Autocatalytic base editing for RNA-responsive translational control.

Genetic circuits that control transgene expression in response to pre-defined transcriptional cues would enable the development of smart therapeutics. To this end, here we engineer programmable single-transcript RNA sensors in which adenosine deaminases acting on RNA (ADARs) autocatalytically convert target hybridization into a translational output. Dubbed DART VADAR (Detection and Amplification of RNA Triggers via ADAR), our system amplifies the signal from editing by endogenous ADAR through a positive feedback loop. Amplification is mediated by the expression of a hyperactive, minimal ADAR variant and its recruitment to the edit site via an orthogonal RNA targeting mechanism. This topology confers high dynamic range, low background, minimal off-target effects, and a small genetic footprint. We leverage DART VADAR to detect single nucleotide polymorphisms and modulate translation in response to endogenous transcript levels in mammalian cells.

Cultivated and Wild Grapevines in Tennessee Possess Overlapping but Distinct Virus Populations.

Viruses and viroids prevalent in a population of 42 wild grapevines (i.e., free-living, uncultivated grapevines; Vitis spp.) were compared with those in a population of 85 cultivated grapevines collected in Tennessee, United States by RNA sequencing analysis of pools of ribosomal RNA-depleted total RNA. The sequences of 10 viruses (grapevine fleck virus, grapevine leafroll-associated virus 2, grapevine rupestris stem pitting-associated virus, grapevine Syrah virus 1, grapevine vein-clearing virus, grapevine virus B, grapevine virus E, tobacco ringspot virus, tomato ringspot virus, and a novel nano-like virus) and two viroids (hop stunt viroid and grapevine yellow speckle viroid 1) were detected in both grapevine populations. Sequences of four viruses (grapevine associated tymo-like virus, grapevine leafroll-associated virus 3, grapevine red blotch virus, and grapevine virus H) were identified only from cultivated grapevines. High, moderate, and low numbers of sequence reads were identified only from wild grapevines for a novel caulimovirus, an enamovirus, and alfalfa mosaic virus, respectively. The presence of most virus sequences and both viroids was verified independently in the original samples by reverse-transcription PCR followed by Sanger sequencing. Comparison of viral sequences shared by both populations showed that cultivated and wild grapevines harbored distinct sequence variants, which suggests that there was limited virus movement between the two populations. Collectively, this study represents the first unbiased survey of viruses and viroids in both cultivated and wild grapevines within a defined geographic region.

Evidence for the splicing of grablovirus transcripts reveals a putative novel open reading frame.

Grapevine red blotch virus (GRBV) is type member of the newly identified genus Grablovirus. It possesses a single-stranded circular DNA genome of around 3200 nucleotides encoding three open reading frames (ORFs) in both the virion sense, the V1 (CP), V2 and V3, and complementary sense, C1 (RepA), C2 and C3. As shown for members of the genus Mastrevirus, the C1 and C2 ORFs are predicted to fuse through splicing to form a replication-associated protein (Rep). Data obtained using high-throughput sequencing (RNA-Seq) of three RNA-enriched populations, extracted from GRBV-infected grapevine (Vitis vinifera), confirmed the presence of the predicted C1-C2 intron (nts 2288-2450), but in addition identified a larger virion-sense intron (nts 251-589) spanning the V2 ORF. Evidence for both introns in a number of isolates was supported by bioinformatic analysis of publicly available datasets (n=20). These observations were further supported by RT-PCR analyses in both GRBV-infected grapevine and transient expression assays where GRBV genome segments were agro-inoculated onto Nicotiana benthamiana. The donor site of the virion-sense intron is located within two small ORFs, V0 and V02, while the acceptor site is two-thirds along the V2 ORF. Splicing at these positions is predicted to delete the N terminus of the encoded V2 protein. Comparative analyses of full-length GRBV sequences and the related tentative grabloviruses Prunus geminivirus A and wild Vitis virus 1 support the existence of both introns and V0. The probable regulatory role of these introns in the GRBV infection cycle is discussed.

A Small RNA-Mediated Regulatory Network in Arabidopsis thaliana Demonstrates Connectivity Between phasiRNA Regulatory Modules and Extensive Co-Regulation of Transcription by miRNAs and phasiRNAs.

Gene regulation involves the orchestrated action of multiple regulators to fine-tune the expression of genes. Hierarchical interactions and co-regulation among regulators are commonly observed in biological systems, leading to complex regulatory networks. Small RNA (sRNAs) have been shown to be important regulators of gene expression due to their involvement in multiple cellular processes. In plants, microRNA (miRNAs) and phased small interfering RNAs (phasiRNAs) correspond to two well-characterized types of sRNAs involved in the regulation of posttranscriptional gene expression, although information about their targets and interactions with other gene expression regulators is limited. We describe an extended sRNA-mediated regulatory network in Arabidopsis thaliana that provides a reference frame to understand sRNA biogenesis and activity at the genome-wide level. This regulatory network combines a comprehensive evaluation of phasiRNA production and sRNA targets supported by degradome data. The network includes ~17% of genes in the A. thaliana genome, representing ~50% annotated gene ontology (GO) functional categories. Approximately 14% of genes with GO annotations corresponding to regulation of gene expression were found to be under sRNA control. The unbiased bioinformatic approach used to produce the network was able to detect 107 PHAS loci (regions of phasiRNA production), 5,047 active phasiRNAs (~70% of which were non-canonical), and reconstruct 17 regulatory modules resulting from complex regulatory interactions between different sRNA-regulatory pathways. Known regulatory modules like miR173-TAS-PPR/TPR and miR390-TAS3-ARF/F-box were faithfully reconstructed and expanded, illustrating the accuracy and sensitivity of the methods and providing confidence for the validity of findings of previously unrecognized modules. The network presented here includes a 2X increase in the number of identified PHAS loci, a large complement (~70%) of non-canonical phasiRNAs, and the most comprehensive evaluation of sRNA cleavage activity in A. thaliana to date. Structural analysis showed similarities to networks of other biological systems and demonstrated connectivity between phasiRNA regulatory modules with extensive co-regulation of transcripts by miRNAs and phasiRNAs. The described regulatory network provides a reference that will facilitate global analyses of individual plant regulatory programs such as those that control homeostasis, development, and responses to biotic and abiotic environmental changes.

The complete nucleotide sequence and genomic characterization of grapevine asteroid mosaic associated virus.

In analyzing grapevine clones infected with grapevine red blotch associated virus, we identified a small number of isometric particles of approximately 30nm in diameter from an enriched fraction of leaf extract. A dominant protein of 25kDa was isolated from this fraction using SDS-PAGE and was identified by mass spectrometry as belonging to grapevine asteroid mosaic associated virus (GAMaV). Using a combination of three methods RNA-Seq, sRNA-Seq, and Sanger sequencing of RT- and RACE-PCR products, we obtained a full-length genome sequence consisting of 6719 nucleotides without the poly(A) tail. The virus possesses all of the typical conserved functional domains concordant with the genus Marafivirus and lies evolutionarily between citrus sudden death associated virus and oat blue dwarf virus. A large shift in RNA-Seq coverage coincided with the predicted location of the subgenomic RNA involved in coat protein (CP) expression. Genus wide sequence alignments confirmed the cleavage motif LxG(G/A) to be dominant between the helicase and RNA dependent RNA polymerase (RdRp), and the RdRp and CP domains. A putative overlapping protein (OP) ORF lacking a canonical translational start codon was identified with a reading frame context more consistent with the putative OPs of tymoviruses and fig fleck associated virus than with those of marafiviruses. BLAST analysis of the predicted GAMaV OP showed a unique relatedness to the OPs of members of the genus Tymovirus.