Structural characterization of human importin alpha 7 in its cargo-free form at 2.5 Å resolution

Shuttling of macromolecules between nucleus and cytoplasm is a tightly regulated process mediated through specific interactions between cargo and nuclear transport proteins. In the classical nuclear import pathway, importin alpha recognizes cargo exhibiting a nuclear localization signal, and this complex is transported through the nuclear pore complex by importin beta. Humans possess seven importin alpha isoforms that can be grouped into three subfamilies, with many cargoes displaying specificity towards these importin alpha isoforms. The cargo binding sites within importin alpha isoforms are highly conserved in sequence, suggesting that specificity potentially relies on structural differences. Structures of some importin alpha isoforms, both in cargo-bound and free states, have been previously solved. However, there are currently no known structures of cargo free importin alpha isoforms within subfamily 3 (importin alpha 5, 6, 7). Here, we present the first crystal structure of human importin alpha 7 lacking the IBB domain solved at 2.5 Å resolution. The structure reveals a typical importin alpha architecture comprised of ten armadillo repeats and is most structurally conserved with importin alpha 5. Very little difference in structure was observed between the cargo-bound and free states, implying that importin alpha 7 does not undergo conformational change when binding cargo. These structural insights provide a strong platform for further evaluation of structure–function relationships and understanding how isoform specificity within the importin alpha family plays a role in nuclear transport in health and disease.

YB-1 Phosphorylation at Serine 209 Inhibits Its Nuclear Translocation

YB-1 is a multifunctional DNA- and RNA-binding protein involved in cell proliferation, differentiation, and migration. YB-1 is a predominantly cytoplasmic protein that is transported to the nucleus in certain conditions, including DNA-damaging stress, transcription inhibition, and viral infection. In tumors, YB-1 nuclear localization correlates with high aggressiveness, multidrug resistance, and a poor prognosis. It is known that posttranslational modifications can regulate the nuclear translocation of YB-1. In particular, well-studied phosphorylation at serine 102 (S102) activates YB-1 nuclear import. Here, we report that Akt kinase phosphorylates YB-1 in vitro at serine 209 (S209), which is located in the vicinity of the YB-1 nuclear localization signal. Using phosphomimetic substitutions, we showed that S209 phosphorylation inhibits YB-1 nuclear translocation and prevents p-S102-mediated YB-1 nuclear import.

Mobile Host mRNAs Are Translated to Protein in the Associated Parasitic Plant Cuscuta campestris

Cuscuta spp. are obligate parasites that connect to host vascular tissue using a haustorium. In addition to water, nutrients, and metabolites, a large number of mRNAs are bidirectionally exchanged between Cuscuta spp. and their hosts. This trans-specific movement of mRNAs raises questions about whether these molecules function in the recipient species. To address the possibility that mobile mRNAs are ultimately translated, we built upon recent studies that demonstrate a role for transfer RNA (tRNA)-like structures (TLSs) in enhancing mRNA systemic movement. C. campestris was grown on Arabidopsis that expressed a β-glucuronidase (GUS) reporter transgene either alone or in GUS-tRNA fusions. Histochemical staining revealed localization in tissue of C. campestris grown on Arabidopsis with GUS-tRNA fusions, but not in C. campestris grown on Arabidopsis with GUS alone. This corresponded with detection of GUS transcripts in Cuscuta on Arabidopsis with GUS-tRNA, but not in C. campestris on Arabidopsis with GUS alone. Similar results were obtained with Arabidopsis host plants expressing the same constructs containing an endoplasmic reticulum localization signal. In C. campestris, GUS activity was localized in the companion cells or phloem parenchyma cells adjacent to sieve tubes. We conclude that host-derived GUS mRNAs are translated in C. campestris and that the TLS fusion enhances RNA mobility in the host-parasite interactions.

Duck Plague Virus pUL48 Protein Activates the Immediate-Early Gene to Initiate the Transcription of the Virus Gene

Duck plague caused by the duck plague virus (DPV) is an infectious disease that seriously harms the waterfowl breeding industry. The VP16 protein of α herpesvirus can bind to specific cis-acting elements upstream of the promoter of the immediate-early (IE, α) gene to promote the transcription of the IE gene, so it is also called the trans-inducer of IE gene (α-TIF). However, no studies on DPV α-TIF have been reported. This study investigated the DPV pUL48, a homolog of HSV-1 VP16, transcriptional activation region, target sequence, and viral protein affecting its transcriptional activation using a dual-luciferase reporter gene detection system, and pUL48 was identified as the α-TIF of DPV. (1) The regulation of pUL48 on DPV different gene promoters showed that pUL48 could activate all the promoters of IE genes (ICP4, ICP22, and ICP27) but not the promoters of early and late genes. (2) The activity of pUL48 to ICP4 and ICP22 promoters with different upstream lengths showed that pUL48 activated ICP4 and ICP22 promoters by acting on TAATGA (T) TAT element upstream of ICP4 promoter and TAATTATAT element upstream of ICP22 promoter, respectively. (3) Transcriptional activation of IE gene by truncated proteins of different lengths at the N-terminal of pUL48 was detected. The results showed that the transcriptional activation domain of pUL48 was amino acids 1–60 at the N-terminal, and amino acids 1–20 was its core region. In addition, it was found that pUL14, pUL46, and pUL47 significantly promoted the transcriptional activation of pUL48. The effects of loss of pUL47 and its nuclear localization signal on the nuclear entry and transcriptional activation function of pUL48 were further examined. The results showed that pUL47 could promote the nuclear entry of pUL48 through its nuclear localization signal at positions 40–50 and 768–777 amino acids, thus, enhancing the transcriptional activation function of pUL48 and synergistic promotion of viral gene transcription.

A Deletion of the Nuclear Localization Signal Domain in the Fus Protein Induces Stable Post-stress Cytoplasmic Inclusions in SH-SY5Y Cells

Mutations in Fused-in-Sarcoma (FUS) gene involving the nuclear localization signal (NLS) domain lead to juvenile-onset Amyotrophic Lateral Sclerosis (ALS). The mutant protein mislocalizes to the cytoplasm, incorporating it into Stress Granules (SG). Whether SGs are the first step to the formation of stable FUS-containing aggregates is still unclear. In this work, we used acute and chronic stress paradigms to study the SG dynamics in a human SH-SY5Y neuroblastoma cell line carrying a deletion of the NLS domain of the FUS protein (homozygous: ΔNLS–/–; heterozygous: ΔNLS+/–). Wild-type (WT) cells served as controls. We evaluated the subcellular localization of the mutant protein through immunoblot and immunofluorescence, in basal conditions and after acute stress and chronic stress with sodium arsenite (NaAsO2). Cells were monitored for up to 24 h after rescue. FUS was expressed in both nucleus and cytoplasm in the ΔNLS+/– cells, whereas it was primarily cytoplasmic in the ΔNLS–/–. Acute NaAsO2 exposure induced SGs: at rescue,>90% of ΔNLS cells showed abundant FUS-containing if compared to less than 5% of the WT cells. The proportion of FUS-positive SGs remained 15–20% at 24 h in mutant cells. Cycloheximide did not abolish the long-lasting SGs in mutant cells. Chronic exposure to NaAsO2 did not induce significant SGs formation. A wealth of research has demonstrated that ALS-associated FUS mutations at the C-terminus facilitate the incorporation of the mutant protein into SGs. We have shown here that mutant FUS-containing SGs tend to fail to dissolve after stress, facilitating a liquid-to-solid phase transition. The FUS-containing inclusions seen in the dying motor neurons might therefore directly derive from SGs. This might represent an attractive target for future innovative therapies.

Mechanically induced nuclear shuttling of β-catenin requires co-transfer of actin

Mesenchymal stem cells (MSC) respond to environmental forces with both cytoskeletal re-structuring and activation of protein chaperones of mechanical information, β-catenin and Yes-Associated Protein 1 (YAP1). To function, MSCs must differentiate between dynamic forces such as cyclic strains of extracellular matrix due to physical activity and static strains due to ECM stiffening. To delineate how MSCs recognize and respond differently to both force types, we compared effects of dynamic (200 cycles x 2%) and static (1 x 2% hold) strain on nuclear translocation of β-catenin and YAP1 at 3h after force application. Dynamic strain induced nuclear accumulation of β-catenin, and increased cytoskeletal actin structure and cell stiffness, but had no effect on nuclear YAP1 levels. Critically, both nuclear actin and nuclear stiffness increased along with dynamic strain-induced β-catenin transport. Augmentation of cytoskeletal structure using either static strain or lysophosphatidic acid (LPA) did not increase nuclear content of β-catenin or actin, but induced robust nuclear increase in YAP1. As actin binds β-catenin, we considered whether β-catenin, which lacks a nuclear localization signal, was dependent on actin to gain entry to the nucleus. Knockdown of cofilin-1 (Cfl1) or importin-9 (Ipo9), which co-mediate nuclear transfer of G-actin, prevented dynamic strain-mediated nuclear transfer of both β-catenin and actin. In sum, dynamic strain induction of actin re-structuring promotes nuclear transport of G-actin, concurrently supporting nuclear access of β-catenin via mechanisms utilized for actin transport. Thus, dynamic and static strain activate alternative mechanoresponses reflected by differences in the cellular distributions of actin, β-catenin and YAP1.

SAFA initiates innate immunity against cytoplasmic RNA virus SFTSV infection

Nuclear scaffold attachment factor A (SAFA) is a novel RNA sensor involved in sensing viral RNA in the nucleus and mediating antiviral immunity. Severe fever with thrombocytopenia syndrome virus (SFTSV) is a bunyavirus that causes SFTS with a high fatality rate of up to 30%. It remains elusive whether and how cytoplasmic SFTSV can be sensed by the RNA sensor SAFA. Here, we demonstrated that SAFA was able to detect SFTSV infection and mediate antiviral interferon and inflammatory responses. Transcription and expression levels of SAFA were strikingly upregulated under SFTSV infection. SAFA was retained in the cytoplasm by interaction with SFTSV nucleocapsid protein (NP). Importantly, SFTSV genomic RNA was recognized by cytoplasmic SAFA, which recruited and promoted activation of the STING-TBK1 signaling axis against SFTSV infection. Of note, the nuclear localization signal (NLS) domain of SAFA was important for interaction with SFTSV NP and recognition of SFTSV RNA in the cytoplasm. In conclusion, our study reveals a novel antiviral mechanism in which SAFA functions as a novel cytoplasmic RNA sensor that directly recognizes RNA virus SFTSV and mediates an antiviral response.

Mitochondrial Localization Signal of Porcine Circovirus Type 2 Capsid Protein Plays a Critical Role in Cap-Induced Apoptosis

Porcine circovirus 2 (PCV2), considered one of the most globally important porcine pathogens, causes postweaning multisystemic wasting syndrome (PMWS). This virus is localized in the mitochondria in pigs with PMWS. Here, we identified, for the first time, a mitochondrial localization signal (MLS) in the PCV2 capsid protein (Cap) at the N-terminus. PK-15 cells showed colocalization of the MLS-EGFP fusion protein with mitochondria. Since the PCV2 Cap also contained a nuclear localization signal (NLS) that mediated entry into the nucleus, we inferred that the subcellular localization of the PCV2 Cap is inherently complex and dependent on the viral life cycle. Furthermore, we also determined that deletion of the MLS attenuated Cap-induced apoptosis. More importantly, the MLS was essential for PCV2 replication, as absence of the MLS resulted in failure of virus rescue from cells infected with infectious clone DNA. In conclusion, the MLS of the PCV2 Cap plays critical roles in Cap-induced apoptosis, and MLS deletion of Cap is lethal for virus rescue.