Terminal Nucleotide Research Articles

Recurrent Neurodevelopmentally Associated Variants of the Pre-mRNA Splicing Factor U2AF2 Alter RNA Binding Affinities and Interactions.

De novo mutations affecting the pre-mRNA splicing factor U2AF2 are associated with developmental delays and intellectual disabilities, yet the molecular basis is unknown. Here, we demonstrated by fluorescence anisotropy RNA binding assays that recurrent missense mutants (Arg149Trp, Arg150His, or Arg150Cys) decreased the binding affinity of U2AF2 for a consensus splice site RNA. Crystal structures at 1.4 Å resolutions showed that Arg149Trp or Arg150His disrupted hydrogen bonds between U2AF2 and the terminal nucleotides of the RNA site. Reanalysis of publicly available RNaseq data confirmed that U2AF2 depletion altered splicing of transcripts encoding RNA binding proteins (RBPs). These results confirmed that the impaired RNA interactions of Arg149Trp and Arg150His U2AF2 variants could contribute to dysregulating an RBP-governed neurodevelopmental program of alternative splicing.

2'-O-methyltransferase-deficient yellow fever virus: Restricted replication in the midgut and secondary tissues of Aedes aegypti mosquitoes severely limits dissemination.

The RNA genome of orthoflaviviruses encodes a methyltransferase within the non-structural protein NS5, which is involved in 2'-O-methylation of the 5'-terminal nucleotide of the viral genome resulting in a cap1 structure. While a 2'-O-unmethylated cap0 structure is recognized in vertebrates by the RNA sensor RIG-I, the cap1 structure allows orthoflaviviruses to evade the vertebrate innate immune system. Here, we analyzed whether the cap0 structure is also recognized in mosquitoes. Replication analyses of 2'-O-methyltransferase deficient yellow fever virus mutants (YFV NS5-E218A) of the vaccine 17D and the wild-type Asibi strain in mosquito cells revealed a distinct downregulation of the cap0 viruses. Interestingly, the level of inhibition differed for various mosquito cells. The most striking difference was found in Aedes albopictus-derived C6/36 cells with YFV-17D cap0 replication being completely blocked. Replication of YFV-Asibi cap0 was also suppressed in mosquito cells but to a lower extent. Analyses using chimeras between YFV-17D and YFV-Asibi suggest that a synergistic effect of several mutations across the viral genome accompanied by a faster initial growth rate of YFV-Asibi cap1 correlates with the lower level of YFV-Asibi cap0 attenuation. Viral growth analyses in Dicer-2 knockout cells demonstrated that Dicer-2 is entirely dispensable for attenuating the YFV cap0 viruses. Translation of a replication-incompetent cap0 reporter YFV-17D genome was reduced in mosquito cells, indicating a cap0 sensing translation regulation mechanism. Further, oral infection of Aedes aegypti mosquitoes resulted in lower infection rates for YFV-Asibi cap0. The latter is related to lower viral loads found in the midguts, which largely diminished dissemination to secondary tissues. After intrathoracic infection, YFV-Asibi cap0 replicated slower and to decreased amounts in secondary tissues compared to YFV-Asibi cap1. These results suggest the existence of an ubiquitously expressed innate antiviral protein recognizing 5'-terminal RNA cap-modifications in mosquitoes, both in the midgut as well as in secondary tissues.

Metformin enhances the survival ability of ovarian granulosa cells in polycystic ovary syndrome by promoting LINC00548 to suppress androgen receptor/klotho pathway.

Metformin (Met) has been reported to play the key role in the pathogenesis of polycystic ovary syndrome (PCOS). However, the precise mechanisms underlying the actions of Met in PCOS remain incompletely understood. This study aimed to confirm the mechanism of Met interacting with a long non-coding RNA LINC00548 in PCOS. Ovarian granulosa cells (OGCs) were incubated 500 nM dihydrotestosterone (DHT) to construct PCOS in vitro model and then were treated 20 μM Met. A series of cell experiments including Cell Counting Kit-8, Terminal uridine nucleotide end labeling, and flow cytometry were performed to confirm the changes of OGC survival ability. Quantitative real-time polymerase chain reaction was conducted to determine the levels of LINC00548, whereas Western blotting was applied to determine the levels of androgen receptor (AR) and klotho. Met improved the cell viability and suppressed cell apoptosis in DHT-treated OGCs. LINC00548 downregulated in DHT-treated OGCs was upregulated by Met, and its overexpression further enhanced the positive effects of Met on the survival ability of DHT-treated OGCs. In addition, Met could induce the upregulation of LINC00548 to suppress the activation of AR/klotho pathway in DHT-treated OGCs. Overall, this study discovers that Met enhances the survival ability of OGCs in PCOS by elevating LINC00548 expression to suppress AR/klotho pathway.

Characterization of Hibiscus Chlorotic Ringspot Virus-Derived vsiRNAs from Infected Hibiscus rosa-sinensis in China.

Lots of progress have been made about pathogen system of Hibiscus rosa-sinensis and hibiscus chlorotic ringspot virus (HCRSV), however, interactions between H. rosa-sinensis and HCRSV remain largely unknown. Hereon, firstly, HCRSV infection in H. rosa-sinensis from Zhangzhou city of China was confirmed by traditional electron microscopy, modern reverse transcription polymerase chain reaction and RNA-seq methods. Secondly, sequence feature analysis showed the full-length sequence of HCRSV-ZZ was 3,909 nucleotides (nt) in length and had a similar genomic structure with other carmovirus. It contains a 5' untranslated region (UTR), followed by seven open reading frames encoding for P28, P23, P81, P8, P9, P38, and P25, and the last a 3-terminal UTR. Thirdly, HCRSV- ZZ-derived vsiRNAs were identified and characterized for the first time from disease H. rosa-sinensis through sRNA-seq to reveal interactions between pathogen ant plant host. It was shown that the majority of HCRSV-ZZ-derived vsiRNAs were 21 nt, 22 nt, and 20 nt, with 21 nt being most abundant. The 5'-terminal nucleotide of HCRSV-ZZ vsiRNAs preferred U and C. HCRSV-ZZ vsiRNAs derived predominantly (72%) from the viral genome positive-strand RNA. The distribution of HCRSV-ZZ vsiRNAs along the viral genome is generally even, with some hot spots and cold spots forming in local regions. These hot spots and cold spots could be corresponded to the regions of stem loop secondary structures forming in HCRSV-ZZ genome by nucleotide paring. Taken together, our findings certify HCRSV infection in H. rosa-sinensis and provide an insight into interaction between HCRSV and H. rosa-sinensis and contribute to the prevention and treatment of this virus.

AFITbin: a metagenomic contig binning method using aggregate l-mer frequency based on initial and terminal nucleotides

BackgroundUsing next-generation sequencing technologies, scientists can sequence complex microbial communities directly from the environment. Significant insights into the structure, diversity, and ecology of microbial communities have resulted from the study of metagenomics. The assembly of reads into longer contigs, which are then binned into groups of contigs that correspond to different species in the metagenomic sample, is a crucial step in the analysis of metagenomics. It is necessary to organize these contigs into operational taxonomic units (OTUs) for further taxonomic profiling and functional analysis. For binning, which is synonymous with the clustering of OTUs, the tetra-nucleotide frequency (TNF) is typically utilized as a compositional feature for each OTU.ResultsIn this paper, we present AFIT, a new l-mer statistic vector for each contig, and AFITBin, a novel method for metagenomic binning based on AFIT and a matrix factorization method. To evaluate the performance of the AFIT vector, the t-SNE algorithm is used to compare species clustering based on AFIT and TNF information. In addition, the efficacy of AFITBin is demonstrated on both simulated and real datasets in comparison to state-of-the-art binning methods such as MetaBAT 2, MaxBin 2.0, CONCOT, MetaCon, SolidBin, BusyBee Web, and MetaBinner. To further analyze the performance of the purposed AFIT vector, we compare the barcodes of the AFIT vector and the TNF vector.ConclusionThe results demonstrate that AFITBin shows superior performance in taxonomic identification compared to existing methods, leveraging the AFIT vector for improved results in metagenomic binning. This approach holds promise for advancing the analysis of metagenomic data, providing more reliable insights into microbial community composition and function.AvailabilityA python package is available at: https://github.com/SayehSobhani/AFITBin.

Synonymous variant at the terminal nucleotide in exon 3 of F7 causes abnormal splicing: A case report.

Synonymous variants are non-pathogenic due to non-substitution of amino acids. However, synonymous exonic terminal nucleotide substitutions may affect splicing. Splicing variants are easily analyzed at RNA level for genes expressed in blood cells. Minigene analysis provides another method for splicing variant analysis of genes that are poorly or not expressed in peripheral blood. Whole exome sequencing was performed to screen for potential pathogenic mutations in the proband, which were validated within the family by Sanger sequencing. The pathogenicity of the synonymous mutation was analyzed using the minigene technology. The proband harbored the compound heterogeneous variants c. [291G >A; 572-50C >T] and c.681 + 1G >T in F7, of which the synonymous variant c.291G >A was located at the terminal position of exon 3. Minigene analysis revealed exon3 skipping due to this mutation, which may have subsequently affected protein sequence, structure, and function. Our finding confirmed the pathogenicity of c.291G >A, thus extending the pathogenic mutation spectrum of F7, and providing insights for effective reproductive counseling.

Structure and Stability of Ago2 MID-Nucleotide Complexes: All-in-One (Drop) His6-SUMO Tag Removal, Nucleotide Binding, and Crystal Growth.

The middle (MID) domain of eukaryotic Argonaute (Ago) proteins and archaeal and bacterial homologues mediates the interaction with the 5'-terminal nucleotide of miRNA and siRNA guide strands. The MID domain of human Ago2 (hAgo2) is comprised of 139 amino acids with a molecular weight of 15.56 kDa. MID adopts a Rossman-like beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-alpha4 fold with a nucleotide specificity loop between beta3 and alpha3. Multiple crystal structures of nucleotides bound to hAgo2 MID have been reported, whereby complexes were obtained by soaking ligands into crystals of MID domain alone. This protocol describes a simplified one-step approach to grow well-diffracting crystals of hAgo2 MID-nucleotide complexes by mixing purified His6-SUMO-MID fusion protein, Ulp1 protease, and excess nucleotide in the presence of buffer and precipitant. The crystal structures of MID complexes with UMP, UTP and 2'-3' linked α-L-threofuranosyl thymidine-3'-triphosphate (tTTP) are presented. This article also describes fluorescence-based assays to measure dissociation constants (Kd) of MID-nucleotide interactions for nucleoside 5'-monophosphates and nucleoside 3',5'-bisphosphates. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Crystallization of Ago2 MID-nucleotide complexes Basic Protocol 2: Measurement of dissociation constant Kd between Ago2 MID and nucleotides.

Identification of the Coexisting Virus-Derived siRNA in Maize and Rice Infected by Rice Black-Streaked Dwarf Virus.

Rice black-streaked dwarf virus is transmitted by small brown planthoppers, which causes maize rough dwarf disease and rice black-streaked dwarf disease. This virus leads to slow growth or death of the host plants. During the coevolutionary arms race between viruses and plants, virus-derived small interfering RNAs (vsiRNAs) challenge the plant's defense response and inhibit host immunity through the RNA silencing system. However, it is currently unknown if rice black-streaked dwarf virus can produce the same siRNAs to mediate the RNA silencing in different infected species. In this study, four small RNA libraries and four degradome libraries were constructed by extracting total RNAs from the leaves of the maize (Zea mays) inbred line B73 and japonica rice (Oryza sativa) variety Nipponbare exposed to feeding by viruliferous and nonviruliferous small brown planthoppers. We analyzed the characteristics of small RNAs and explored virus-derived siRNAs in small RNA libraries through high-throughput sequencing. On analyzing the characteristics of small RNA, we noted that the size distributions of small RNAs were mainly 24 nt (19.74 to 62.00%), whereas those of vsiRNAs were mostly 21 nt (41.06 to 41.87%) and 22 nt (39.72 to 42.26%). The 5'-terminal nucleotides of vsiRNAs tended to be adenine or uracil. Exploring the distribution of vsiRNA hot spots on the viral genome segments revealed that the frequency of hotspots in B73 was higher than those in Nipponbare. Meanwhile, hotspots in the S9 and S10 virus genome segments were distributed similarly in both hosts. In addition, the target genes of small RNA were explored by degradome sequencing. Analyses of the regulatory pathway of these target genes unveiled that viral infection affected the ribosome-related target genes in maize and the target genes in the metabolism and biosynthesis pathways in rice. Here, 562 and 703 vsiRNAs were separately obtained in maize and rice and 73 vsiRNAs named as coexisting vsiRNAs (co-vsiRNAs) were detected in both hosts. Stem-loop PCR and real-time quantitative PCR confirmed that co-vsiRNA 3.1 and co-vsiRNA 3.5, derived from genome segment S3, simultaneously play a role in maize and rice and inhibited host gene expression. The study revealed that rice black-streaked dwarf virus can produce the same siRNAs in different species and provides a new direction for developing new antiviral strategies.

Simulations predict preferred Mg2+ coordination in a nonenzymatic primer-extension reaction center

The mechanism by which genetic information was copied prior to the evolution of ribozymes is of great interest because of its importance to the origin of life. The most effective known process for the nonenzymatic copying of an RNA template is primer extension by a two-step pathway in which 2-aminoimidazole-activated nucleotides first react with each other to form an imidazolium-bridged intermediate that subsequently reacts with the primer. Reaction kinetics, structure-activity relationships, and X-ray crystallography have provided insight into the overall reaction mechanism, but many puzzles remain. In particular, high concentrations of Mg2+ are required for efficient primer extension, but the mechanism by which Mg2+ accelerates primer extension remains unknown. By analogy with the mechanism of DNA and RNA polymerases, a role for Mg2+ in facilitating the deprotonation of the primer 3′-hydroxyl is often assumed, but no catalytic metal ion is seen in crystal structures of the primer-extension complex. To explore the potential effects of Mg2+ binding in the reaction center, we performed atomistic molecular dynamics simulations of a series of modeled complexes in which a Mg2+ ion was placed in the reaction center with inner-sphere coordination with different sets of functional groups. Our simulations suggest that coordination of a Mg2+ ion with both O3′ of the terminal primer nucleotide and the pro-Sp nonbridging oxygen of the reactive phosphate of an imidazolium-bridged dinucleotide would help to pre-organize the structure of the primer/template substrate complex to favor the primer-extension reaction. Our results suggest that the catalytic metal ion may play an important role in overcoming electrostatic repulsion between a deprotonated O3′ and the reactive phosphate of the bridged dinucleotide and lead to testable predictions of the mode of Mg2+ binding that is most relevant to catalysis of primer extension.

Barley AGO4 proteins show overlapping functionality with distinct small RNA-binding properties in heterologous complementation

Key messageBarley AGO4 proteins complement expressional changes of epigenetically regulated genes in Arabidopsis ago4-3 mutant and show a distinct affinity for the 5′ terminal nucleotide of small RNAs, demonstrating functional conservation and divergence.The function of Argonaute 4 (AGO4) in Arabidopsis thaliana has been extensively characterized; however, its role in monocots, which have large genomes abundantly supplemented with transposable elements (TEs), remains elusive. The study of barley AGO4 proteins can provide insights into the conserved aspects of RNA-directed DNA methylation (RdDM) and could also have further applications in the field of epigenetics or crop improvement. Bioinformatic analysis of RNA sequencing data identified two active AGO4 genes in barley, HvAGO4a and HvAGO4b. These genes function similar to AtAGO4 in an Arabidopsis heterologous complementation system, primarily binding to 24-nucleotide long small RNAs (sRNAs) and triggering methylation at specific target loci. Like AtAGO4, HvAGO4B exhibits a preference for binding sRNAs with 5′ adenine residue, while also accepting 5′ guanine, uracil, and cytosine residues. In contrast, HvAGO4A selectively binds only sRNAs with a 5′ adenine residue. The diverse binding capacity of barley AGO4 proteins is reflected in TE-derived sRNAs and in their varying abundance. Both barley AGO4 proteins effectively restore the levels of extrachromosomal DNA and transcript abundancy of the heat-activated ONSEN retrotransposon to those observed in wild-type Arabidopsis plants. Our study provides insight into the distinct binding specificities and involvement in TE regulation of barley AGO4 proteins in Arabidopsis by heterologous complementation.

Comprehensive Comparison and Critical Assessment of RNA-Specific Force Fields.

Molecular dynamics simulations play a pivotal role in elucidating the dynamic behaviors of RNA structures, offering a valuable complement to traditional methods such as nuclear magnetic resonance or X-ray. Despite this, the current precision of RNA force fields lags behind that of protein force fields. In this work, we systematically compared the performance of four RNA force fields (ff99bsc0χOL3, AMBERDES, ff99OL3_CMAP1, AMBERMaxEnt) across diverse RNA structures. Our findings highlight significant challenges in maintaining stability, particularly with regard to cross-strand and cross-loop hydrogen bonds. Furthermore, we observed the limitations in accurately describing the conformations of nonhelical structural motif, terminal nucleotides, and also base pairing and base stacking interactions by the tested RNA force fields. The identified deficiencies in existing RNA force fields provide valuable insights for subsequent force field development. Concurrently, these findings offer recommendations for selecting appropriate force fields in RNA simulations.

Molecular Insights into the Specific Targeting of c-MYC G-Quadruplex by Thiazole Peptides.

Stabilization of a G-quadruplex (G4) in the promotor of the c-MYC proto-oncogene leads to inhibition of gene expression, and it thus represents a potentially attractive new strategy for cancer treatment. However, most G4 stabilizers show little selectivity among the many G4s present in the cellular complement of DNA and RNA. Intriguingly, a crescent-shaped cell-penetrating thiazole peptide, TH3, preferentially stabilizes the c-MYC G4 over other promotor G4s, but the mechanisms leading to this selective binding remain obscure. To investigate these mechanisms at the atomic level, we performed an in silico comparative investigation of the binding of TH3 and its analogue TH1 to the G4s from the promotors of c-MYC, c-KIT1, c-KIT2, and BCL2. Molecular docking and molecular dynamics simulations, combined with in-depth analyses of non-covalent interactions and bulk and per-nucleotide binding free energies, revealed that both TH3 and TH1 can induce the formation of a sandwich-like framework through stacking with both the top and bottom G-tetrads of the c-MYC G4 and the adjacent terminal capping nucleotides. This framework produces enhanced binding affinities for c-MYC G4 relative to other promotor G4s, with TH3 exhibiting an outstanding binding priority. Van der Waals interactions were identified to be the key factor in complex formation in all cases. Collectively, our findings fully agree with available experimental data. Therefore, the identified mechanisms leading to specific binding of TH3 towards c-MYC G4 provide valuable information to guide the development of new selective G4 stabilizers.

SARS-CoV-2 RNA-Dependent RNA Polymerase Follows Asynchronous Translocation Pathway for Viral Transcription and Replication.

Translocation is one essential step for the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) to exert viral replication and transcription. Although cryo-EM structures of SARS-CoV-2 RdRp are available, the molecular mechanisms of dynamic translocation remain elusive. Herein, we constructed a Markov state model based on extensive molecular dynamics simulations to elucidate the translocation dynamics of the SARS-CoV-2 RdRp. We identified two intermediates that pinpoint the rate-limiting step of translocation and characterize the asynchronous movement of the template-primer duplex. The 3'-terminal nucleotide in the primer strand lags behind due to the uneven distribution of protein-RNA interactions, while the translocation of the template strand is delayed by the hurdle residue K500. Even so, the two strands share the same "ratchet" to stabilize the polymerase in the post-translocation state, suggesting a Brownian-ratchet model. Overall, our study provides intriguing insights into SARS-CoV-2 replication and transcription, which would open a new avenue for drug discoveries.

Nano3'RACE: A Method to Analyze Poly(A) Tail Length and Nucleotide Additions at the 3' Extremity of Selected mRNAs Using Nanopore Sequencing.

Deadenylation is a major process that regulates gene expression by shaping the length of mRNA poly(A) tails. Deadenylation is controlled by factors in trans that recruit or impede deadenylases, by theincorporation of non-adenosines during poly(A) tail synthesis, and by the posttranscriptional addition of 3' nucleotides to poly(A) tails. Deciphering the regulation of poly(A) tail shortening requires both transcriptome-wide approaches and more targeted methodologies, allowing deep analyses of specific mRNAs. In this chapter, we present Nano3'RACE, a nanopore-based cDNA sequencing method that allows in-depth analysis to precisely measure poly(A) tail length and detect 3' terminal nucleotide addition, such as uridylation, for mRNAs of interest.

Conformational Plasticity of Parallel G-Quadruplex─Implications on Duplex-Quadruplex Motifs.

DNA G-quadruplexes are essential motifs in molecular biology performing a wide range of functions enabled by their unique and diverse structures. In this study, we focus on the conformational plasticity of the most abundant and biologically relevant parallel G-quadruplex topology. A multipronged approach of structure survey, solution-state NMR spectroscopy, and molecular dynamics simulations unravels subtle yet essential features of the parallel G-quadruplex topology. Stark differences in flexibility are observed for the nucleotides depending upon their positioning in the tetrad planes that are intricately correlated with the conformational sampling of the propeller loop. Importantly, the terminal nucleotides in the 5'-end versus the 3'-end of the parallel quadruplex display differential dynamics that manifests their ability to accommodate a duplex on either end of the G-quadruplex. The conformational plasticity characterized in this study provides essential cues toward biomolecular processes such as small molecular binding, intermolecular quadruplex stacking, and implications on how a duplex influences the structure of a neighboring quadruplex.

RNA Polymerase Ribozyme That Recognizes the Template-Primer Complex through Tertiary Interactions.

RNA enzymes (ribozymes) often rely on specific base-pairing interactions to engage RNA substrates, which limits the substrate sequence generality of these enzymes. An RNA polymerase ribozyme that was previously optimized by directed evolution to operate in a more efficient and sequence-general manner can now recognize the RNA template, RNA primer, and incoming nucleoside 5'-triphosphate (NTP) entirely through tertiary interactions. As with proteinaceous polymerases, these tertiary interactions are largely agnostic to the sequence of the template, which is an essential property for the unconstrained transmission of genetic information. The polymerase ribozyme exhibits Michaelis-Menten saturation kinetics, with a catalytic rate of 0.1-1 min-1 and a Km of 0.1-1 μM. Earlier forms of the polymerase did not exhibit a saturable substrate binding site, but this property emerged over the course of directed evolution as the ribozyme underwent a structural rearrangement of its catalytic center. The optimized polymerase makes tertiary contacts with both the template and primer, including a critical interaction at the C2' position of the template nucleotide that opposes the 3'-terminal nucleotide of the primer. UV cross-linking studies paint a picture of how several portions of the ribozyme, including regions that were remodeled by directed evolution, come together to position the template, primer, and NTP within the active site for RNA polymerization.

Terminal bridging of siRNA duplex at the ribose 2′ position controls strand bias and target sequence preference

Small interfering RNA (siRNA) and short hairpin RNA (shRNA) are widely used as RNA interference (RNAi) reagents. Recently, truncated shRNAs that trigger RNAi in a Dicer-independent manner have been developed. We generated a novel class of RNAi reagent, designated enforced strand bias (ESB) RNA, in which an siRNA duplex was chemically bridged between the 3' terminal overhang region of the guide strand and the 5' terminal nucleotide of the passenger strand. ESB RNA, which is chemically bridged at the 2' positions of ribose (2'-2' ESB RNA), functions in a Dicer-independent manner and was highly effective at triggering RNAi without the passenger strand-derived off-target effect. In addition, the 2'-2' ESB RNA exhibited a unique target sequence preference that differs from siRNA and silenced target sequences that could not be effectively suppressed by siRNA. Our results indicate that ESB RNA has the potential to be an effective RNAi reagent even when the target sequence is not suitable for siRNA.

Structural basis for sequence-specific recognition of guide and target strands by the Archaeoglobus fulgidus Argonaute protein

Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos). Being the structural core of RNA interference machinery, they use guide RNA molecules for RNA targeting. Prokaryotic Argonautes (pAgos) are more diverse, both in terms of structure (there are eAgo-like ‘long’ and truncated ‘short’ pAgos) and mechanism, as many pAgos are specific for DNA, not RNA guide and/or target strands. Some long pAgos act as antiviral defence systems. Their defensive role was recently demonstrated for short pAgo-encoding systems SPARTA and GsSir2/Ago, but the function and action mechanisms of all other short pAgos remain unknown. In this work, we focus on the guide and target strand preferences of AfAgo, a truncated long-B Argonaute protein encoded by an archaeon Archaeoglobus fulgidus. We demonstrate that AfAgo associates with small RNA molecules carrying 5′-terminal AUU nucleotides in vivo, and characterize its affinity to various RNA and DNA guide/target strands in vitro. We also present X-ray structures of AfAgo bound to oligoduplex DNAs that provide atomic details for base-specific AfAgo interactions with both guide and target strands. Our findings broaden the range of currently known Argonaute-nucleic acid recognition mechanisms.

Structure-Based Regulatory Role for the 5′UTR of RCNMV RNA2

Red clover necrotic mosaic virus (RCNMV) is a segmented positive-strand RNA virus consisting of RNA1 and RNA2. Previous studies demonstrated that efficient translation of RCNMV RNA2 requires de novo synthesis of RNA2 during infections, suggesting that RNA2 replication is required for its translation. We explored a potential mechanism underlying the regulation of replication-associated translation of RNA2 by examining RNA elements in its 5' untranslated region (5'UTR). Structural analysis of the 5'UTR suggested that it can form two mutually exclusive configurations: a more thermodynamically stable conformation, termed the 5'-basal stem structure (5'BS), in which 5'-terminal sequences are base paired, and an alternative conformation, where the 5'-end segment is single stranded. Functional mutational analysis of the 5'UTR structure indicated that (i) 43S ribosomal subunits enter at the very 5'-end of RNA2; (ii) the alternative conformation, containing unpaired 5'-terminal nucleotides, mediates efficient translation; (iii) the 5'BS conformation, with a paired 5'-end segment, supresses translation; and (iv) the 5'BS conformation confers stability to RNA2 from 5'-to-3' exoribonuclease Xrn1. Based on our results, we suggest that during infections, newly synthesized RNA2s transiently adopt the alternative conformation to allow for efficient translation, then refold into the 5'BS conformation, which supresses translation and promotes efficient RNA2 replication. The potential advantages of this proposed 5'UTR-based regulatory mechanism for coordinating RNA2 translation and replication are discussed.

Enzymatic reactions of AGO4 in RNA-directed DNA methylation: siRNA duplex loading, passenger strand elimination, target RNA slicing, and sliced target retention.

RNA-directed DNA methylation in plants is guided by 24-nt siRNAs generated in parallel with 23-nt RNAs of unknown function. We show that 23-nt RNAs function as passenger strands during 24-nt siRNA incorporation into AGO4. The 23-nt RNAs are then sliced into 11- and 12-nt fragments, with 12-nt fragments remaining associated with AGO4. Slicing recapitulated with recombinant AGO4 and synthetic RNAs reveals that siRNAs of 21-24 nt, with any 5'-terminal nucleotide, can guide slicing, with sliced RNAs then retained by AGO4. In vivo, RdDM target locus RNAs that copurify with AGO4 also display a sequence signature of slicing. Comparing plants expressing slicing-competent versus slicing-defective AGO4 shows that slicing elevates cytosine methylation levels at virtually all RdDM loci. We propose that siRNA passenger strand elimination and AGO4 tethering to sliced target RNAs are distinct modes by which AGO4 slicing enhances RNA-directed DNA methylation.