Linfeng Huang

An atypical RNA polymerase involved in RNA silencing shares small subunits with RNA polymerase II

Genetic evidence indicates that plant-specific homologs of DNA-dependent RNA polymerase (Pol) II large subunits form Pol IV and Pol V complexes involved in small interfering RNA production and RNA-directed DNA methylation. Here we describe evidence that Pol V contains subunits shared with Pol II, but that RNA polymerase II subunit (RPB)-4 is missing from Pol V and that RPB5 is present as a Pol V–specific isomer, RPB5b. Pol V also has other proteins that are not present in Pol II, consistent with a role of this complex as an effector of silencing.

Efficient and specific gene knockdown by small interfering RNAs produced in bacteria

Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells and may be used for therapeutic purposes to knock down genes implicated in disease. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in Escherichia coli. This method relies on ectopic expression of p19, an siRNA-binding protein found in a plant RNA virus. When expressed in E. coli, p19 stabilizes an ∼21-nt siRNA-like species produced by bacterial RNase III. When mammalian cells are transfected by them, siRNAs that were generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene reproducibly knock down target gene expression by ∼90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes.

Heterogeneity in codon usages of sobemovirus genes

Summary.When conventional phylogenetic trees were built using 14 genome sequences of 9 sobemoviruses, two main lineages were apparent: monocot-infecting viruses and dicot-infecting viruses. To investigate whether members of the genus Sobemovirus originated from monocot hosts or from dicot hosts, we constructed relationship trees based on Relative Synonymous Codon Usage (RSCU) of the viruses. The RSCU relationship trees grouped the monocot-infecting and dicot-infecting viruses even better than the genome phylogenetic trees. The RSCU approach also enabled direct comparisons among viral and host species. When host species were added into the RSCU tree, the viral species clustered with the monocot hosts, indicating codon usage homologies to monocots. The stability of the RSCU tree was improved when RSCU values were calculated for individual viral open reading frames (ORFs). Most interestingly, the codon usages of the viral ORF-2 that encodes the replicase showed affinity to that of the plants whereas codon usages of the other viral ORFs were not relevant to the host species. All ORF-2s from 3 monocot viruses and 4 out of 6 dicot viruses had greater RSCU affinities to sequences of ORFs in monocot than to dicot hosts, possibly indicating that ORF-2, and therefore the replicase module of sobemovirus has a monocot origin.

Nucleotide bias of DCL and AGO in plant anti-virus gene silencing

Plant Dicer-like (DCL) and Argonaute (AGO) are the key enzymes involved in anti-virus post-transcriptional gene silencing (AV-PTGS). Here we show that AV-PTGS exhibited nucleotide preference by calculating a relative AV-PTGS efficiency on processing viral RNA substrates. In comparison with genome sequences of dicot-infecting Turnip mosaic virus (TuMV) and monocot-infecting Cocksfoot streak virus (CSV), viral-derived small interfering RNAs (vsiRNAs) displayed positive correlations between AV-PTGS efficiency and G+C content (GC%). Further investigations on nucleotide contents revealed that the vsiRNA populations had G-biases. This finding was further supported by our analyses of previously reported vsiRNA populations in diverse plant-virus associations, and AGO associated Arabidopsis endogenous siRNA populations, indicating that plant AGOs operated with G-preference. We further propose a hypothesis that AV-PTGS imposes selection pressure(s) on the evolution of plant viruses. This hypothesis was supported when potyvirus genomes were analysed for evidence of GC elimination, suggesting that plant virus evolution to have low GC% genomes would have a unique function, which is to reduce the host AV-PTGS attack during infections.

Production of highly potent recombinant siRNAs in Escherichia coli.

We recently invented a method to produce highly potent siRNAs in Escherichia coli, based on the serendipitous discovery that ectopic expression of p19, a plant viral siRNA-binding protein, stabilizes otherwise unstable bacterial siRNAs, which we named pro-siRNAs for prokaryotic siRNAs. We present a detailed protocol describing how to produce pro-siRNAs for efficiently knocking down any gene, beginning with the design of a pro-siRNA expression plasmid and ending with siRNA purification. This protocol uses one plasmid to co-express a recombinant His-tagged p19 protein and a long hairpin RNA containing sense and antisense sequences of the target gene. pro-siRNAs are isolated and purified using nickel beads and HPLC, using methods used to produce recombinant proteins. Once a pro-siRNA plasmid is obtained, production of purified pro-siRNAs takes a few days. The pro-siRNA technique provides a reliable and renewable source of siRNAs, and it can be implemented in any laboratory whose members are skilled in routine molecular biology techniques.

The complete genome sequence, organization and affinities of carrot red leaf virus

Summary.A sequence of 5723 nucleotides (GenBank accession number: AY695933) is reported for the RNA genome of an isolate of Carrot red leaf virus (CtRLV). The sequence is predicted to contain six large open reading frames and non coding sequences of 28 nucleotides at the 5′ end, 110 nucleotides at the 3′ end, and 215 nucleotides between the two main blocks of coding sequences. The 5′ coding region encodes two polypeptides with calculated molecular masses (Mr) of 28.6 kDa (P0) and 68.2 kDa (P1) that overlap in different reading frames. Circumstantially, the third ORF in the 5′ block is putatively translated by frameshift read-through to yield a polypeptide (P1 + P2) with a calculated Mr of 116.9 kDa. Frameshifting is predicted at a “shifty” sequence (GGGAAAC; nt 1523–1529) also found in most members of the genus Polerovirus. The C-terminal region of the 116.9 kDa polypeptide includes the consensus sequence for the viral RNA-directed RNA polymerase. The 3′ block of coding sequence defines three putative polypeptides of: 23.0 kDa (P3), 21.3 kDa (P4, in a different reading frame) and 77.2 kDa (P3 + P5, by read-through of P3) respectively. From the genome structure of CtRLV, it is suggested that this virus belongs to the genus Polerovirus, rather than either the genus Luteovirus or the genus Enamovirus.

Analyses on mutation patterns, detection of population bottlenecks, and suggestion of deleterious-compensatory evolution among members of the genus Potyvirus

Summary.Viruses of the family Potyviridae exhibited a robust single-nucleotide polymorphism profile at the between-species level, conforming to the neutral theory rule. However, the ratios of nonsynonymous to synonymous mutations (Ka/Ks) were relatively greater between-species than within-species in viral cistrons examined from members of the genus Potyvirus, indicating a relaxation on constraint. Judged by the McDonald and Kreitman’s test, the fixation frequencies for nonsynonymous mutations across the genomes of closely related potyviruses were greater than expected, suggesting population bottlenecks at speciation. These mutation patterns are best explained by a deleterious-compensatory model.

Milligram scale production of potent recombinant small interfering RNAs in Escherichia coli : KAUR et al.

Small interfering RNAs (siRNAs) are invaluable research tools for studying gene functions in mammalian cells. siRNAs are mainly produced by chemical synthesis or by enzymatic digestion of double‐stranded RNA (dsRNA) produced in vitro. Recently, bacterial cells, engineered with ectopic plant viral siRNA binding protein p19, have enabled the production of “recombinant” siRNAs (pro‐siRNAs). Here, we describe an optimized methodology for the production of milligram amount of highly potent recombinant pro‐siRNAs from Escherichia coli cells. We first optimized bacterial culture medium and tested new designs of pro‐siRNA production plasmid. Through the exploration of multiple pro‐siRNA related factors, including the expression of p19 protein, (dsRNA) generation method, and the level of RNase III, we developed an optimal pro‐siRNA production plasmid. Together with a high–cell density fed‐batch fermentation method in a bioreactor, we have achieved a yield of ~10 mg purified pro‐siRNA per liter of bacterial culture. The pro‐siRNAs produced by the optimized method can achieve high efficiency of gene silencing when used at low nanomolar concentrations. This new method enables fast, economical, and renewable production of pure and highly potent bioengineered pro‐siRNAs at the milligram level. Our study also provides important insights into the strategies for optimizing the production of RNA products in bacteria, which is an under‐explored field.