findings suggest that each protein has one or more unique activities, in addition to the DNA pairing revealed in vitro, that contribute to DNA repair in the more complex cellular milieu. The SFPQ•NONO complex contains an RNA binding domain, and prior work has demonstrated diverse roles in RNA metabolism. It is thus plausible that the additional repair function of NONO, revealed in cell-based assays, could involve RNA interaction.

Protein⁻Protein Interactions with Connexin 43: Regulation and Function.

Connexins are integral membrane building blocks that form gap junctions, enabling direct cytoplasmic exchange of ions and low-molecular-mass metabolites between adjacent cells. In the heart, gap junctions mediate the propagation of cardiac action potentials and the maintenance of a regular beating rhythm. A number of connexin interacting proteins have been described and are known gap junction regulators either through direct effects (e.g., kinases) or the formation of larger multifunctional complexes (e.g., cytoskeleton scaffold proteins). Most connexin partners can be categorized as either proteins promoting coupling by stimulating forward trafficking and channel opening or inhibiting coupling by inducing channel closure, internalization, and degradation. While some interactions have only been implied through co-localization using immunohistochemistry, others have been confirmed by biophysical methods that allow detection of a direct interaction. Our understanding of these interactions is, by far, most well developed for connexin 43 (Cx43) and the scope of this review is to summarize our current knowledge of their functional and regulatory roles. The significance of these interactions is further exemplified by demonstrating their importance at the intercalated disc, a major hub for Cx43 regulation and Cx43 mediated effects.

A conserved charged single α-helix with a putative steric role in paraspeckle formation.

Paraspeckles are subnuclear particles involved in the regulation of mRNA expression. They are formed by the association of DBHS family proteins and the NEAT1 long noncoding RNA. Here, we show that a recently identified structural motif, the charged single α-helix, is largely conserved in the DBHS family. Based on the available structural data and a previously suggested multimerization scheme of DBHS proteins, we built a structural model of a (PSPC1/NONO)(n) multimer that might have relevance in paraspeckle formation. Our model contains an extended coiled-coil region that is followed by and partially overlaps with the predicted charged single α-helix. We suggest that the charged single α-helix can act as an elastic ruler governing the exact positioning of the dimeric core structures relative to each other during paraspeckle assembly along the NEAT1 noncoding RNA.

Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA.

Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.

Vesiculoviral matrix (M) protein occupies nucleic acid binding site at nucleoporin pair (Rae1 • Nup98).

mRNA export factor 1 (Rae1) and nucleoporin 98 (Nup98) are host cell targets for the matrix (M) protein of vesicular stomatitis virus (VSV). How Rae1 functions in mRNA export and how M protein targets both Rae1 and Nup98 are not understood at the molecular level. To obtain structural insights, we assembled a 1:1:1 complex of M•Rae1•Nup98 and established a crystal structure at 3.15-Å resolution. We found that the M protein contacts the Rae1•Nup98 heterodimer principally by two protrusions projecting from the globular domain of M like a finger and thumb. Both projections clamp to the side of the ß-propeller of Rae1, with the finger also contacting Nup98. The most prominent feature of the finger is highly conserved Methionine 51 (Met51) with upstream and downstream acidic residues. The complementary surface on Rae1 displays a deep hydrophobic pocket, into which Met51 fastens like a bolt, and a groove of basic residues on either side, which bond to the acidic residues of the finger. Notably, the M protein competed for in vitro binding of various oligonucleotides to Rae1•Nup98. We localized this competing activity of M to its finger using a synthetic peptide. Collectively, our data suggest that Rae1 serves as a binding protein for the phosphate backbone of any nucleic acid and that the finger of M mimics this ligand. In the context of mRNA export, we propose that a given mRNA segment, after having been deproteinated by helicase, is transiently reproteinated by Nup98-tethered Rae1. We suggest that such repetitive cycles provide cytoplasmic stopover sites required for ratcheting mRNA across the nuclear pore.

The Dynamic Multisite Interactions between Two Intrinsically Disordered Proteins.

Protein interactions involving intrinsically disordered proteins (IDPs) comprise a variety of binding modes, from the well-characterized folding upon binding to dynamic fuzzy complexes. To date, most studies concern the binding of an IDP to a structured protein, while the interaction between two IDPs is poorly understood. In this study, NMR, smFRET, and molecular dynamics (MD) simulation are combined to characterize the interaction between two IDPs, the C-terminal domain (CTD) of protein 4.1G and the nuclear mitotic apparatus (NuMA) protein. It is revealed that CTD and NuMA form a fuzzy complex with remaining structural disorder. Multiple binding sites on both proteins were identified by molecular dynamics and mutagenesis studies. This study provides an atomic scenario in which two IDPs bearing multiple binding sites interact with each other in dynamic equilibrium. The combined approach employed here could be widely applicable for investigating IDPs and their dynamic interactions.

Intrinsic Disorder to Order Transitions in the Scaffold Phosphoprotein P from the Respiratory Syncytial Virus RNA Polymerase Complex.

Intrinsic disorder is at the center of biochemical regulation and is particularly overrepresented among the often multifunctional viral proteins. Replication and transcription of the respiratory syncytial virus (RSV) relies on a RNA polymerase complex with a phosphoprotein cofactor P as the structural scaffold, which consists of a four-helix bundle tetramerization domain flanked by two domains predicted to be intrinsically disordered. Because intrinsic disorder cannot be reduced to a defined atomic structure, we tackled the experimental dissection of the disorder-order transitions of P by a domain fragmentation approach. P remains as a tetramer above 70 °C but shows a pronounced reversible secondary structure transition between 10 and 60 °C. While the N-terminal module behaves as a random coil-like IDP in a manner independent of tetramerization, the isolated C-terminal module displays a cooperative and reversible metastable transition. When linked to the tetramerization domain, the C-terminal module becomes markedly more structured and stable, with strong ANS binding. Therefore, the tertiary structure in the C-terminal module is not compact, conferring "late" molten globule-like IDP properties, stabilized by interactions favored by tetramerization. The presence of a folded structure highly sensitive to temperature, reversibly and almost instantly formed and broken, suggests a temperature sensing activity. The marginal stability allows for exposure of protein binding sites, offering a thermodynamic and kinetic fine-tuning in order-disorder transitions, essential for the assembly and function of the RSV RNA polymerase complex.

Structure, Dynamics, and Interaction of p54(nrb)/NonO RRM1 with 5' Splice Site RNA Sequence.

p54(nrb)/NonO is a nuclear RNA-binding protein involved in many cellular events such as pre-mRNA processing, transcription, and nuclear retention of hyper-edited RNAs. In particular, it participates in the splicing process by directly binding the 5' splice site of pre-mRNAs. The protein also concentrates in a nuclear body called paraspeckle by binding a G-rich segment of the ncRNA NEAT1. The N-terminal section of p54(nrb)/NonO contains tandem RNA recognition motifs (RRMs) preceded by an HQ-rich region including a threonine residue (Thr15) whose phosphorylation inhibits its RNA binding ability, except for G-rich RNAs. In this work, our goal was to understand the rules that characterize the binding of the p54(nrb)/NonO RRMs to their RNA target. We have done in vitro RNA binding experiments which revealed that only the first RRM of p54(nrb)/NonO binds to the 5' splice site RNA. We have then determined the structure of the p54(nrb)/NonO RRM1 by liquid-state NMR which revealed the presence of a canonical fold (ß1α1ß2ß3α2ß4) and the conservation of aromatic amino acids at the protein surface. We also investigated the dynamics of this domain by NMR. The p54(nrb)/NonO RRM1 displays some motional properties that are typical of a well-folded protein with some regions exhibiting more flexibility (loops and ß-strands). Furthermore, we determined the affinity of p54(nrb)/NonO RRM1 interaction to the 5' splice site RNA by NMR and fluorescence quenching and mapped its binding interface by NMR, concluding in a classical nucleic acid interaction. This study provides an improved understanding of the molecular basis (structure and dynamics) that governs the binding of the p54(nrb)/NonO RRM1 to one of its target RNAs.

Furospinosulin-1, Marine Spongean Furanosesterterpene, Suppresses the Growth of Hypoxia-Adapted Cancer Cells by Binding to Transcriptional Regulators p54(nrb) and LEDGF/p75.

Hypoxia-adapted cancer cells in tumors contribute to the pathological progression of cancer. Cancer research has therefore focused on the identification of molecules responsible for hypoxia adaptation in cancer cells, as well as the development of new compounds with action against hypoxia-adapted cancer cells. The marine natural product furospinosulin-1 (1) has displayed hypoxia-selective growth inhibition against cultured cancer cells, and has shown in vivo anti-tumor activity, although its precise mode of action and molecular targets remain unclear. In this study, we found that 1 is selectively effective against hypoxic regions of tumors, and that it directly binds to the transcriptional regulators p54(nrb) and LEDGF/p75, which have not been previously identified as mediators of hypoxia adaptation in cancer cells.

Mitochondrial and nucleolar localization of cysteine desulfurase Nfs and the scaffold protein Isu in Trypanosoma brucei.

Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.