Structure and activation mechanism of the yeast RNA Pol II CTD kinase CTDK-1 complex.

The C-terminal domain (CTD) kinase I (CTDK-1) complex is the primary RNA Polymerase II (Pol II) CTD Ser2 kinase in budding yeast. CTDK-1 consists of a cyclin-dependent kinase (CDK) Ctk1, a cyclin Ctk2, and a unique subunit Ctk3 required for CTDK-1 activity. Here, we present a crystal structure of CTDK-1 at 1.85-Å resolution. The structure reveals that, compared to the canonical two-component CDK-cyclin system, the third component Ctk3 of CTDK-1 plays a critical role in Ctk1 activation by stabilizing a key element of CDK regulation, the T-loop, in an active conformation. In addition, Ctk3 contributes to the assembly of CTDK-1 through extensive interactions with both Ctk1 and Ctk2. We also demonstrate that CTDK-1 physically and genetically interacts with the serine/arginine-like protein Gbp2. Together, the data in our work reveal a regulatory mechanism of CDK complexes.

Mechanisms of nuclear mRNA export: A structural perspective.

Export of mRNA from the nucleus to the cytoplasm is a critical process for all eukaryotic gene expression. As mRNA is synthesized, it is packaged with a myriad of RNA-binding proteins to form ribonucleoprotein particles (mRNPs). For each step in the processes of maturation and export, mRNPs must have the correct complement of proteins. Much of the mRNA export pathway revolves around the heterodimeric export receptor yeast Mex67•Mtr2/human NXF1•NXT1, which is recruited to signal the completion of nuclear mRNP assembly, mediates mRNP targeting/translocation through the nuclear pore complex (NPC), and is displaced at the cytoplasmic side of the NPC to release the mRNP into the cytoplasm. Directionality of the transport is governed by at least two DEAD-box ATPases, yeast Sub2/human UAP56 in the nucleus and yeast Dbp5/human DDX19 at the cytoplasmic side of the NPC, which respectively mediate the association and dissociation of Mex67•Mtr2/NXF1•NXT1 onto the mRNP. Here we review recent progress from structural studies of key constituents in different steps of nuclear mRNA export. These findings have laid the foundation for further studies to obtain a comprehensive mechanistic view of the mRNA export pathway.

Cellular mRNA export factor UAP56 recognizes nucleic acid binding site of influenza virus NP protein.

Influenza A virus nucleoprotein (NP) is a structural component that encapsulates the viral genome into the form of ribonucleoprotein complexes (vRNPs). Efficient assembly of vRNPs is critical for the virus life cycle. The assembly route from RNA-free NP to the NP-RNA polymer in vRNPs has been suggested to require a cellular factor UAP56, but the mechanism is poorly understood. Here, we characterized the interaction between NP and UAP56 using recombinant proteins and showed that UAP56 features two NP binding sites. In addition to the UAP56 core comprised of two RecA domains, we identified the N-terminal extension (NTE) of UAP56 as a previously unknown NP binding site. In particular, UAP56-NTE recognizes the nucleic acid binding region of NP. This corroborates our observation that binding of UAP56-NTE and RNA to NP is mutually exclusive. Collectively, our results reveal the molecular basis for how UAP56 acts on RNA-free NP, and provide new insights into NP-mediated influenza genome packaging.

Tel2 structure and function in the Hsp90-dependent maturation of mTOR and ATR complexes.

We reported previously that the stability of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs) depends on their interaction with Tel2, the ortholog of yeast Tel2 and Caenorhabditis elegans Clk-2. Here we provide evidence that Tel2 acts with Hsp90 in the maturation of PIKK complexes. Quantitative immunoblotting showed that the abundance of Tel2 is low compared with the PIKKs, and Tel2 preferentially bound newly synthesized ATM, ATR, mTOR, and DNA-PKcs. Tel2 complexes contained, in addition to Tti1-Tti2, the Hsp90 chaperone, and inhibition of Hsp90 interfered with the interaction of Tel2 with the PIKKs. Analysis of in vivo labeled nascent protein complexes showed that Tel2 and Hsp90 mediate the formation of the mTOR TORC1 and TORC2 complexes and the association of ATR with ATRIP. The structure of yeast Tel2, reported here, shows that Tel2 consists of HEAT-like helical repeats that assemble into two separate α-solenoids. Through mutagenesis, we identify a surface patch of conserved residues involved in binding to the Tti1-Tti2 complex in vitro. In vivo, mutation of this conserved patch affects cell growth, levels of PIKKs, and ATM/ATR-mediated checkpoint signaling, highlighting the importance of Tti1-Tti2 binding to the function of Tel2. Taken together, our data suggest that the Tel2-Tti1-Tti2 complex is a PIKK-specific cochaperone for Hsp90.

Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.

Cryo-EM structure of the CBC-ALYREF complex.

In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5' end with a 7-methylguanosine (m 7 G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism including transcription, splicing, polyadenylation and export. It promotes mRNA export through direct interaction with ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5' end of mRNA. However, the molecular mechanism for CBC mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with a mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contacts with both the NCBP1 and NCBP2 subunits of the CBC. Comparison of CBC-ALYREF to other CBC and ALYREF containing cellular complexes provides insights into the coordinated events during mRNA transcription, splicing, and export.

Structural basis for high-order complex of SARNP and DDX39B to facilitate mRNP assembly.

mRNA in eukaryotic cells is packaged into highly compacted ribonucleoprotein particles (mRNPs) in the nucleus and exported to the cytoplasm for translation. mRNP packaging and export require the evolutionarily conserved transcription-export (TREX) complex. TREX facilitates loading of various RNA-binding proteins on mRNA through the action of its DDX39B subunit. SARNP (Tho1 [transcriptional defect of Hpr1 by overexpression 1] in yeast) is shown to interact with DDX39B and affect mRNA export. The molecular mechanism of how SARNP recognizes DDX39B and functions in mRNP assembly is unclear. Here, we determine the crystal structure of a Tho1/DDX39B/RNA complex, revealing a multivalent interaction mediated by tandem DDX39B interacting motifs in SARNP/Tho1. The high-order complex of SARNP and DDX39B is evolutionarily conserved, and human SARNP can engage with five DDX39B molecules. RNA sequencing (RNA-seq) from SARNP knockdown cells shows the most affected RNAs in export are GC rich. Our work suggests the role of the high-order SARNP/DDX39B/RNA complex in mRNP assembly and export.

Cryo-EM structure of the yeast TREX complex and coordination with the SR-like protein Gbp2.

The evolutionarily conserved TRanscript-EXport (TREX) complex plays central roles during mRNP (messenger ribonucleoprotein) maturation and export from the nucleus to the cytoplasm. In yeast, TREX is composed of the THO sub-complex (Tho2, Hpr1, Tex1, Mft1, and Thp2), the DEAD box ATPase Sub2, and Yra1. Here we present a 3.7 Šcryo-EM structure of the yeast THO•Sub2 complex. The structure reveals the intimate assembly of THO revolving around its largest subunit Tho2. THO stabilizes a semi-open conformation of the Sub2 ATPase via interactions with Tho2. We show that THO interacts with the serine-arginine (SR)-like protein Gbp2 through both the RS domain and RRM domains of Gbp2. Cross-linking mass spectrometry analysis supports the extensive interactions between THO and Gbp2, further revealing that RRM domains of Gbp2 are in close proximity to the C-terminal domain of Tho2. We propose that THO serves as a landing pad to configure Gbp2 to facilitate its loading onto mRNP.

Nsp1 protein of SARS-CoV-2 disrupts the mRNA export machinery to inhibit host gene expression.

The ongoing unprecedented severe acute respiratory syndrome caused by the SARS-CoV-2 outbreak worldwide has highlighted the need for understanding viral-host interactions involved in mechanisms of virulence. Here, we show that the virulence factor Nsp1 protein of SARS-CoV-2 interacts with the host messenger RNA (mRNA) export receptor heterodimer NXF1-NXT1, which is responsible for nuclear export of cellular mRNAs. Nsp1 prevents proper binding of NXF1 to mRNA export adaptors and NXF1 docking at the nuclear pore complex. As a result, a significant number of cellular mRNAs are retained in the nucleus during infection. Increased levels of NXF1 rescues the Nsp1-mediated mRNA export block and inhibits SARS-CoV-2 infection. Thus, antagonizing the Nsp1 inhibitory function on mRNA export may represent a strategy to restoring proper antiviral host gene expression in infected cells.

Structural basis for influenza virus NS1 protein block of mRNA nuclear export.

Influenza viruses antagonize key immune defence mechanisms via the virulence factor non-structural protein 1 (NS1). A key mechanism of virulence by NS1 is blocking nuclear export of host messenger RNAs, including those encoding immune factors1-3; however, the direct cellular target of NS1 and the mechanism of host mRNA export inhibition are not known. Here, we identify the target of NS1 as the mRNA export receptor complex, nuclear RNA export factor 1-nuclear transport factor 2-related export protein 1 (NXF1-NXT1), which is the principal receptor mediating docking and translocation of mRNAs through the nuclear pore complex via interactions with nucleoporins4,5. We determined the crystal structure of NS1 in complex with NXF1-NXT1 at 3.8 Å resolution. The structure reveals that NS1 prevents binding of NXF1-NXT1 to nucleoporins, thereby inhibiting mRNA export through the nuclear pore complex into the cytoplasm for translation. We demonstrate that a mutant influenza virus deficient in binding NXF1-NXT1 does not block host mRNA export and is attenuated. This attenuation is marked by the release of mRNAs encoding immune factors from the nucleus. In sum, our study uncovers the molecular basis of a major nuclear function of influenza NS1 protein that causes potent blockage of host gene expression and contributes to inhibition of host immunity.

[Expression of human novel gene CT120 in lung cancer and its effects on cell growth].

BACKGROUND & OBJECTIVE: A novel membrane-associated gene CT120 was isolated from chromosome 17p13.3 locus in our laboratory. Its mRNA was not expressed in human normal lung tissues, but was abundant in human lung cancer cell line SPC-A-1. This study was designed to investigate the differential expression patterns of CT120 in different lung cancer and noncancerous tissues using immunohistochemistry and to explore the effects of ectopic expression and overexpression of CT120 on cell growth in vitro and in vivo. METHODS: A polypeptide at the C-terminus of CT120 was selected by bioinformatics, then was synthesized and conjugated to KLH (a high molecular carrier). The chicken anti-CT120 antibody IgY was prepared with the synthesized antigen and was used to determine the different expression patterns of CT120 in various tumor cell lines and in lung cancer and noncancerous tissues. The effects of ectopic expression of CT120 on NIH/3T3 cell growth were investigated through colony formation analysis. The effect of overexpression of CT120 on the cell growth of A549 was analyzed using growth curve assay and tumor formation assay of transfected cells in nude mice. RESULTS: The novel gene CT120 expressed in various tumor cell lines and expressed remarkably higher in lung cancers than in noncancerous tissues as well as normal lung tissues. Also, it promoted the proliferation of NIH/3T3 and A549 cells in vitro and in vivo. CONCLUSION: CT120 gene may be a novel candidate gene closely related to lung carcinogenesis.

Molecular cloning and characterization of CT120, a novel membrane-associated gene involved in amino acid transport and glutathione metabolism.

Within the minimum LOH region on chromosome 17p13.3 deleted in hepatocellular carcinoma, a novel human plasma membrane-associated gene, named CT120, was isolated from a human kidney cDNA library using electronical cloning and RACE. The novel gene CT120 consists of 2145bp and encodes a protein with 257 amino acids. Database search revealed that homologs of CT120 exist in different organisms from plant to animal kingdoms, which suggests that CT120 is a highly conserved gene during biological evolution. Different expression patterns of CT120 were observed in many different human normal tissues and in various human tumor cell lines. Transcript of CT120 was not detectable in normal lung tissue, but was abundant in SPC-A-1 (human epithelial-like lung adenocarcinoma) cell line, suggesting that CT120 may be involved in lung cancer development. Subcellular localization analysis showed that CT120 is a novel membrane-associated protein. CT120 can interact with SLC3A2 (member 2 of solute carrier family 3) and GGTL3B (isoform of gamma-glutamyltranspeptidase-like 3) in eukaryotic cells by yeast two-hybrid screen and co-immunoprecipitation assay, which suggested that CT120 may assume very essential physiological functions involved in amino acid transport and glutathione metabolism.