Systematic identification of endogenous RNA polymeraseⅢpromoters for efficient RNA guidebased genome editing technologies in maize

Single-guide RNA(sg RNA) is one of the two core components of the CRISPR(clustered regularly interspaced short palindromic repeat)/Cas(CRISPR-associated) genome-editing technology. We established an in vitro Traffic Light Reporter(TLR) system, which is designated as the same colors as traffic lights such as green, red and yellow were produced in cells. The TLR can be readily used in maize mesophyll protoplast for a quick test of promoter activity. The TLR assay indicates the variation in transcription activities of the seven Pol III promoters, from 3.4%(U6-1) to over 21.0%(U6-6). The U6-2 promoter, which was constructed to drive sg RNA expression targeting the Zm Wx1 gene, yielded mutation efficiencies ranging from 48.5% to 97.1%. Based on the reported and unpublished data, the in vitro TLR assay results were confirmed to be a readily system and may be extended to other plant species amenable to efficient genome editing via CRISPR/Cas. Our efforts provide an efficient method of identifying native Pol III-recognized promoters for RNA guide-based genome-editing systems in maize.

Hordatines as a Potential Inhibitor of COVID-19 Main Protease and RNA Polymerase: An In-Silico Approach

Total 40 natural compounds were selected to perform the molecular docking studies to screen and identify the potent antiviral agents specifically for Severe Acute Respiratory Syndrome Coronavirus 2 that causes coronavirus disease 2019(COVID-19).The key targets of COVID-19,protease(PDB ID:7BQY)and RNA polymerase(PDB ID:7bV2)were used to dock our target compounds by Molecular Operating Environment(MOE)version 2014.09.We used 3 different conformations of protease target(6M0K,6Y2F and 7BQY)and two different score functions to strengthen the probability of inhibitors discovery.After an extensive screening analysis,20 compounds exhibit good binding affinities to one or both COVID-19 targets.7 out of 20 compounds were predicted to overcome the activity of both targets.The top 7 hits are,flacourticin(3),sagerinic acid(16),hordatine A(23),hordatine B(24),N-feruloyl tyramine dimer(25),bisavenanthramides B-5(29)and vulnibactins(40).According to our results,all these top hits was found to have a better binding scores than remdesivir,the native ligand in RNA polymerase target(PDB ID:7bV2).Hordatines are phenolic compounds present in barley,were found to exhibit the highest binding affinity to both protease and polymerase through forming strong hydrogen bonds with the catalytic residues,as well as significant interactions with other receptor-binding residues.These results probably provided an excellent lead candidate for the development of therapeutic drugs against COVID-19.Eventually,animal experiment and accurate clinical trials are needed to confirm the preventive potentials of these compounds.

Brownian ratchet mechanism of translocation in T7 RNA polymerase facilitated by a post-translocation energy bias arising from the conformational change of the enzyme

T7 RNA polymerase can transcribe DNA to RNA by translocating along the DNA. Structural studies suggest that the pivoting rotation of the O helix in the fingers domain may drive the movement of the O helix C-terminal Tyr639 from pre- to post-translocation positions. In a series of all-atom molecular dynamics simulations, we show that the movement of Tyr639 is not tightly coupled to the rotation of the O helix, and that the two processes are only weakly dependent on each other. We also show that the internal potential of the enzyme itself generates a small difference in free energy （△E） between the post- and pre-translocation positions of Tyr639. The calculated value of △E is consistent with that obtained from single-molecule experimental data. These findings lend support to a model in which the translocation takes place via a Brownian ratchet mechanism, with the small free energy bias △E arising from the conformational change of the enzyme itself.

An intermediate state of T7 RNA polymerase provides another pathway of nucleotide selection

Phage T7 RNA polymerase is a single-subunit transcription enzyme, transcribing template DNA to RNA. Nucleoside triphosphate （NTP） selection and translocation are two critical steps of the transcription elongation. Here, using all-atom molecular dynamics simulations, we found that between pre- and post-translocation states of T7 RNA polymerase an intermediate state exists, where the O helix C-terminal residue tyrosine 639, which plays important roles in translocation, locates between its pre- and post-translocation positions and the side chain of the next template DNA nucleotide has moved into the active site. NTP selection in this intermediate state was studied, revealing that the selection in the intermediate state can be achieved relying on the effect of Watson-Crick interaction between NTP and template DNA nucleotide, effect of stability of the components near the active site such as the nascent DNA-RNA hybrid and role of tyrosine 639. This indicates that another NTP-selection pathway can also exist besides the main pathway where NTP selection begins at the post-translocation state upon the entry of NTE

Probing conformational change of T7 RNA polymerase and DNA complex by solid-state nanopores

Proteins are crucial to most biological processes, such as enzymes, and in various catalytic processes a dynamic motion is required. The dynamics of protein are embodied as a conformational change, which is closely related to the flexibility of protein. Recently, nanopore sensors have become accepted as a low cost and high throughput method to study the features of proteins. In this article, we used a SiN nanopore device to study the flexibility of T7 RNA polymerase（RNAP） and its complex with DNA promoter. By calculating full-width at half-maximum（FWHM） of Gaussian fits to the blockade histograms, we found that T7 RNAP becomes more flexible after binding DNA promoter. Moreover, the distribution of fractional current blockade suggests that flexibility alters due to a breath-like change of the volume.

Analysis of RNA-Dependent RNA Polymerase Sequence of Infectious Flacherie Virus Isolated in China and Its Expression in BmN Cells

Full gene sequence of RNA-dependent RNA polymerase （RdRp） from Bombyx mori infectious flacherie virus isolated in Zhejiang Province, China （Zhejiang01/CHN/2002） was cloned. The sequence was 1 920 nucleotides in length coding 639 amino acid residues. Sequences comparison of RdRp showed Zhejiang01/CHN/2002 was 99.7% nucleotide sequence and 99.1% amino acids sequence homology with Japanese strain. The RdRp sequence was aligned with 8 representative picorna（-like） viruses and 8 highly conserved regions were detected. The result indicated their relevance function. Phylogenetic tree of 14 picorna（-like） viruses which RdRp presumed protein sequences revealed that the viruses from Iflavirus genus formed an independent clade. The RdRp was successfully expressed in BmN cells using Bac-to-Bac expression system.

Crystal structures of RNA-dependent RNA polymerases from Jingmen tick virus and Alongshan virus

Jingmenviruses are a group of flavi-like viruses with segmented genome and have been found in various types of hosts,including humans,cattle,monkeys,bats,rodents,sheep,ticks,mosquitoes and nematodes.Jingmenviruses,including the Jingmen tick virus(JMTV)and Alongshan virus(ALSV),have been associated with febrile illness and flu-like symptoms in humans.Viral polymerase plays critical roles in genome replication and transcription and is an ideal target for antiviral drugs.Here,we determined the crystal structures of RNA-dependent RNA polymerase(RdRp)domains of JMTV and ALSV at 2.6Åand 3.2Åresolutions,respectively.The overall structures of JMTV and ALSV RdRp domains are similar to those from the typical unsegmented viruses in Flaviviridae family,especially the Flavivirus genus.JMTV and ALSV RdRps can be divided into three subdomains and the catalytical Motif A-G are conserved like the typical flaviviruses,whereas the zinc-binding pockets are absent from the JMTV and ALSV RdRps.The 50-ends of jingmenvirus genomes are varied in length and sequence,and a highly conserved 8-nucleotide element located on the tip of stem loop A was identified and shown to be required for binding with RdRp and performing de novo replication activity.These findings provide important structural insights into RdRp of segmented flavivirus and reveal the key region of virus genome responsible for replication initiation,which would promote molecular understanding of segmented flavivirus replication and the structure-based design of antiviral drugs against flaviviruses.

Unusual substructure conformations observed in crystal structures of a dicistrovirus RNA-dependent RNA polymerase suggest contribution of the N-terminal extension in proper folding

The Dicistroviridae is a virus family that includes many insect pathogens.These viruses contain a positive-sense RNA genome that is replicated by the virally encoded RNA-dependent RNA polymerase(RdRP)also named 3D^(pol).Compared with the Picornaviridae RdRPs such as poliovirus(PV)3D^(pol),the Dicistroviridae representative Israeli acute paralysis virus(IAPV)3D^(pol) has an additional N-terminal extension(NE)region that is about 40-residue in length.To date,both the structure and catalytic mechanism of the Dicistroviridae RdRP have remain elusive.Here we reported crystal structures of two truncated forms of IAPV 3D^(pol),namelyΔ85 andΔ40,both missing the NE region,and the 3D^(pol) protein in these structures exhibited three conformational states.The palm and thumb domains of these IAPV 3D^(pol) structures are largely consistent with those of the PV 3D^(pol) structures.However,in all structures,the RdRP fingers domain is partially disordered,while different conformations of RdRP substructures and interactions between them are also present.In particular,a large-scale conformational change occurred in the motif B-middle finger region in one protein chain of theΔ40 structure,while a previously documented alternative conformation of motif A was observed in all IAPV structures.These experimental data on one hand show intrinsic conformational variances of RdRP substructures,and on the other hand suggest possible contribution of the NE region in proper RdRP folding in IAPV.

Equine ANP32 proteins support influenza A virus RNA polymerase activity

Host ANP32 family proteins are crucial for maintaining the activity of influenza RNA polymerase and play an important role in the cross-species transmission of influenza viruses.To date,the molecular properties of equine ANP32(eqANP32)protein are poorly understood,particularly the mechanisms that affect equine influenza virus(EIV)RNA polymerase activity.Here,we found that there are six alternative splicing variants of equine ANP32A(eqANP32A)with different levels of expression.Further studies showed that these six splicing variants of eqANP32A supported the activity of EIV RNA polymerase to varying degrees,with the variant eqANP32A_X2 having the highest expression abundance and exhibiting the highest support of polymerase activity.Sequence analysis demonstrated that the differences in the N-Cap regions of the six splicing variants significantly affected their N-terminal conformation,but did not affect their ability to bind RNA polymerase.We also demonstrated that there is only one transcript of eqANP32B,and that this transcript showed only very low support to the EIV RNA polymerase.This functional defect in eqANP32B is caused by the sequence of the 110–259 amino acids at its Cterminus.Our results indicated that it is the eqANP32A_X2 protein that mainly determines the efficiency of the EIV replication in horses.In conclusion,our study parsed the molecular properties of eqANP32 family proteins and revealed the sequence features of eqANP32A and eqANP32B,suggesting for the first time that the N-Cap region of ANP32A protein also plays an important role in supporting the activity of the influenza virus polymerase.

Structures of EV71 RNA-dependent RNA polymerase in complex with substrate and analogue provide a drug target against the hand-foot-and-mouth disease pandemic in China

Enterovirus 71(EV71),one of the major causative agents for hand-foot-and-mouth disease(HFMD),has caused more than 100 deaths among Chinese children since March 2008.The EV71 genome encodes an RNAdependent RNA polymerase(RdRp),denoted 3D^(pol),which is central for viral genome replication and is a key target for the discovery of specific antiviral therapeutics.Here we report the crystal structures of EV71 RdRp(3D^(pol))and in complex with substrate guanosine-5'-triphosphate and analog 5-bromouridine-5'-triphosphate best to 2.4Åresolution.The structure of EV71 RdRp(3D^(pol))has a wider open thumb domain compared with the most closely related crystal structure of poliovirus RdRp.And the EV71 RdRp(3D^(pol))complex with GTP or Br-UTP bounded shows two distinct movements of the polymerase by substrate or analogue binding.The model of the complex with the template:primer derived by superimposition with foot-and-mouth disease virus(FMDV)3D/RNA complex reveals the likely recognition and binding of template:primer RNA by the polymerase.These results together provide a molecular basis for EV71 RNA replication and reveal a potential target for anti-EV71 drug discovery.

RNA Polymerase V Functions in Arabidopsis Interphase Heterochromatin Organization Independently of the 24-nt siRNA-Directed DNA Methylation Pathway

In Arabidopsis, pericentromeric repeats, retroelements, and silenced rRNA genes are assembled into heterochromatin within nuclear structures known as chromocenters. The mechanisms governing higher-order heterochromatin organization are poorly understood but 24-nt small interfering RNAs （siRNAs） are known to play key roles in heterochromatin formation. Nuclear RNA polymerase IV （Pol IV）, RNA-DEPENDENT RNA POLYMERASE 2 （RDR2）, and DICER-LIKE 3 （DCL3） are required for biogenesis of 24-nt siRNAs that associate with ARGONAUTE 4 （AGO4）. Nuclear RNA polymerase V （Pol V） collaborates with DRD1 （DEFICIENT IN RNA-DEPENDENT DNA METHYLATION 1） to generate transcripts at heterochromatic loci that are hypothesized to bind to siRNA-AGO4 complexes and subsequently recruit the de-novo DNA methylation and/or histone modifying machinery. Here, we report that decondensation of the major pericentromeric repeats and depletion of the heterochromatic mark histone H3 lysine 9 dimethylation at chromocenters occurs specifically in pol V and drdl mutants. Disruption of pericentromeric repeats condensation is coincident with transcriptional reactivation of specific classes of pericentromeric 180-bp repeats. We further demonstrate that Pol V functions independently of Pol IV, RDR2, and DCL3-mediated siRNA production to affect interphase heterochromatin organization, possibly by involving RNAs that recruit structural or chromatin-modifying proteins.

Improved plasmid-based recovery of coxsackievirus A16 infectious clone driven by human RNA polymerase I promoter

Dear Editor,Coxsackievirus A16(CA16)is one of the major viral pathogens associated with hand,foot,and mouth disease.CA16 belongs to the Enterovirus genus of the Picornaviridae family and possesses a single-stranded positivesense RNA genome(Mao et al.,2014).Reverse genetics is an important tool for CA16 research.Previously,a reverse genetics T7 polymerase-based system was de-

Crystal structure of the coxsackievirus A16 RNA-dependent RNA polymerase elongation complex reveals novel features in motif A dynamics

Dear Editor,Coxsackievirus A16(CV A16)and enterovirus 71(EV71)are currently the two primary causative agents of handfoot-and-mouth disease(HFMD)(Solomon et al.,2010;Mao et al.,2014),threatening health of children worldwide.They both belong to the Enterovirus genus of

SARS-CoV-2 spike protein and RNA dependent RNA polymerase as targets for drug and vaccine development: A review

The present pandemic has posed a crisis to the economy of the world and the health sector.Therefore,the race to expand research to understand some good molecular targets for vaccine and therapeutic development for SARS-CoV-2 is inevitable.The newly discovered coronavirus 2019(COVID-19)is a positive sense,single-stranded RNA,and enveloped virus,assigned to the beta CoV genus.The virus(SARS-CoV-2)is more infectious than the previously detected coronaviruses(MERS and SARS).Findings from many studies have revealed that S protein and RdRp are good targets for drug repositioning,novel therapeutic development(antibodies and small molecule drugs),and vaccine discovery.Therapeutics such as chloroquine,convalescent plasma,monoclonal antibodies,spike binding peptides,and small molecules could alter the ability of S protein to bind to the ACE-2 receptor,and drugs such as remdesivir(targeting SARS-CoV-2 RdRp),favipir,and emetine could prevent SASR-CoV-2 RNA synthesis.The novel vaccines such as mRNA1273(Moderna),3LNP-mRNAs(Pfizer/BioNTech),and ChAdOx1-S(University of Oxford/Astra Zeneca)targeting S protein have proven to be effective in combating the present pandemic.Further exploration of the potential of S protein and RdRp is crucial in fighting the present pandemic.

Conserved Curvature of RNA Polymerase Ⅰ Core Promoter Beyond rRNA Genes: The Case of the Tritryps

In trypanosomatids, the RNA polymerase I （RNAPI）-dependent promoters controlling the ribosomal RNA （rRNA） genes have been well identified. Although the RNAPI transcription machinery recognizes the DNA conformation instead of the DNA sequence of promoters, no con- formational study has been reported for these promoters. Here we present the in silico analysis of the intrinsic DNA curvature of the rRNA gene core promoters in Trypanosoma brucei, Trypanosoma cruzi, and Leiskmania major. We found that, in spite of the absence of sequence conservation, these promoters hold conformational properties similar to other eukaryotic rRNA promoters. Our results also indicated that the intrinsic DNA curvature pattern is conserved within the Leishmania genus and also among strains of T. cruzi and T. brucei. Furthermore, we analyzed the impact of point mutations on the intrinsic curvature and their impact on the promoter activity. Furthermore, we found that the core promoters of protein-coding genes transcribed by RNAPI in T. brucei show the same conserved conformational characteristics. Overall, our results indicate that DNA intrinsic curvature of the rRNA gene core promoters is conserved in these ancient eukaryotes and such con- served curvature might be a requirement of RNAPI machinery for transcription of not only rRNA genes but also protein-coding genes.

Theoretical analysis of RNA polymerase fidelity: a steady-state copolymerization approach

The fidelity of DNA transcription catalyzed by RNA polymerase(RNAP)has long been an important issue in biology.Experiments have revealed that RNAP can incorporate matched nucleotides selectively and proofread the incorporated mismatched nucleotides.However,systematic theoretical researches on RNAP fidelity are still lacking.In the last decade,several theories on RNA transcription have been proposed,but they only handled highly simplified models without considering the high-order neighbor effects and the oligonucleotides cleavage both of which are critical for the overall fidelity.In this paper,we regard RNA transcription as a binary copolymerization process and calculate the transcription fidelity by the steady-state copolymerization theory recently proposed by us for DNA replication.With this theory,the more realistic models considering higher-order neighbor effects,oligonucleotides cleavage,multi-step incorporation and multi-step cleavage can be rigorously handled.

Functional implications of C-terminus of TBX5 with high homology to C-terminal domain of yeast DNA-directed RNA polymerase Ⅱ largest subunit

TBX5, as a member of the T-box-containing transcription factor family, encodes a protein of 518 amino acids and is expressed in the embryonic heart and developing limb tissues.1 The coding region of TBX5 cDNA is 1.5 kb with eight exons including the N-terminal portion, the DNA binding domain and C-terminal region. We reported that the abnormality in transcription level of the TBX5 gene might be the mechanism underlying human simple congenital heart disease in the absence of TBX5 mutations.

Human cytomegalovirus RNA2.7 inhibits RNA polymeraseⅡ(PolⅡ)Serine-2 phosphorylation by reducing the interaction between PolⅡand phosphorylated cyclin-dependent kinase 9(pCDK9)

Human cytomegalovirus(HCMV)is a ubiquitous pathogen belongs to betaherpesvirus subfamily.RNA2.7 is a highly conserved long non-coding RNA accounting for more than 20%of total viral transcripts.In our study,functions of HCMV RNA2.7 were investigated by comparison of host cellular transcriptomes between cells infected with HCMV clinical strain and RNA2.7 deleted mutant.It was demonstrated that RNA polymeraseⅡ(PolⅡ)-dependent host gene transcriptions were significantly activated when RNA2.7 was removed during infection.A145 nt-in-length motif within RNA2.7 was identified to inhibit the phosphorylation of PolⅡSerine-2(PolⅡS2)by reducing the interaction between PolⅡand phosphorylated cyclin-dependent kinase 9(pCDK9).Due to the loss of PolⅡS2 phosphorylation,cellular DNA pre-replication complex(pre-RC)factors,including Cdt1 and Cdc6,were significantly decreased,which prevented more cells from entering into S phase and facilitated viral DNA replication.Our results provide new insights of HCMV RNA2.7 functions in regulation of host cellular transcription.

RNA-DEPENDENT RNA POLYMERASE ASSOCIATED WITH WHEAT ROSETTE STUNT VIRIONS

Wheat rosette stunt virus （WRSV） contains an RNA-dependent RNA polymerase. The RNA polymerase activity is associated with the viral nucleocapsid （NP）.In vitro transcription of purified NP required all of the four nucleoside triphosphates and Mg2+.There was a need of a proper salt concentration and some reducing reagents in the system for increasing the RNA polymerase activity.The optimum temperature for in vitro transcription was around 25℃.Within the first 90 min of in vitro reaction, incorporation rose linearly with the time course of incubation. The experiments of ribonuclease and deoxyribonuclease treatments showed that single-stranded RNAs were synthesized in vitro by the RNA polymerase.Two fractions of WRSV-NP could be separated by SDS-dissociation and ultracentrifugation.The supernatant fraction contained three structural proteins of NP: L, N and NS;and the pellet fraction contained the viral RNA.When the supernatant proteins and the viral RNA were mixed together, RNA polymerase activity

Potential and application of abortive transcripts as a novel molecular marker of cancers

Abortive transcript(AT)is a 2-19 nt long non-coding RNA that is produced in the abortive initiation stage.Abortive initiation was found to be closely related to RNA polymerase through in vitro experiments.Therefore,the distribution of AT length and the scale of abortive initiation are correlated to the promoter,discriminator,and transcription initiation sequence,and can be affected by transcription elongation factors.AT plays an important role in the occurrence and development of various diseases.Here we summarize the discovery of AT,the factors responsible for AT formation,the detection methods and biological functions of AT,to provide new clues for finding potential targets in the early diagnosis and treatment of cancers.