Transcription strategy in a Closterovirus: a novel 5'-proximal controller element of Citrus Tristeza Virus produces 5'- and 3'-terminal subgenomic RNAs and differs from 3' open reading frame controller elements.
ABSTRACT: Citrus tristeza virus (CTV) produces more than thirty 3'- or 5'-terminal subgenomic RNAs (sgRNAs) that accumulate to various extents during replication in protoplasts and plants. Among the most unusual species are two abundant populations of small 5'-terminal sgRNAs of approximately 800 nucleotides (nt) termed low-molecular-weight tristeza (LMT1 and LMT2) RNAs. Remarkably, CTV replicons with all 10 3' genes deleted produce only the larger LMT1 RNAs. These 5'-terminal positive-sense sgRNAs do not have corresponding negative strands and were hypothesized to be produced by premature termination during plus-strand genomic RNA synthesis. We characterized a cis-acting element that controls the production of the LMT1 RNAs. Since manipulation of this cis-acting element in its native position (the L-ProI region of replicase) was not possible because the mutations negatively affect replication, a region (5'TR) surrounding the putative termination sites (nt approximately 550 to 1000) was duplicated in the 3' end of a CTV replicon to allow characterization. The duplicated sequence continued to produce a 5'-terminal plus-strand sgRNA, here much larger ( approximately 11 kb), apparently by termination. Surprisingly, a new 3'-terminal sgRNA was observed from the duplicated 5'TR. A large 3'-terminal sgRNA resulting from the putative promoter activity of the native 5'TR was not observed, possibly because of the down-regulation of a promoter approximately 19 kb from the 3' terminus. However, we were able to observe a sgRNA produced from the native 5'TR of a small defective RNA, which placed the native 5'TR closer to the 3' terminus, demonstrating sgRNA promoter activity of the native 5'TR. Deletion mutagenesis mapped the promoter and the terminator activities of the 5'TR (in the 3' position in the CTV replicon) to a 57-nt region, which was folded by the MFOLD computer program into two stem-loops. Mutations in the putative stem-loop structures equally reduced or prevented production of both the 3'- and 5'-terminal sgRNAs. These mutations, when introduced in frame in the native 5'TR, similarly abolished the synthesis of the LMT1 RNAs and presumably the large 3'-terminal sgRNA while having no impact on replication, demonstrating that neither 5'- nor 3'-terminal sgRNA is necessary for replication of the replicon or full-length CTV in protoplasts. Differences between the 5'TR, which produced two plus-strand sgRNAs, and the cis-acting elements controlling the 3' open reading frames, which produced additional minus-strand sgRNAs corresponding to the 3'-terminal mRNAs, suggest that the different sgRNA controller elements had different origins in the modular evolution of closteroviruses.
Project description:Citrus tristeza virus (CTV), a member of the Closteroviridae, has a positive-sense RNA genome of about 20 kb organized into 12 open reading frames (ORFs). The last 10 ORFs are expressed through 3'-coterminal subgenomic RNAs (sgRNAs) regulated in both amounts and timing. Additionally, relatively large amounts of complementary sgRNAs are produced. We have been unable to determine whether these sgRNAs are produced by internal promotion from the full-length template minus strand or by transcription from the minus-stranded sgRNAs. Understanding the regulation of 10 sgRNAs is a conceptual challenge. In analyzing commonalities of a replicase complex in producing so many sgRNAs, we examined initiating nucleotides of the sgRNAs. We mapped the 5' termini of intermediate- (CP and p13) and low- (p18) produced sgRNAs that, like the two highly abundant sgRNAs (p20 and p23) previously mapped, all initiate with an adenylate. We then examined modifications of the initiation site, which has been shown to be useful in defining mechanisms of sgRNA synthesis. Surprisingly, mutation of the initiating nucleotide of the CTV sgRNAs did not prevent sgRNA accumulation. Based on our results, the CTV replication complex appears to initiate sgRNA synthesis with purines, preferably with adenylates, and is able to initiate synthesis using a nucleotide a few positions 5' or 3' of the native initiation nucleotide. Furthermore, the context of the initiation site appears to be a regulatory mechanism for levels of sgRNA production. These data do not support either of the established mechanisms for synthesis of sgRNAs, suggesting that CTV sgRNA production utilizes a different mechanism.
Project description:CRISPR-Cas9 is widely applied for genome engineering in various organisms. The assembly of single guide RNA (sgRNA) with the Cas9 protein may limit the Cas9/sgRNA effector complex function. We developed a FRET-based assay for detection of CRISPR-Cas9 complex binding to its targets and used this assay to investigate the kinetics of Cas9 assembly with a set of structurally distinct sgRNAs. We find that Cas9 and isolated sgRNAs form the effector complex efficiently and rapidly. Yet, the assembly process is sensitive to the presence of moderate concentrations of non-specific RNA competitors, which considerably delay the Cas9/sgRNA complex formation, while not significantly affecting already formed complexes. This observation suggests that the rate of sgRNA loading into Cas9 in cells can be determined by competition between sgRNA and intracellular RNA molecules for the binding to Cas9. Non-specific RNAs exerted particularly large inhibitory effects on formation of Cas9 complexes with sgRNAs bearing shortened 3'-terminal segments. This result implies that the 3'-terminal segment confers sgRNA the ability to withstand competition from non-specific RNA and at least in part may explain the fact that use of sgRNAs truncated for the 3'-terminal stem loops leads to reduced activity during genomic editing.
Project description:Hibiscus chlorotic ringspot virus (HCRSV), which belongs to the genus Carmovirus, generates two 3'-coterminal subgenomic RNAs (sgRNAs) of 1.4 kb and 1.7 kb. Transcription start sites of the two sgRNAs were identified at nucleotides (nt) 2178 and 2438, respectively. The full promoter of sgRNA1, a 118-base sequence, is localized between positions +6 and -112 relative to its transcription start site (+1). Similarly, a 132-base sequence, from +6 to -126, defines the sgRNA2 promoter. Computer analysis revealed that both sgRNA promoters share a similar two-stem-loop (SL1 + SL2) structure, immediately upstream of the transcription start site. Mutational analysis of the primary sequence and secondary structures showed further similarities between the two subgenomic promoters. The basal portion of SL2, encompassing the transcription start site, was essential for transcription activity in each promoter, while SL1 and the upper portion of SL2 played a role in transcription enhancement. Both the 5' untranslated region (UTR) and the last 87 nt at the 3' UTR of HCRSV genomic RNA are likely to be the putative genomic plus-strand and minus-strand promoters, respectively. They function well as individual sgRNA promoters to produce ectopic subgenomic RNAs in vivo but not to the same levels of the actual sgRNA promoters. This suggests that HCRSV sgRNA promoters share common features with the promoters for genomic plus-strand and minus-strand RNA synthesis. To our knowledge, this is the first demonstration that both the 5' UTR and part of the 3' UTR can be duplicated and function as sgRNA promoters within a single viral genome.
Project description:The expression of the genomic information of severe acute respiratory syndrome coronavirus (SARS CoV) involves synthesis of a nested set of subgenomic RNAs (sgRNAs) by discontinuous transcription. In SARS CoV-infected cells, 10 sgRNAs, including 2 novel ones, were identified, which were predicted to be functional in the expression of 12 open reading frames located in the 3' one-third of the genome. Surprisingly, one new sgRNA could lead to production of a truncated spike protein. Sequence analysis of the leader-body fusion sites of each sgRNA showed that the junction sequences and the corresponding transcription-regulatory sequence (TRS) are unique for each species of sgRNA and are consistent after virus passages. For the two novel sgRNAs, each used a variant of the TRS that has one nucleotide mismatch in the conserved hexanucleotide core (ACGAAC) in the TRS. Coexistence of both plus and minus strands of SARS CoV sgRNAs and evidence for derivation of the sgRNA core sequence from the body core sequence favor the model of discontinuous transcription during minus-strand synthesis. Moreover, one rare species of sgRNA has the junction sequence AAA, indicating that its transcription could result from a noncanonical transcription signal. Taken together, these results provide more insight into the molecular mechanisms of genome expression and subgenomic transcription of SARS CoV.
Project description:The complete nucleotide sequences of genomic RNA1 (9,407 nucleotides [nt]) and RNA2 (8,223 nt) of Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) were determined, revealing that SPCSV possesses the second largest identified positive-strand single-stranded RNA genome among plant viruses after Citrus tristeza virus. RNA1 contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyltransferase, helicase, and polymerase domains. RNA2 contains the Closteroviridae hallmark gene array represented by a heat shock protein homologue (Hsp70h), a protein of 50 to 60 kDa depending on the virus, the major coat protein, and a divergent copy of the coat protein. This grouping resembles the genome organization of Lettuce infectious yellows virus (LIYV), the only other crinivirus for which the whole genomic sequence is available. However, in striking contrast to LIYV, the two genomic RNAs of SPCSV contained nearly identical 208-nt-long 3' terminal sequences, and the ORF for a putative small hydrophobic protein present in LIYV RNA2 was found at a novel position in SPCSV RNA1. Furthermore, unlike any other plant or animal virus, SPCSV carried an ORF for a putative RNase III-like protein (ORF2 on RNA1). Several subgenomic RNAs (sgRNAs) were detected in SPCSV-infected plants, indicating that the sgRNAs formed from RNA1 accumulated earlier in infection than those of RNA2. The 5' ends of seven sgRNAs were cloned and sequenced by an approach that provided compelling evidence that the sgRNAs are capped in infected plants, a novel finding for members of the Closteroviridae.
Project description:CRISPRi, an adapted CRISPR-Cas9 system, is proposed to act as a strand-specific roadblock to repress transcription in eukaryotic cells using guide RNAs (sgRNAs) to target catalytically inactive Cas9 (dCas9) and offers an alternative to genetic interventions for studying pervasive antisense transcription. Here, we successfully use click chemistry to construct DNA templates for sgRNA expression and show, rather than acting simply as a roadblock, sgRNA/dCas9 binding creates an environment that is permissive for transcription initiation/termination, thus generating novel sense and antisense transcripts. At HMS2 in Saccharomyces cerevisiae, sgRNA/dCas9 targeting to the non-template strand for antisense transcription results in antisense transcription termination, premature termination of a proportion of sense transcripts and initiation of a novel antisense transcript downstream of the sgRNA/dCas9-binding site. This redefinition of the transcriptional landscape by CRISPRi demonstrates that it is not strand-specific and highlights the controls and locus understanding required to properly interpret results from CRISPRi interventions.
Project description:BACKGROUND:The non-translated regions at the genome ends of RNA viruses serve diverse functions and can exhibit various levels of nucleotide (nt) heterogeneity. However, the extent of nt heterogeneity at the extreme termini of Citrus tristeza virus (CTV) genomes has not been comprehensively documented. This study aimed to characterize two widely prevalent CTV genotypes, T36-CA and T30-CA, from California that have not been sequenced or analyzed substantially. The information obtained will be used in our ongoing effort to construct the infectious complementary (c) DNA clones of these viruses. METHODS:The terminal nts of the viral genomes were identified by sequencing cDNA clones of the plus- and/or minus-strand of the viral double-stranded (ds) RNAs generated using 5' and 3' rapid amplification of cDNA ends. Cloned cDNAs corresponding to the complete genome sequences of both viruses were generated using reverse transcription-polymerase chain reactions, sequenced, and subjected to phylogenetic analysis. RESULTS:Among the predominant terminal nts identified, some were identical to the consensus sequences in GenBank, while others were different or unique. Remarkably, one of the predominant 5' nt variants of T36-CA contained the consensus nts "AATTTCAAA" in which a highly conserved cytidylate, seen in all other full-length T36 sequences, was absent. As expected, but never systematically verified before, unique variants with additional nt (s) incorporated upstream of the 5' terminal consensus nts of T36-CA and T30-CA were also identified. In contrast to the extreme 5' terminal nts, those at the extreme 3' termini of T36-CA and T30-CA were more conserved compared to the reference sequences, although nt variants were also found. Notably, an additional thymidylate at the extreme 3' end was identified in many T36-CA sequences. Finally, based on pairwise comparisons and phylogenetic analysis with multiple reference sequences, the complete sequences of both viruses were found to be highly conserved with those of the respective genotypes. CONCLUSIONS:The extreme terminal nts in the T36-CA and T30-CA genomes were identified, revealing new insights on the heterogeneity of these CTV genomic regions. T36-CA and T30-CA were the first and the second genotypes, respectively, of CTV originating from California to be completely sequenced and analyzed.
Project description:Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) is the causal agent of devastating epidemics that changed the course of the citrus industry. Adapted to replicate in phloem cells of a few species within the family Rutaceae and to transmission by a few aphid species, CTV and citrus probably coevolved for centuries at the site of origin of citrus plants. CTV dispersal to other regions and its interaction with new scion varieties and rootstock combinations resulted in three distinct syndromes named tristeza, stem pitting and seedling yellows. The first, inciting decline of varieties propagated on sour orange, has forced the rebuilding of many citrus industries using tristeza-tolerant rootstocks. The second, inducing stunting, stem pitting and low bearing of some varieties, causes economic losses in an increasing number of countries. The third is usually observed by biological indexing, but rarely in the field. CTV polar virions are composed of two capsid proteins and a single-stranded, positive-sense genomic RNA (gRNA) of approximately 20 kb, containing 12 open reading frames (ORFs) and two untranslated regions (UTRs). ORFs 1a and 1b, encoding proteins of the replicase complex, are directly translated from the gRNA, and together with the 5' and 3'UTRs are the only regions required for RNA replication. The remaining ORFs, expressed via 3'-coterminal subgenomic RNAs, encode proteins required for virion assembly and movement (p6, p65, p61, p27 and p25), asymmetrical accumulation of positive and negative strands during RNA replication (p23), or suppression of post-transcriptional gene silencing (p25, p20 and p23), with the role of proteins p33, p18 and p13 as yet unknown. Analysis of genetic variation in CTV isolates revealed (1) conservation of genomes in distant geographical regions, with a limited repertoire of genotypes, (2) uneven distribution of variation along the gRNA, (3) frequent recombination events and (4) different selection pressures shaping CTV populations. Measures to control CTV damage include quarantine and budwood certification programmes, elimination of infected trees, use of tristeza-tolerant rootstocks, or cross protection with mild isolates, depending on CTV incidence and on the virus strains and host varieties predominant in each region. Incorporating resistance genes into commercial varieties by conventional breeding is presently unfeasible, whereas incorporation of pathogen-derived resistance by plant transformation has yielded variable results, indicating that the CTV-citrus interaction may be more specific and complex than initially thought. A deep understanding of the interactions between viral proteins and host and vector factors will be necessary to develop reliable and sound control measures.
Project description:Several pieces of evidence suggest that small RNA degradation products together with tRNase ZL appear to form another layer of the whole gene regulatory network. The degraded RNA such as a 5'-half-tRNA and an rRNA fragment function as small guide RNA (sgRNA) to guide the enzyme to target RNA. We were curious whether there exist RNAs in plasma that can function as sgRNAs for tRNase ZL, whether these RNAs are working as signaling molecules between cells to fulfill physiological roles, and whether there are any differences in plasma sgRNA species and levels between normal and pathological conditions. Here, we analyzed small plasma RNAs from three healthy persons and three multiple myeloma patients for potential sgRNAs by deep sequencing. We also examined small RNAs from peripheral blood mononuclear cells (PBMC) of three healthy persons and three myeloma patients and from various cultured human cell lines for sgRNAs. We found that read-number distribution patterns of plasma and PBMC RNAs differ between persons in the range of 5-40 nt and that there are many RNA species that exist significantly more or less abundantly in the plasma or PBMC of the myeloma patients than those of the healthy persons. Furthermore, we found that there are many potential sgRNAs in the 5-40-nt RNAs and that, among them, a 31-nt RNA fragment derived from 94-nt Y4-RNA, which can function as a 5'-half-tRNA-type sgRNA, is overwhelmingly abundant in the plasma of 2/3 of the examinees. These observations suggest that the gene regulatory network via tRNase ZL and sgRNA may be extended intercellularly.
Project description:Cas9 nucleases can be programmed with single guide RNAs (sgRNAs) to mediate gene editing. High CRISPR/Cas9-mediated gene knockout efficiencies are essential for genetic screens and critically depend on the properties of the sgRNAs used. The specificity of an sgRNA is defined by its targeting sequence. Here, we discovered that two short sequence motifs at the 3' end of the targeting sequence are almost exclusively present in inefficient sgRNAs of published sgRNA-activity datasets. By specific knock-in of sgRNA target sequences with or without these motifs and quantitative measurement of knockout efficiency, we show that the presence of these motifs in sgRNAs per se results in a 10-fold reduction of gene knockout frequencies. Mechanistically, the cause of the low efficiency differs between the two motifs. These sequence motifs are relevant for future sgRNA design approaches and studies of Cas9-DNA interactions.