End Of RNA Research Articles

Discovery and Genome Characterization of a Closterovirus from Wheat Plants with Yellowing Leaf Symptoms in Japan.

Many aphid-borne viruses are important pathogens that affect wheat crops worldwide. An aphid-transmitted closterovirus named wheat yellow leaf virus (WYLV) was found to have infected wheat plants in Japan in the 1970s; however, since then, its viral genome sequence and occurrence in the field have not been investigated. We observed yellowing leaves in the 2018/2019 winter wheat-growing season in an experimental field in Japan where WYLV was detected five decades ago. A virome analysis of those yellow leaf samples lead to the discovery of a closterovirus together with a luteovirus (barley yellow dwarf virus PAV variant IIIa). The complete genomic sequence of this closterovirus, named wheat closterovirus 1 isolate WL19a (WhCV1-WL19a), consisted of 15,452 nucleotides harboring nine open reading frames. Additionally, we identified another WhCV1 isolate, WL20, in a wheat sample from the winter wheat-growing season of 2019/2020. A transmission test indicated that WhCV1-WL20 was able to form typical filamentous particles and transmissible by oat bird-cherry aphid (Rhopalosiphum pad). Sequence and phylogenetic analyses showed that WhCV1 was distantly related to members of the genus Closterovirus (family Closteroviridae), suggesting that the virus represents a novel species in the genus. Furthermore, the characterization of WhCV1-WL19a-derived small RNAs using high-throughput sequencing revealed highly abundant 22-nt-class small RNAs potentially derived from the 3'-terminal end of the WhCV1 negative-strand genomic RNA, indicating that this terminal end of the WhCV1 genome is likely particularly targeted for the synthesis of viral small RNAs in wheat plants. Our results provide further knowledge on closterovirus diversity and pathogenicity and suggest that the impact of WhCV1 on wheat production warrants further investigations.

Virtual Screening of Hepatitis B Virus Pre-Genomic RNA as a Novel Therapeutic Target.

The global burden imposed by hepatitis B virus (HBV) infection necessitates the discovery and design of novel antiviral drugs to complement existing treatments. One attractive and underexploited therapeutic target is ε, an ~85-nucleotide (nt) cis-acting regulatory stem-loop RNA located at the 3'- and 5'-ends of the pre-genomic RNA (pgRNA). Binding of the 5'-end ε to the viral polymerase protein (P) triggers two early events in HBV replication: pgRNA and P packaging and reverse transcription. Our recent solution nuclear magnetic resonance spectroscopy structure of ε permits structure-informed drug discovery efforts that are currently lacking for P. Here, we employ a virtual screen against ε using a Food and Drug Administration (FDA)-approved compound library, followed by in vitro binding assays. This approach revealed that the anti-hepatitis C virus drug Daclatasvir is a selective ε-targeting ligand. Additional molecular dynamics simulations demonstrated that Daclatasvir targets ε at its flexible 6-nt priming loop (PL) bulge and modulates its dynamics. Given the functional importance of the PL, our work supports the notion that targeting ε dynamics may be an effective anti-HBV therapeutic strategy.

Versatile strategy using vaccinia virus-capping enzyme to synthesize functional 5' cap-modified mRNAs.

The potential of synthetic mRNA as a genetic carrier has increased its application in scientific fields. Because the 5' cap regulates the stability and translational activity of mRNAs, there are concerted efforts to search for and synthesize chemically-modified 5' caps that improve the functionality of mRNA. Here, we report an easy and efficient method to synthesize functional mRNAs by modifying multiple 5' cap analogs using a vaccinia virus-capping enzyme. We show that this enzyme can introduce a variety of GTP analogs to the 5' end of RNA to generate 5' cap-modified mRNAs that exhibit different translation levels. Notably, some of these modified mRNAs improve translation efficiency and can be conjugated to chemical structures, further increasing their functionality. Our versatile method to generate 5' cap-modified mRNAs will provide useful tools for RNA therapeutics and biological research.

RNA structure directs the targeting specificity of Cas13d.

Type VI CRISPR enzymes exclusively bind and cleave target RNAs, and are widely used for gene regulation, RNA tracking, and diagnostics. However, a systematic understanding of their RNA binding specificity is lacking. Here, we describe RNA-CHAMP, a massively parallel platform that repurposes used next-generation DNA sequencing chips to measures the binding affinity for >10,000 RNA targets containing structural perturbations, mismatches, insertions, and deletions relative to the guide RNA. We profile the RNA binding specificity of Cas13d, a compact and widely used RNA nuclease. Contrary to other type VI CRISPR enzymes, Cas13d does not have a protospacer flanking sequence (PFS) preference. Cas13d tolerates mismatches, insertions, and deletions, but is exquisitely sensitive to secondary structure within the target RNA. Basepairing in the distal region of the target RNA strongly decreases Cas13d binding, whereas structuring the first six nucleotides inhibits nuclease activity without impacting binding. A biophysical model built from these data reveals that target recognition begins at the distal end of unstructured target RNAs and proceeds to the proximal end with limited RNA melting. Using this model, we design a series of partially mismatched guide RNAs that modulate nuclease activity to detect single nucleotide polymorphisms (SNPs) in circulating SARS-CoV-2 variants. This work describes the key determinants of RNA targeting by a type VI CRISPR enzyme to improve RNA targeting and CRISPR diagnostics. More broadly, RNA-CHAMP provides a quantitative platform for systematically measuring protein-RNA interactions.

Transfer RNA Modification Enzymes with a Thiouridine Synthetase, Methyltransferase and Pseudouridine Synthase (THUMP) Domain and the Nucleosides They Produce in tRNA.

The existence of the thiouridine synthetase, methyltransferase and pseudouridine synthase (THUMP) domain was originally predicted by a bioinformatic study. Since the prediction of the THUMP domain more than two decades ago, many tRNA modification enzymes containing the THUMP domain have been identified. According to their enzymatic activity, THUMP-related tRNA modification enzymes can be classified into five types, namely 4-thiouridine synthetase, deaminase, methyltransferase, a partner protein of acetyltransferase and pseudouridine synthase. In this review, I focus on the functions and structures of these tRNA modification enzymes and the modified nucleosides they produce. Biochemical, biophysical and structural studies of tRNA 4-thiouridine synthetase, tRNA methyltransferases and tRNA deaminase have established the concept that the THUMP domain captures the 3'-end of RNA (in the case of tRNA, the CCA-terminus). However, in some cases, this concept is not simply applicable given the modification patterns observed in tRNA. Furthermore, THUMP-related proteins are involved in the maturation of other RNAs as well as tRNA. Moreover, the modified nucleosides, which are produced by the THUMP-related tRNA modification enzymes, are involved in numerous biological phenomena, and the defects of genes for human THUMP-related proteins are implicated in genetic diseases. In this review, these biological phenomena are also introduced.

Structural insights into viral genome replication by the severe fever with thrombocytopenia syndrome virus L protein.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA-dependent RNA polymerase (RdRp), is responsible for catalysing viral genome replication and transcription. Here, we present 5 cryo-electron microscopy (cryo-EM) structures of the L protein in several states of the genome replication process, from pre-initiation to late-stage elongation, at a resolution of up to 2.6 Å. We identify how the L protein binds the 5' viral RNA in a hook-like conformation and show how the distal 5' and 3' RNA ends form a duplex positioning the 3' RNA terminus in the RdRp active site ready for initiation. We also observe the L protein stalled in the early and late stages of elongation with the RdRp core accommodating a 10-bp product-template duplex. This duplex ultimately splits with the template binding to a designated 3' secondary binding site. The structural data and observations are complemented by in vitro biochemical and cell-based mini-replicon assays. Altogether, our data provide novel key insights into the mechanism of viral genome replication by the SFTSV L protein and will aid drug development against segmented negative-strand RNA viruses.

Enzymatic synthesis of RNA standards for mapping and quantifying RNA modifications in sequencing analysis.

Synthesis of RNA standards that contain an internal site-specific modification is important for mapping and quantification of the modified nucleotide in sequencing analysis. While RNA containing a site-specific modification can be readily synthesized by solid-state coupling for less than 100-mer nucleotides, longer RNA must be synthesized by enzymatic ligation in the presence of a DNA splint. However, long RNAs have structural heterogeneity, and those generated by in vitro transcription have 3'-end sequence heterogeneity, which together substantially reduce the yield of ligation. Here we describe a method of 3-part splint ligation that joins an in vitro transcribed left-arm RNA, an in vitro transcribed right-arm RNA, and a chemically synthesized modification-containing middle RNA, with an efficiency higher than previously reported. We report that the improved efficiency is largely attributed to the inclusion of a pair of DNA disruptors proximal to the ligation sites, and to a lesser extent to the homogeneous processing of the 3'-end of the left-arm RNA. The yields of the ligated long RNA are sufficiently high to afford purification to homogeneity for practical RNA research. We also verify the sequence accuracy at each ligation junction by nanopore sequencing.

Genome-wide Mapping of 5'-monophosphorylated Ends of Mammalian Nascent RNA Transcripts.

In eukaryotic cells, RNA biogenesis generally requires processing of the nascent transcript as it is being synthesized by RNA polymerase. These processing events include endonucleolytic cleavage, exonucleolytic trimming, and splicing of the growing nascent transcript. Endonucleolytic cleavage events that generate an exposed 5'-monophosphorylated (5'-PO4) end on the growing nascent transcript occur in the maturation of rRNAs, tRNAs, and mRNAs. These 5'-PO4 ends can be a target of further processing or be subjected to 5'-3' exonucleolytic digestion that may result in termination of transcription. Here, we describe how to identify 5'-PO4 ends of intermediates in nascent RNA metabolism. We capture these species via metabolic labeling with bromouridine followed by immunoprecipitation and specific ligation of 5'-PO4 RNA ends with the 3'-hydroxyl group of a 5' adaptor (5'-PO4 Bru-Seq) using RNA ligase I. These ligation events are localized at single nucleotide resolution via highthroughput sequencing, which identifies the position of 5'-PO4 groups precisely. This protocol successfully detects the 5'monophosphorylated ends of RNA processing intermediates during production of mature ribosomal, transfer, and micro RNAs. When combined with inhibition of the nuclear 5'-3' exonuclease Xrn2, 5'-PO4 Bru-Seq maps the 5' splice sites of debranched introns and mRNA and tRNA 3' end processing sites cleaved by CPSF73 and RNaseZ, respectively. Key features • Metabolic labeling for brief periods with bromouridine focuses the analysis of 5'-PO4 RNA ends on the population of nascent transcripts that are actively transcribed. • Detects 5'-PO4 RNA ends on nascent transcripts produced by all RNA polymerases. • Detects 5'-PO4 RNA ends at single nucleotide resolution.

TRNA-derived small RNA 3′U-tRFValCAC promotes tumour migration and early progression in ovarian cancer

IntroductionDespite recent advances in epithelial ovarian cancer (EOC) management, the highly heterogenous histological/molecular tumour background and patients' treatment response obstructs personalised prognosis and therapeutics. Herein, we have studied the role and clinical utility of the novel subclass of tRNA-derived small RNA fragments emerging via 3′-trailer processing of pre-tRNAs (3′U-tRFs) in EOC. MethodsSK-OV-3 and OVCAR-3 cells were used for in vitro study. Following transfection, cell growth and migration were assessed by CCK8 and wound healing assays, respectively. 3′U-tRFs levels were assessed by reverse transcription quantitative PCR (RT-qPCR), following 3′-end RNA polyadenylation. A screening (OVCAD, n = 100) and institutionally independent validation (TU Munich, n = 103) cohorts were employed for survival analysis using disease progression and patients' death as clinical end-points. Bootstrap analysis was performed for internal validation, and decision curve analysis was used to evaluate clinical benefit on disease prognosis. ResultsFollowing primary clinical assessment, target prediction and gene ontology analyses, the 3′U-tRFValCAC (derived from pre-tRNAValCAC) was highlighted to regulate cell proliferation and adhesion, and to correlate with inferior patients' outcome. 3′U-tRFValCAC transfection of SK-OV-3 and OVCAR-3 cells resulted in significantly increased cell growth and migration, in a dose-dependent manner. Elevated tumour 3′U-tRFValCAC levels were associated with significantly higher risk for early progression and worse survival following first-line platinum-based chemotherapy, independently of patients' clinicopathological data, chemotherapy response, and residual tumour. Interestingly, 3′U-tRFValCAC-fitted multivariate models improved risk stratification and provided superior clinical net benefit in prediction of treatment outcome compared to disease established markers. Conclusions3′U-tRFValCAC promotes tumour cell growth and migration and supports modern risk stratification and prognosis in EOC.

MiRador: a fast and precise tool for the prediction of plant miRNAs.

Plant microRNAs (miRNAs) are short, noncoding RNA molecules that restrict gene expression via posttranscriptional regulation and function in several essential pathways, including development, growth, and stress responses. Accurately identifying miRNAs in populations of small RNA sequencing libraries is a computationally intensive process that has resulted in the misidentification of inaccurately annotated miRNA sequences. In recent years, criteria for miRNA annotation have been refined with the aim to reduce these misannotations. Here, we describe miRador, a miRNA identification tool that utilizes the most up-to-date, community-established criteria for accurate identification of miRNAs in plants. We combined target prediction and Parallel Analysis of RNA Ends (PARE) data to assess the precision of the miRNAs identified by miRador. We compared miRador to other commonly used miRNA prediction tools and found that miRador is at least as precise as other prediction tools while being substantially faster than other tools. miRador should be broadly useful for the plant community to identify and annotate miRNAs in plant genomes.

Multi-Omic Analysis of 253 Untreated Patients with Waldenström's Macroglobulinemia Reveals Clinically and Genomically Distinct Disease Subtypes and a Model for Disease Progression

Background: Waldenström's Macroglobulinemia (WM) is characterized by recurring mutations in MYD88 (95-97%) and CXCR4 (40%). While these genomic insights have heralded new insights into disease pathogenesis and treatment, a multi-omic approach in a large population of WM patients may further clarify underlying disease biology. Methods: Stranded 150bp paired end RNA sequencing was performed on CD19+ bone marrow (BM) biopsies from 253 treatment-naive WM patients along with paired CD19+CD27- and CD19+CD27+ peripheral blood (PB) cells from 13 healthy donors. For 216 patients, whole exome sequencing of CD19+ BM WM cells with CD19- PB controls was also performed. All p-values were subjected to correction using the false discovery rate. Results: Baseline characteristics were typical of treatment-naive patients. Their median age was 66 years; BM disease involvement 50% ; and serum IgM 3,108 mg/dL. The median follow-up was 7.5 years. MYD88 mutations were identified in 250 (98.8%) patients. Exploratory analysis of MYD88 mutated patients revealed three distinct groups of patients that were observed in a hierarchical clustering analysis shown in Fig 1A. Gene set enrichment analysis revealed distinct B-cell Like (BCL) and Plasma Cell Like (PCL) patient groupings and a third non-BCL/PCL patient group that clustered independently. Non-BCL/PCL patients were more likely to be asymptomatic (36/84; 43%) versus those in the BCL (62/96; 64.6%) and PCL (48/68; 70.6%) groups (p<0.005). Among BCL and PCL grouped patients, important differences in disease presentation were noted including BM involvement (50% vs. 70%; p<0.001), adenopathy (18.6% vs. 48.4%; p<0.002), CD10 (1.1% vs. 13.4%; p=0.012), CD23 (39.4% vs. 22.4%; p=0.09) and CD5 (19.1% vs. 6%; p=0.09) expression, respectively. CXCR4 mutations were identified in 76/97 (78.4%) of BCL versus 6/69 (8.7%) of PCL patients (p<0.001). Amplifications in chr18q amplifications were also more prevalent in BCL (14/83; 16.9%) versus PCL (1/64; 1.6%) grouped patients (p=0.035). chr6q deletions were observed in 24/83 (29%) of BCL versus 30/64 (47%) PCL patients (p=0.11). BCL patients also exhibited fewer chr6p amplifications, i.e. 3/83 (3.6%) versus 12/64 (18.8%) of PCL patients (p=0.036). Differences in deletions in chr17p (0/83; 0%) vs (6/64; 9.4% (p=0.036); as well as NOTCH1 (0/82; 0%) vs. (6/64; 9.4%) (p=0.084) and EP300 3/82; 3.7%) vs. (11/64; 17.2%) (p=0.084) mutations were also identified in BCL vs PCL patients, respectively. NOTCH pathway mutations were also found in fewer BCL (17/82; 20.7%) versus PCL (30/64; 46.9%) patients (p=0.01). Diffusion mapping and pseudo-time analysis were performed on the top 1,000 high variance genes which bifurcated patients into two distinct populations over time as shown in Fig. 1B, with the point size scaled for BM involvement. By diffusion pseudo-time (DPT), patients were stratified into early- and late-stage WM, the former showing lower median BM involvement (30% vs. 70%; p<0.001) and serum IgM (2,666 vs. 3713; p=0.133); higher hemoglobin (12.1g/dL vs. 10.65g/dL; p<0.001), serum IgA (56.5mg/dL vs. 41mg/dL; p=0.018) and IgG (629mg/dL vs. 498mg/dL; p=0.015), and less splenomegaly (3.7% vs. 17.2%; p=0.006). A longer time to first treatment (5.3 vs 0.9 months; (p<0.001) was also observed in those with early- versus late- stage WM, respectively. DPT showed that the non-BCL/PCL patients were more likely to be early stage (68/84; 81%) versus BCL (51/97; 53%) and PCL (30/69; 43%) grouped patients (p<0.001). In addition, hierarchical clustering delineated the non-BCL/PCL group into two subgroups with CXCR4 mutations rates of 30/57 (52.6%) and 2/27 (7.4%), and gene expression similarities that aligned with either the BCL or PCL subtypes, respectively. Taken together, these findings suggest that the non-BCL/PCL group represents an earlier evolving stage of WM that may progress into BCL and PCL like WM disease. Conclusions: In this largest multi-omic investigation undertaken into WM, we identified three distinct WM subtypes with distinct clinical and genomic features. Central these findings were the identification of BCL, PCL, and an earlier non-BCL/PCL evolving stage recognized through DPT modeling. The discovery of the DPT model for WM disease progression provides insight into the biology and targeting of WM progression. Our findings provide a critical framework for multi-omic based study and targeted treatment approach to WM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genome-wide identification of GRF gene family and their contribution to abiotic stress response in pitaya (Hylocereus polyrhizus)

Growth-regulating factors (GRFs) are plant-specific transcription factors identified in many land plants. Recently, their indispensable roles in stress response are highlighted. In present work, 11 HpGRFs were cloned in pitaya. Segmental duplication is considered essential for the expansion of HpGRFs. A phylogenetic tree suggested that GRFs could be divided into eight categories, among which G-I was a Caryophyllales-specific one. The categorization was further evidenced by differences in the gene structure, collinearity, protein domain of HpGRFs. Five miR396 hairpins giving rise to two types of matured miR396s were identified in pitaya via sRNA-Seq in combination with bioinformatic analysis. Parallel analysis of RNA ends proved that HpGRFs except HpGRF5 were degraded by miR396-directed cleavages at the regions which code the conserved WRC motifs of HpGRFs. Multiple cis-regulatory elements were discovered in the promoters of HpGRFs. Among the elements, most are involved in stress and phytohormone response as well as plant growth, indicating a crosstalk between them. Expression analysis showed the responsive patterns of the miR396-GRF module under abiotic stresses. To conclude, our work systematically identified the miR396-targeted HpGRFs in pitaya and confirmed their involvement in stress response, providing novel insights into the comprehensive understanding of the stress resistance of pitaya.

Mechanistic Insights into the Protection Effect of Argonaute-RNA Complex on the HCV Genome.

While host miRNA usually plays an antiviral role, the relentless tides of viral evolution have carved out a mechanism to recruit host miRNA as a viral protector. By complementing miR-122 at the 5' end of the genome, the hepatitis C virus (HCV) gene can form a complex with Argonaute 2 (Ago2) protein to protect the 5' end of HCV RNA from exonucleolytic attacks. Experiments showed that the disruption of the stem-loop 1(SL1) structure and the 9th nucleotide (T9) of HCV site 1 RNA could enhance the affinity of the Ago2 protein to the HCV site 1 RNA (target RNA). However, the underlying mechanism of how the conformation and dynamics of the Ago2: miRNA: target RNA complex is affected by the SL1 and T9 remains unclear. To address this, we performed large-scale molecular dynamics simulations on the AGO2-miRNA complex binding with the WT target, T9-abasic target and SL1-disruption target, respectively. The results revealed that the T9 and SL1 structures could induce the departing motion of the PAZ, PIWI and N domains, propping up the mouth of the central groove which accommodates the target RNA, causing the instability of the target RNA and disrupting the Ago2 binding. The coordinated motion among the PAZ, PIWI and N domains were also weakened by the T9 and SL1 structures. Moreover, we proposed a new model wherein the Ago2 protein could adopt a more constraint conformation with the proximity and more correlated motions of the PAZ, N and PIWI domains to protect the target RNA from dissociation. These findings reveal the mechanism of the Ago2-miRNA complex's protective effect on the HCV genome at the atomic level, which will offer guidance for the design of drugs to confront the protection effect and engineering of Ago2 as a gene-regulation tool.

Small RNAs target native and cross-kingdom transcripts on both sides of the wheat stripe rust interaction

The wheat stripe rust fungus (Puccinia striiformis f.sp. tritici) threatens global wheat production. Small RNAs (sRNAs) modulate plant defense induction, and RNA exchange between host and microbe causes cross-kingdom gene silencing, but few examples are known in rust fungi. This study combined sRNA, parallel analysis of RNA ends, and gene expression data to discover sRNA-target pairs on each side of the interaction. Specific wheat 24 nt sRNAs were suppressed, while particular 35 nt fragments were strongly induced upon infection. Wheat sRNAs cleaved fungal transcripts coding for a ribosomal protein and a glycosyl hydrolase effector. Fungal microRNA-like and phased 21 nt sRNAs originated from long inverted repeats near protein coding genes. Fungal sRNAs targeted native transcripts: transposons and kinases; and cross-kingdom transcripts: a wheat nucleotide-binding domain leucine-rich repeat receptor (NLR) and multiple defense-related transcription factor families. This work sheds light on host-microbe coevolution and delivers prospects for developing pathogen control biotechnology.

An atypical RNA quadruplex marks RNAs as vectors for gene silencing.

The addition of poly(UG) ('pUG') repeats to 3' termini of mRNAs drives gene silencing and transgenerational epigenetic inheritance in the metazoan Caenorhabditis elegans. pUG tails promote silencing by recruiting an RNA-dependent RNA polymerase (RdRP) that synthesizes small interfering RNAs. Here we show that active pUG tails require a minimum of 11.5 repeats and adopt a quadruplex (G4) structure we term the pUG fold. The pUG fold differs from known G4s in that it has a left-handed backbone similar to Z-RNA, no consecutive guanosines in its sequence, and three G quartets and one U quartet stacked non-sequentially. The compact pUG fold binds six potassium ions and brings the RNA ends into close proximity. The biological importance of the pUG fold is emphasized by our observations that porphyrin molecules bind to the pUG fold and inhibit both gene silencing and binding of RdRP. Moreover, specific 7-deaza substitutions that disrupt the pUG fold neither bind RdRP nor induce RNA silencing. These data define the pUG fold as a previously unrecognized RNA structural motif that drives gene silencing. The pUG fold can also form internally within larger RNA molecules. Approximately 20,000 pUG-fold sequences are found in noncoding regions of human RNAs, suggesting that the fold probably has biological roles beyond gene silencing.

BRAT1 links Integrator and defective RNA processing with neurodegeneration

Mutations in BRAT1, encoding BRCA1-associated ATM activator 1, have been associated with neurodevelopmental and neurodegenerative disorders characterized by heterogeneous phenotypes with varying levels of clinical severity. However, the underlying molecular mechanisms of disease pathology remain poorly understood. Here, we show that BRAT1 tightly interacts with INTS9/INTS11 subunits of the Integrator complex that processes 3’ ends of various noncoding RNAs and pre-mRNAs. We find that Integrator functions are disrupted by BRAT1 deletion. In particular, defects in BRAT1 impede proper 3’ end processing of UsnRNAs and snoRNAs, replication-dependent histone pre-mRNA processing, and alter the expression of protein-coding genes. Importantly, impairments in Integrator function are also evident in patient-derived cells from BRAT1 related neurological disease. Collectively, our data suggest that defects in BRAT1 interfere with proper Integrator functions, leading to incorrect expression of RNAs and proteins, resulting in neurodegeneration.

Mature Rotavirus Particles Contain Equivalent Amounts of 7meGpppG-Capped and Noncapped Viral Positive-Sense RNAs.

Viruses have evolved different strategies to overcome their recognition by the host innate immune system. The addition of caps at their 5' RNA ends is an efficient mechanism not only to ensure escape from detection by the innate immune system but also to ensure the efficient synthesis of viral proteins. Rotavirus mRNAs contain a type 1 cap structure at their 5' end that is added by the viral capping enzyme VP3, which is a multifunctional protein with all the enzymatic activities necessary to add the cap and also functions as an antagonist of the 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway. Here, the relative abundances of capped and noncapped viral RNAs during the replication cycle of rotavirus were determined. We found that both classes of rotaviral plus-sense RNAs (+RNAs) were encapsidated and that they were present in a 1:1 ratio in the mature infectious particles. The capping of viral +RNAs was dynamic, since different ratios of capped and noncapped RNAs were detected at different times postinfection. Similarly, when the relative amounts of capped and uncapped viral +RNAs produced in an in vitro transcription system were determined, we found that the proportions were very similar to those in the mature viral particles and in infected cells, suggesting that the capping efficiency of VP3, both in vivo and in vitro, might be close to 50%. Unexpectedly, when the effect of simultaneously knocking down the expression of VP3 and RNase L on the cap status of viral +RNAs was evaluated, we found that, even though at late times postinfection there was an increased proportion of capped viral RNAs in infected cells, the viral particles isolated from this condition contained equal ratios of capped and noncapped viral RNA, suggesting that there might be selective packaging of capped and noncapped RNAs. IMPORTANCE Rotaviruses have a genome composed of 11 segments of double-stranded RNA. Whether all 5' ends of the positive-sense genomic RNAs contained in the mature viral particles are modified by a cap structure is unknown. In this work, we characterized the relative proportions of capped and noncapped viral RNAs in rotavirus-infected cells and in viral particles by using a direct quantitative assay. We found that, independent of the relative proportions of capped/noncapped RNAs present in rotavirus-infected cells, there were similar proportions of these two kinds of 5'-modified positive-sense RNAs in the viral particles.

Parallel analysis of RNA ends reveals global microRNA-mediated target RNA cleavage in maize.

MicroRNAs (miRNAs) are endogenous 20-24-nucleotide non-coding RNAs that play important regulatory roles in many biological processes in eukaryotes. miRNAs modulate the expression of target genes at the post-transcriptional level by transcript cleavage or translational inhibition. The identification of miRNA target genes has been extensively investigated in Arabidopsis and rice, but an in-depth global analysis of miRNA-mediated target regulation is still lacking in maize. Here, we report a transcriptome-wide identification of miRNA targets by analyzing parallel analysis of RNA ends (PARE) datasets derived from nine different tissues at five developmental stages of the maize (Zea mays L.) B73 cultivar. In total, 246 targets corresponding to 60 miRNAs from 25 families were identified, including transcription factors and other genes. In addition, PARE analysis revealed that miRNAs guide specific target transcript cleavage in a tissue-preferential manner. Primary transcripts of MIR159c and MIR169e were found to be cleaved by mature miR159 and miR169, respectively, indicating a negative-feedback regulatory mechanism in miRNA biogenesis. Moreover, several miRNA-target gene pairs involved in seed germination were identified and experimentally validated. Our PARE analyses generated a wide and detailed miRNA-target interaction atlas, which provides a valuable resource for investigating the roles of miRNAs and their targets in maize.

Identifying inhibitors of NSP16-NSP10 of SARS-CoV-2 from large databases

The COVID-19 pandemic, which has already claimed millions of lives, continues to pose a serious threat to human health, requiring the development of new effective drugs. Non-structural proteins of SARS-CoV-2 play an important role in viral replication and infection. Among them, NSP16 (non-structured protein 16) and its cofactor NSP10 (non-structured protein 10) perform C2'-O methylation at the 5' end of the viral RNA, which promotes efficient virus replication. Therefore, the NSP16-NSP10 complex becomes an attractive target for drug development. Using a multi-step virtual screening protocol which includes Lipinski’s rule, docking, steered molecular dynamics and umbrella sampling, we searched for potential inhibitors from the PubChem and anti-HIV databases. It has been shown that CID 135566620 compound from PubChem is the best candidate with an inhibition constant in the sub-μM range. The Van der Waals interaction was found to be more important than the electrostatic interaction in the binding affinity of this compound to NSP16-NSP10. Further in vitro and in vivo studies are needed to test the activity of the identified compound against COVID-19. Communicated by Ramaswamy H. Sarma

Importance of RNA length for in vitro encapsidation by the nucleoprotein of human respiratory syncytial virus

Respiratory syncytial virus has a negative-sense single-stranded RNA genome constitutively encapsidated by the viral nucleoprotein N, forming a helical nucleocapsid which is the template for viral transcription and replication by the viral polymerase L. Recruitment of L onto the nucleocapsid depends on the viral phosphoprotein P, which is an essential L cofactor. A prerequisite for genome and antigenome encapsidation is the presence of the monomeric, RNA-free, neosynthesized N protein, named N0. Stabilization of N0 depends on the binding of the N-terminal residues of P to its surface, which prevents N oligomerization. However, the mechanism involved in the transition from N0-P to nucleocapsid assembly, and thus in the specificity of viral genome encapsidation, is still unknown. Furthermore, the specific role of N oligomerization and RNA in the morphogenesis of viral factories, where viral transcription and replication occur, have not been elucidated although the interaction between P and N complexed to RNA has been shown to be responsible for this process. Here, using a chimeric protein comprising N and the first 40 N-terminal residues of P, we succeeded in purifying a recombinant N0-like protein competent for RNA encapsidation in vitro. Our results showed the importance of RNA length for stable encapsidation and revealed that the nature of the 5′ end of RNA does not explain the specificity of encapsidation. Finally, we showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories. Together, our findings provide insight into respiratory syncytial virus viral genome encapsidation specificity.