Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression Here we investigate the requirements for structure and delivery of the interfering RNA To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference The effects of this interference were evident in both the injected animals and their progeny Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III–like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3 ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA–protein complex.

/pdf/rna-interference-is-mediated-by-21-and-22-nucleotide-rnas-1tlpza9thy.pdf

RNA interference is mediated by 21- and 22-nucleotide RNAs

Two small temporal RNAs (stRNAs), lin-4 and let-7, control developmental timing in Caenorhabditis elegans. We find that these two regulatory RNAs are members of a large class of 21- to 24-nucleotide noncoding RNAs, called microRNAs (miRNAs). We report on 55 previously unknown miRNAs in C. elegans. The miRNAs have diverse expression patterns during development: a let-7 paralog is temporally coexpressed with let-7; miRNAs encoded in a single genomic cluster are coexpressed during embryogenesis; and still other miRNAs are expressed constitutively throughout development. Potential orthologs of several of these miRNA genes were identified in Drosophila and human genomes. The abundance of these tiny RNAs, their expression patterns, and their evolutionary conservation imply that, as a class, miRNAs have broad regulatory functions in animals.

/pdf/an-abundant-class-of-tiny-rnas-with-probable-regulatory-vtv94uvmvm.pdf

An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans

In a diverse group of organisms that includes Caenorhabditis elegans, Drosophila, planaria, hydra, trypanosomes, fungi and plants, the introduction of double-stranded RNAs inhibits gene expression in a sequence-specific manner. These responses, called RNA interference or post-transcriptional gene silencing, may provide anti-viral defence, modulate transposition or regulate gene expression. We have taken a biochemical approach towards elucidating the mechanisms underlying this genetic phenomenon. Here we show that 'loss-of-function' phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs. This coincides with a marked reduction in the level of cognate cellular messenger RNAs. Extracts of transfected cells contain a nuclease activity that specifically degrades exogenous transcripts homologous to transfected double-stranded RNA. This enzyme contains an essential RNA component. After partial purification, the sequence-specific nuclease co-fractionates with a discrete, approximately 25-nucleotide RNA species which may confer specificity to the enzyme through homology to the substrate mRNAs.

An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells

Posttranscriptional gene silencing (PTGS) is a nucleotide sequence-specific defense mechanism that can target both cellular and viral mRNAs. Here, three types of transgene-induced PTGS and one example of virus-induced PTGS were analyzed in plants. In each case, antisense RNA complementary to the targeted mRNA was detected. These RNA molecules were of a uniform length, estimated at 25 nucleotides, and their accumulation required either transgene sense transcription or RNA virus replication. Thus, the 25-nucleotide antisense RNA is likely synthesized from an RNA template and may represent the specificity determinant of PTGS.

/pdf/a-species-of-small-antisense-rna-in-posttranscriptional-gene-4x7aphjpq5.pdf

A species of small antisense RNA in posttranscriptional gene silencing in plants.

Gene silencing in plants, in which an endogenous gene is suppressed by introduction of a related transgene, has been used for crop improvement. Observations that viruses are potentially both initiators and targets of gene silencing suggested that this phenomenon may be related to natural defense against viruses. Supporting this idea, it was found that nepovirus infection of nontransgenic plants induces a resistance mechanism that is similar to transgene-induced gene silencing.

https://science.sciencemag.org/content/sci/276/5318/1558.full.pdf

A similarity between viral defense and gene silencing in plants.

Previously, it was shown that the upper leaves of plants infected with nepoviruses and caulimoviruses are symptom free and contain reduced levels of virus. These leaves are said to be recovered. Recovery is associated with RNA-mediated cross-protection against secondary virus infection. Here, by analyzing plants infected with viruses that are quite distinct from the nepovirus or caulimovirus groups, we demonstrate that this RNA-mediated defense is a general response to virus infection. Upon infection with a tobravirus, plants exhibited RNA-mediated cross-protection and recovery, as occurs in nepovirus-infected plants. However, upon infection with a potexvirus, plants exhibited RNA-mediated cross-protection without recovery. In both instances, a transient gene expression assay showed that RNA-mediated cross-protection was functionally equivalent to post-transcriptional gene silencing. Combined, these data provide direct evidence that post-transcriptional gene silencing of nuclear genes is a manifestation of a natural defense mechanism that is induced by a wide range of viruses.

Gene silencing without DNA. rna-mediated cross-protection between viruses

RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires Met1 for maintenance

Disclosed are nucleic acid vectors which comprise: (a) a transfer nucleotide sequence comprising (i) a plant active promoter, operably linked to (ii) a recombinant tobacco rattle virus (TRV) cDNA (preferably derived from TRV RNA2) which includes at least cis acting elements permitting replication of the cDNA; a subgenomic promoter operably linked to a sequence encoding a TRV coat protein; and a heterologous nucleotide sequence which is foreign to the virus;(b) border sequences which permit the transfer of the transfer nucleotide sequence into a plant genome Such vectors may be used as expression vectors or for achieving viral induced gene silencing (VIGS) of a target gene, wherein the heterologous nucleotide sequence is a targeting sequence which corresponding to that gene Example vectors include pTV00 and vectors which are derived from PTV00 and have the characteristics thereof Also disclosed are associated processes, methods, viruses or viral particle, kits, host cells and plant tissues

Frank G. Ratcliff

Papers

A similarity between viral defense and gene silencing in plants.

Gene silencing without DNA. rna-mediated cross-protection between viruses

RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires Met1 for maintenance

Recombinant plant viral vectors

Activation of osmotically-activated potassium transporters after injection of mRNA from A6 cells in Xenopus oocytes