Division of Labor in an Oligomer of the DEAD-Box RNA Helicase Ded1p.

Most aspects of RNA metabolism involve DEAD-box RNA helicases, enzymes that bind and remodel RNA and RNA-protein complexes in an ATP-dependent manner. Here we show that the DEAD-box helicase Ded1p oligomerizes in the cell and in vitro, and unwinds RNA as a trimer. Two protomers bind the single-stranded region of RNA substrates and load a third protomer to the duplex, which then separates the strands. ATP utilization differs between the strand-separating protomer and those bound to the single-stranded region. Binding of the eukaryotic initiation factor 4G to Ded1p interferes with oligomerization and thereby modulates unwinding activity and RNA affinity of the helicase. Our data reveal a strict division of labor between the Ded1p protomers in the oligomer. This mode of oligomerization fundamentally differs from other helicases. Oligomerization represents a previously unappreciated level of regulation for DEAD-box helicase activities.

Nitrogen-doped graphene aerogel as both a sulfur host and an effective interlayer for high-performance lithium-sulfur batteries.

Lithium-sulfur batteries have attracted great concern because of the high theoretical capacity of sulfur (1675 mA h g-1). However, the poor electrical conductivity and volumetric expansion of sulfur along with the dissolution of lithium polysulfides largely limit their practical application. In this study, nitrogen-doped graphene aerogel (NGA) with high nitrogen content and porosity is used as a host for the impregnation of sulfur. The effects of sulfur impregnation on the specific surface area, pore volume, and microstructure of NGA supported sulfur composite (S@NGA) are well investigated. Furthermore, NGA is also processed into a NGA film, which is sandwiched between a separator and S@NGA cathode. The lithium-sulfur battery with such a configuration delivers a high reversible capacity of 1514 mA h g-1 at 0.1 C, excellent rate performance (822 mA h g-1 at 2.0 C), and good cycling stability (946 mA h g-1 at 0.5 C even after 100 cycles). The enhanced electrochemical performance can be ascribed to the introduction of the NGA interlayer, the unique interconnected porous structure, and strong interaction between the three-dimensional nitrogen-doped graphene network and the homogeneously dispersed sulfur and/or lithium polysulfides.

Preparation and characterization of a composite hydrogel with graphene oxide as an acid catalyst.

In this study, a facile method for synthesizing a novel graphene oxide/pyrrole-formaldehyde (GOP-1) composite hydrogel was developed via in situ polymerization of pyrrole and formaldehyde in the presence of graphene oxide sheets without any additional catalyst. During the polymerization, graphene oxide can act as a two-dimensional template to regulate the aggregation state of polymer and as an acid catalyst to accelerate the reaction rate of pyrrole and formaldehyde. The morphology and microstructure were investigated by scanning electron microscopy, transmission electron microscopy, and X-ray diffraction, respectively. The chemical properties were analyzed via X-ray photoelectron spectroscopy, infrared spectroscopy, and Raman spectroscopy. The freeze-dried GOP-1 composite hydrogel exhibited a large specific surface area, high nitrogen content, and three-dimensional network structure. Based on the above features, the freeze-dried GOP-1 composite hydrogel used as a gas adsorbent showed a high carbon dioxide uptake capacity at 1.0 bar and 273 K (11.1 wt%), in sharp contrast to that of graphene oxide (7.4 wt%). Furthermore, the as-prepared composite hydrogel may possess attractive potential in the fields of electrode material, tissue engineering, and water treatment.

The DEAD-box protein Ded1 unwinds RNA duplexes by a mode distinct from translocating helicases.

Helicases unwind RNA or DNA duplexes and displace proteins from nucleic acids in an ATP-dependent fashion. To unwind duplexes, helicases typically load onto one of the two nucleic acid strands, usually at a single-stranded region, and then translocate on this strand in a unidirectional fashion, thereby displacing the complementary DNA or RNA. Here we show that the DEAD-box RNA helicase Ded1 unwinds duplexes in a different manner. Ded1 uses the single-stranded region to gain access to the duplex. Strand separation is directly initiated from the duplex region and no covalent connection between the single strand and the duplex region is required. This new type of helicase activity explains observations with other DEAD-box proteins and may be the prototype for duplex-unwinding reactions in RNA metabolism.

Hospital biosecurity capacitation: Analysis and recommendations for the prevention and control of COVID-19.

The outbreak of the coronavirus disease 2019 (COVID-19) in December 2019 highlighted several concerns regarding hospital biosafety capacitation in the People's Republic of China, although the epidemic is now under control. This study examined the primary problems related to hospital biosecurity, including the absence of a hospital emergency system, inadequate management and control of nosocomial infection, limited hospital laboratory capacity, and poor hospital admission capacity. Accordingly, this study puts forward the following countermeasures and suggestions for hospitals to deal with future biosecurity events, such as a major epidemic: first, biosecurity management systems and emergency response mechanisms in hospitals need to be set up; second, the investment and guarantee mechanisms for hospital biosecurity construction should be improved; third, the capacity building of biosecurity incident management requires special attention in general hospitals; and finally, comprehensive plans need to be developed for the integrated construction of medical treatment and prevention facilities through disease-control systems.

DEAD-box-protein-assisted RNA structure conversion towards and against thermodynamic equilibrium values.

RNAs in biological processes often interconvert between defined structures. These RNA structure conversions are assisted by proteins and are frequently coupled to ATP hydrolysis. It is not well understood how proteins coordinate RNA structure conversions and which role ATP hydrolysis has in these processes. Here, we have investigated in vitro how the DEAD-box ATPase Ded1 facilitates RNA structure conversions in a simple model system. We find that Ded1 assists RNA structure conversions via two distinct pathways. One pathway requires ATP hydrolysis and involves the complete disassembly of the RNA strands. This pathway represents a kinetically controlled steady state between the RNA structures, which allows formation of less stable from more stable RNA conformations and thus RNA structure conversion against thermodynamic equilibrium values. The other pathway is ATP-independent and proceeds via multipartite intermediates that are stabilized by Ded1. Our results provide a basic mechanistic framework for protein-assisted RNA structure conversions that illuminates the role of ATP hydrolysis and reveal an unexpected diversity of pathways.

Involvement of DEAD-box proteins in group I and group II intron splicing. Biochemical characterization of Mss116p, ATP hydrolysis-dependent and -independent mechanisms, and general RNA chaperone activity.

The RNA-catalyzed splicing of group I and group II introns is facilitated by proteins that stabilize the active RNA structure or act as RNA chaperones to disrupt stable inactive structures that are kinetic traps in RNA folding. In Neurospora crassa and Saccharomyces cerevisiae, the latter function is fulfilled by specific DEAD-box proteins, denoted CYT-19 and Mss116p, respectively. Previous studies showed that purified CYT-19 stimulates the in vitro splicing of structurally diverse group I and group II introns, and uses the energy of ATP binding or hydrolysis to resolve kinetic traps. Here, we purified Mss116p and show that it has RNA-dependent ATPase activity, unwinds RNA duplexes in a non-polar fashion, and promotes ATP-independent strand-annealing. Further, we show that Mss116p binds RNA non-specifically and promotes in vitro splicing of both group I and group II intron RNAs, as well as RNA cleavage by the aI5gamma-derived D135 ribozyme. However, Mss116p also has ATP hydrolysis-independent effects on some of these reactions, which are not shared by CYT-19 and may reflect differences in its RNA-binding properties. We also show that a non-mitochondrial DEAD-box protein, yeast Ded1p, can function almost as efficiently as CYT-19 and Mss116p in splicing the yeast aI5gamma group II intron and less efficiently in splicing the bI1 group II intron. Together, our results show that Mss116p, like CYT-19, can act broadly as an RNA chaperone to stimulate the splicing of diverse group I and group II introns, and that Ded1p also has an RNA chaperone activity that can be assayed by its effect on splicing mitochondrial introns. Nevertheless, these DEAD-box protein RNA chaperones are not completely interchangeable and appear to function in somewhat different ways, using biochemical activities that have likely been tuned by coevolution to function optimally on specific RNA substrates.

Amelioration of osteoarthritis by intra-articular hyaluronan synthase 2 gene therapy.

Osteoarthritis (OA) is a chronic, degenerative disorder of multifactorial aetiology, characterized by loss of articular cartilage and periarticular bone remodelling. Goals of managing OA include controlling pain, maintaining and improving function and health-related quality of life, and limiting functional impairment. Although several managements had been proved to ameliorate the symptoms of osteoarthritis, no methods could cure it thoroughly. High-molecular-weight hyaluronan (HMW-HA) is a major component of synovial joint fluids which physically acts as a viscous lubricant for slow joint movements and as an elastic shock absorber during rapid movements. It also has a variety of biologic effects in vivo, such as inhibiting the release of inflammatory factors and suppressing the degradation of cartilage matrix. Intra-articular injection of synthetic HMW-HA has been used as viscosupplement for knee OA and its therapeutic efficacy has been verified. However, repeated injections of HMW-HA which is needed to control symptoms increase the probability of infection and sometimes there will have acute joint pain with effusion, which requires aspiration to exclude sepsis. In order to overcome the disadvantages of repeated injections of HMW-HA, novel strategies should be developed. As HMW-HA is synthesized by hyaluronan synthase-2 (HAS2), we postulate that HAS2 gene could be delivered into intra-articular cells by methods of gene therapy to achieve long-lasting synthesis of HMW-HA. In our opinion, this strategy seems to hold interesting future prospects for the treatment of OA.

Cloning and expression of a novel human galectin cDNA, predominantly expressed in placenta(1).

A novel human galectin cDNA (PPL13) was isolated by screening a human 18-week fetal brain library. The mRNA was predominantly expressed in placenta, while the expression of it was not or barely detectable in heart, brain, lung, liver, skeletal muscle, kidney, and pancreas by Northern blot. COS-7 cells transfected with cDNA encoding human PPL13 sequestered the protein in nuclei although it lacked any known nuclear localization signal. STS of Unigene Hs. 24236 placed the cDNA to human chromosome 19q13.2.

RNA helicases: versatile ATP-driven nanomotors.

RNA helicases are a large family of molecular motors that utilize nucleoside triphosphates to unwind RNA duplexes and to remodel RNA protein complexes. In this review, we discuss the structure and function of RNA helicases with an emphasis on the potential application of these enzymes to control conformational changes in nanoassemblies that contain RNA.

Function of the C-terminal domain of the DEAD-box protein Mss116p analyzed in vivo and in vitro.

The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are general RNA chaperones that function in splicing mitochondrial group I and group II introns and in translational activation. Both proteins consist of a conserved ATP-dependent RNA helicase core region linked to N and C-terminal domains, the latter with a basic tail similar to many other DEAD-box proteins. In CYT-19, this basic tail was shown to contribute to non-specific RNA binding that helps tether the core helicase region to structured RNA substrates. Here, multiple sequence alignments and secondary structure predictions indicate that CYT-19 and Mss116p belong to distinct subgroups of DEAD-box proteins, whose C-terminal domains have a defining extended alpha-helical region preceding the basic tail. We find that mutations or C-terminal truncations in the predicted alpha-helical region of Mss116p strongly inhibit RNA-dependent ATPase activity, leading to loss of function in both translational activation and RNA splicing. These findings suggest that the alpha-helical region may stabilize and/or regulate the activity of the RNA helicase core. By contrast, a truncation that removes only the basic tail leaves high RNA-dependent ATPase activity and causes only a modest reduction in translation and RNA splicing efficiency in vivo and in vitro. Biochemical analysis shows that deletion of the basic tail leads to weaker non-specific binding of group I and group II intron RNAs, and surprisingly, also impairs RNA-unwinding at saturating protein concentrations and nucleotide-dependent tight binding of single-stranded RNAs by the RNA helicase core. Together, our results indicate that the two sub-regions of Mss116p's C-terminal domain act in different ways to support and modulate activities of the core helicase region, whose RNA-unwinding activity is critical for both the translation and RNA splicing functions.

DEAD-box proteins unwind duplexes by local strand separation.

DEAD-box proteins catalyze ATP-driven, local structural changes in RNA or RNA-protein complexes (RNP) during which only few RNA base pairs are separated. It is unclear how duplex unwinding by DEAD-box proteins differs from unwinding by canonical helicases, which can separate many base pairs by directional and processive translocation on the nucleic acid, starting from a helical end. Here, we show that two different DEAD-box proteins, Ded1p and Mss116p, can unwind RNA duplexes from internal as well as terminal helical regions and act on RNA segments as small as two nucleotides flanked by DNA. The data indicate that duplex unwinding by DEAD-box proteins is based on local destabilization of RNA helical regions. No directional movement of the enzymes through the duplex is involved. We propose a three-step mechanism in which DEAD-box proteins unwind duplexes as "local strand separators." This unwinding mode is well-suited for local structural changes in complex RNA or RNP assemblies.

Do DEAD-box proteins promote group II intron splicing without unwinding RNA?

The DEAD-box protein Mss116p promotes group II intron splicing in vivo and in vitro. Here we explore two hypotheses for how Mss116p promotes group II intron splicing: by using its RNA unwinding activity to act as an RNA chaperone or by stabilizing RNA folding intermediates. We show that an Mss116p mutant in helicase motif III (SAT/AAA), which was reported to stimulate splicing without unwinding RNA, retains ATP-dependent unwinding activity and promotes unfolding of a structured RNA. Its unwinding activity increases sharply with decreasing duplex length and correlates with group II intron splicing activity in quantitative assays. Additionally, we show that Mss116p can promote ATP-independent RNA unwinding, presumably via single-strand capture, also potentially contributing to DEAD-box protein RNA chaperone activity. Our findings favor the hypothesis that DEAD-box proteins function in group II intron splicing as in other processes by using their unwinding activity to act as RNA chaperones.

ATP- and ADP-dependent modulation of RNA unwinding and strand annealing activities by the DEAD-box protein DED1.

DEAD-box RNA helicases, which are involved in virtually all aspects of RNA metabolism, are generally viewed as enzymes that unwind RNA duplexes or disrupt RNA-protein interactions in an ATP-dependent manner. Here, we show in vitro that the DEAD-box protein DED1 from Saccharomyces cerevisiae promotes not only RNA unwinding but also strand annealing, the latter in such a profound fashion that the physical limit for a bimolecular association rate constant is approached. We further demonstrate that DED1 establishes an ATP-dependent steady state between unwinding and annealing, which enables the enzyme to modulate the balance between the two opposing activities through ATP and ADP concentrations. The ratio between unwinding and annealing and the degree to which both activities are ATP- and ADP-modulated are strongly influenced by structured as well as unstructured regions in the RNA substrate. Collectively, these findings expand the known functional repertoire of DEAD-box proteins and reveal the capacity of DED1 to remodel RNA in response to ADP and ATP concentrations by facilitating not only disruption but also formation of RNA duplexes.

Cloning and identification of a novel cDNA which may be associated with FKBP25.

A 1209 bp novel cDNA with a putative open reading frame of 627 bp that has an overlapped fragment of 527 bp with FKBP25 was isolated from a human fetal brain library. Northern Blot analysis detected a signal about 1.5 kb that suggests it is a full-length cDNA. Reverse transcription-PCR demonstrates that the transcripts are high in adult brain, testis, ovary, spleen, and fetal brain. Comparing the cDNA with the human genome database identifies a contig that reveals that the gene contains six exons and is located on human chromosome 20p11. The gene encodes a putative protein with an unknown domain conserved in many species and is similar to bacterial histidyl-tRNA synthetase, and so it is named human histidyl-tRNA-synthetase-related gene.

Cloning and expression of a novel retinoblastoma binding protein cDNA, RBBP10.

A 2860-bp cDNA was isolated from a human fetal brain cDNA library by high throughput cDNA sequencing, which encodes a putative protein with 186 amino acids. The putative protein shares 90.7% identity with rat pBOG (3403163) and shares 93.4% identity with human RBBP9 (NP_006597.1). A conserved RB binding domain, L x C x E, located between residue 63 and 68 was recognized. Therefore, it was named RBBP10. Mapviewer analysis locates it on human chromosome 20q11.22. RBBP10 spans about 9.6 kb of the genome and consists of six exons and five introns. RT-PCR revealed that the gene was expressed widely in various human tissues, and the expression level is somewhat higher in tumor tissues than in normal tissues. But subsequent sequencing analysis did notfound any mutation of this in tumor tissues. The COS 7 cell transfected with the ORF of RBBP10 showed that the protein was distributed both in the cytoplasm and in the nucleus. Our results suggest that RBBP10 is the orthologue of the rat BOG gene (AF025819) and a paralogue of human RBBP9 (AF039564).

Cloning and expression pattern of a spermatogenesis-related gene, BEX1, mapped to chromosome Xq22.

Through screening a human fetal brain cDNA library, a cDNA similar to the mouse Bex1 was isolated. This new gene was named brain expressed X-linked protein 1 (BEX1). Northern blot analysis revealed a 1.0 kb transcript highly expressed in brain, pancreas, testis, and ovary, with lower levels present in heart, placenta, liver, kidney, spleen, thymus, prostate, small intestine, colon (no mucus), thyroid, spinal cord, and adrenal gland. No hybridization signal was seen in lung, skeletal muscle, peripheral blood leukocyte, stomach, lymph node, trachea, and bone marrow. The BEX1 gene was localized to chromosome band Xq22 between markers between DXS990 and DXS1059 by screening Stanford radiation hybrid G3 panels. In situ hybridization of mouse testis using BEX1 as a probe detected the signal in the pachytene spermatocytes and spermatids but not in spermatogonia. Furthermore, it was not detected at 6, 9, and 12 days postpartum, was present in low amount on Days 15 and 18 and its expression increased sharply after the initiation of puberty (about 21 days) in mouse testis.

Cloning, expression, and genomic structure of a novel human Rap2 interacting gene (RPIP9).

During a large-scale screen of a human fetal brain cDNA library, a full-length cDNA encoding a novel Rap2 interacting protein was isolated and sequenced. The cDNA is 3397bp long and has a predicted open reading frame encoding a protein of 329 aa. The predicted protein shows high homology to mouse and human RPIP8, and has a RUN domain near its C-terminus. The gene was mapped to human chromosome 7q21-7q22 and has 9 exons and 8 introns. The expression pattern was also detected by cycle-limited reverse polymerase chain reaction (RT-PCR).

Cloning and identification of a cDNA that encodes a novel human protein with thrombospondin type I repeat domain, hPWTSR.

A cDNA was isolated from the fetal brain cDNA library by high throughput cDNA sequencing. The 2390 bp cDNA with an open reading fragment (ORF) of 816 bp encodes a 272 amino acids putative protein with a thrombospondin type I repeat (TSR) domain and a cysteine-rich region at the N-terminus, so it is named hPWTSR. We used Northern blot detected two bands with length of about 3 kb and 4 kb respectively, which expressed in human adult tissues with different intensities. The expression pattern was verified by RT-PCR, revealing that the transcripts were expressed ubiquitously in fetal tissues and human tumor tissues too. However, the transcript was detected neither in ovarian carcinoma GI-102 nor in lung carcinoma LX-1. Blast analysis against NCBI database revealed that the new gene contained at least 5 exons and located in human chromosome 6q22.33. Our results demonstrate that the gene is a novel member of TSR supergene family.