RNA Silencing Research
The basic pathway of RNA silencing is similar in many animals and plants. An RNA dependent RNA polymerase generates double stranded RNA from a single stranded precursor. The double stranded RNA is cleaved into 21-25nt fragments by nucleases of the Dicer family. These RNA fragments, known as mico (mi)RNAs and short interfering (si)RNAs, are separated into single stranded molecules and one of the two strands then becomes bound to an Argonaute nuclease. This RNA then forms base paired duplex structures with longer RNAs and thereby guides Argonaute to its target. Argonautes are sometimes referred to as Slicer. RNA silencing is therefore a combination of Dicing and Slicing.
We describe the mechanism of RNA silencing as part of a DEFRA-funded desk study on risks associated with RNA silencing in transgenic plants: http://www.plantsci.cam.ac.uk/Baulcombe/Schwach/.
 Small interfering (si)RNAs are double-stranded RNA molecules, 20-25 nucleotides in length, with two unpaired bases at the 3’ ends of each strand. They are the mediators of sequence specificity in RNA silencing. |
 When siRNAs are bound to an Argonaute protein they can degrade targeted messenger RNA |
We are currently using three model systems in our research: Arabidopsis is a model higher plant with a completely sequenced genome; tomato is a model crop plant whose genome is being sequenced and for which there are extensive germplasm resources; Chlamydomonas reinhardtii is a model unicellular alga with a completely sequenced genome, a silencing system that is similar in many respects to that of higher plants. We have several projects that are designed to unravel the mechanisms of silencing in these three plants.
 Our three model plants |
Recent and key(*) publications on RNA silencing
Baulcombe, D. (2007). "Amplified Silencing." Science 315: 199-200.
Baumberger, N., C.-H. Tsai, et al. (2007). "The Polerovirus silencing suppressor PO targets argonaute proteins for degradation." Current Biology 17: 1609-1614
*Baurle, I., L. M. A. Smith, et al. (2007). "Widespread role for the flowering time regulators FCA and FPA in siRNA-directed chromatin silencing." Science (in the press)
Boccara, M., A. Sarazin, et al. (2007). "New approaches for the analysis of Arabidopsis thaliana small RNAs." Biochimie doi:10.1016/j.biochi.2007.04.011.
Hernandez-Pinzon, I., N. E. Yelina, et al. (2007). "SDE5, the putative homologue of a human mRNA export factor, is required for transgene silencing and accumulation of trans-acting endogenous siRNA." The Plant Journal 50: 140-148.
*Molnar, A., F. Schwach, et al. (2007). "miRNAs control gene expression in single cell alga Chlamydomonas reinhardtii." Nature 447(doi:10.1038/nature05903): 1126-1129.
Smith, L. M., O. Pontes, et al. (2007). "A novel SNF2 protein associated with nuclear RNA silencing and spread of a silencing signal between cells in Arabidopsis." Plant Cell 19: doi: 10.1105/tpc.107.051540
*Baulcombe, D. (2006). "Short Silencing RNA: The Dark Matter of Genetics?" Cold Spring Harbor Symposia on Quantitative Biology LXXI: 13-20.
*Herr, A. J., A. Molnar, et al. (2006). "Defective RNA processing enhances RNA silencing and accelerates flowering in Arabidopsis." Proceedings Of The National Academy Of Sciences Of The United States Of America 103(41): 14994-15001.
*Baumberger, N. and D. C. Baulcombe (2005). "Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits micro RNAs and short interfering RNAs." Proceedings Of The National Academy Of Sciences Of The United States Of America 102(33): 11928-11933.
Bayne, E. H., D. V. Rakitina, et al. (2005). "Cell-to-cell movement of Potato Potexvirus X is dependent on suppression of RNA silencing." Plant Journal 44: 471-482.
*Herr, A. J., M. B. Jensen, et al. (2005). "RNA polymerase IV directs silencing of endogenous DNA." Science 308: 118-120.
Schwach, F., F. E. Vaistij, et al. (2005). "An RNA-dependent RNA-polymerase prevents meristem invasion by Potato virus X and is required for the activity but not the production of a systemic silencing signal." Plant Physiology 138: 1842-1852.
*Baulcombe, D. (2004). "RNA silencing in plants." Nature 431(7006): 356-363.
Lu, R., I. Malcuit, et al. (2003). "High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance." EMBO Journal 22(21): 5690-5699.
Voinnet, O., S. Rivas, et al. (2003). "An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus." Plant Journal 33(5): 949-956.
*Hamilton, A. J., O. Voinnet, et al. (2002). "Two classes of short interfering RNA in RNA silencing." EMBO Journal 21(17): 4671-4679.
*Jones, L., F. Ratcliff, et al. (2001). "RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires Met1 for maintenance." Current Biology 11: 747-757.
Ratcliff, F., A. M. Martin-Hernandez, et al. (2001). "Tobacco rattle virus as a vector for analysis of gene function by silencing." Plant Journal 25(2): 237-245.
*Dalmay, T., A. J. Hamilton, et al. (2000). "An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus." Cell 101(5): 543-553.
Baulcombe, D. C. (1999). "Fast forward genetics based on virus-induced gene silencing." Current Opinion In Plant Biology 2(2): 109-113.
Hamilton, A. J. and D. C. Baulcombe (1999). "A species of small antisense RNA in post-transcriptional gene silencing in plants." Science 286: 950-952.
Jones, L., A. J. Hamilton, et al. (1999). "RNA-DNA interactions and DNA methylation in post-transcriptional gene silencing." Plant Cell 11: 2291-2302.
Ratcliff, F. G., S. A. MacFarlane, et al. (1999). "Gene silencing without DNA. RNA-mediated cross-protection between viruses." Plant Cell 11(7): 1207-16.
Voinnet, O., Y. M. Pinto, et al. (1999). "Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses." Proceedings Of The National Academy Of Sciences Of The United States Of America 96(24): 14147-14152.
Ruiz, M. T., O. Voinnet, et al. (1998). "Initiation and maintenance of virus-induced gene silencing." Plant Cell 10: 937-946.
*Voinnet, O., P. Vain, et al. (1998). "Systemic spread of sequence-specific transgene RNA degradation is initiated by localised introduction of ectopic promoterless DNA." Cell 95(2): 177-187.
*Ratcliff, F., B. D. Harrison, et al. (1997). "A similarity between viral defense and gene silencing in plants." Science 276: 1558-1560.
Baulcombe, D. C. (1996). "RNA as a target and an initiator of post-transcriptional gene silencing in transgenic plants." Plant Molecular Biology 32(1-2): 79-88.
Mueller, E., J. E. Gilbert, et al. (1995). "Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing." Plant J. 7(6): 1001-1013.
Current projects in the laboratory include:
Characterisation of the complete miRNA and siRNA profile in our three model plants.
These projects have now become possible because recently developed high throughput sequencing technologies have made it possible to determine millions of separate siRNA or miRNA sequences for low cost. We are using this approach to determine which regions of the three model system genomes are transcribed into precursors of siRNA and miRNAs. We can also use this technology to compare siRNA and miRNA profiles of plants subjected to different treatments or in different mutants. There are many thousands of loci with the potential to generate siRNAs and miRNAs in Arabidopsis and Chlamydomonas.
 This is a view from the Arabidopsis genome browser showing the alignment of siRNA and miRNA sequences with 200 kilobase pairs of the genome sequence. Each small arrow represents one siRNA or miRNA sequence and the blue boxes are long RNAs including messenger RNA. The siRNAs and miRNAs clearly align to specific regions of the genome that we refer to as siRNA or miRNA loci. A computational challenge is to develop ways to define these loci, to compare their representation in different datasets and to classify them in terms of their corresponding genomic features. |
The analysis of these large sequence databases requires expertise in computational biology and considerable computational input. For that reason we are establishing a computational biology group in the Plant Science Department in Cambridge who will work with us on these and other projects including systems modelling.
The diversity of RNA silencing pathways.
There are several RNA silencing pathways that are variations on the basic mechanism described above. One approach to understanding this diversity is through the Argonaute proteins. In Arabidopsis there is a ten member multigene family for Argonaute proteins and we are investigating their expression patterns, mutant phenotypes and the characteristics of bound short RNAs. The emerging picture is that Argonaute targets different types on longer RNA because they are not all associated with the same type of 21-25nt RNA. This diversity of Argonaute proteins allows RNA silencing to have many different effects on genetic regulation.
 A phylogenetic tree illustrating the relationship between the ten Argonaute proteins encoded in the Arabidopsis genome. |
RNA silencing in epigenetics.
In plants, fungi and perhaps in animals, a consequence of RNA silencing is epigenetic modification of DNA and chromatin and, consequently, modification of chromosome structure and function. These epigenetic modifications are often associated with changes in gene expression – normally silencing but also with gene activation. We are interested in the mechanism of this epigenetic silencing and its effects on chromosome structure and aspects of growth development and evolution of plants. An intriguing possibility, suggested by our work on RNA-mediated epigenetic silencing, is that heritable epigenetic changes could be mediated by siRNAs that are induced in response to disease, stress or other external stimuli.
Heritable silencing: these plants are viewed under UV light. The progenitor plants have a transgene that causes them to fluoresce green. We silenced this gene in these progenitor plants and then observed that the silencing persisted in several generations of progeny plants. In the absence of green fluorescence these plants fluoresced red due to their chlorophyll. |
One set of projects involves RNA polymerase IV. This protein is similar to the well characterised Polymerases I-III. However, Pol IV is distinct in that it is specifically required for biogenesis of RNA in RNA silencing pathways including those involved in epigenetic processes. We are currently testing the biochemical activity and nature of proteins associated with PolIV and characterising the genetic loci that are affected by PolIV mediated silencing.
An Arabidopsis nucleus reacted with PolIV antibodies (green) or a stain for DNA (DAPI- blue). The images show PolIV is present in localised regions of the chromatin and in a structure in the nucleolus (the region that does not stain for DNA). |
An intercellular and systemic signal of RNA silencing.
Localised induction of RNA silencing may lead to silencing in adjacent cells or even in distant parts of the plant. We infer that the mobile signal is likely an RNA molecule because its effects are highly sequence specific. We are currently using transgene systems to identify the nature of the silencing signal through the characterisation of RNA in phloem tissue and through the analysis of mutants to identify genes required for the spread of silencing. Having characterised the signal we shall assess whether endogenous RNA silencing molecules are also mobile and part of a previously uncharacterised signalling system.
 A signal of RNA silencing is moving out of the main vein of a leaf causing silencing of a green fluorescent protein transgene. The red chlorophyll fluorescence shows through in the silenced regions of the leaf. |
RNA silencing networks.
Long RNA molecules that are targeted by an siRNA-guided Argonaute protein may be degraded or translationally suppressed. They may also be stimulated to become a source of secondary siRNAs. The secondary siRNAs may also guide Argonaute proteins and stimulate further rounds of siRNA production. We are using computer modelling approaches to investigate the potential for these cascades or networks of siRNA and miRNA and we will follow up this analysis with experimental testing of the predicted networks.
 A representation of networks in which siRNAs and miRNAs are represented as red symbols and their precursors and targets as blue or yellow symbols. Such networks could be used by the cell to integrate the expression of many genes. |
RNA silencing and natural genetic variation.
Many of the siRNA loci are associated with transposons and repeated sequence structures in the genome. These genetic elements are highly polymorphic between genotypes and exhibit significant variation between related species. It is conceivable that part of the phenotypic variation between related genotypes or species is due to the effects of these polymorphic siRNA loci. We are exploring this possibility in tomato with the aim of using siRNAs as markers in breeding programmes. It may also be possible to select for variation in small RNA loci or to manipulate the expression of these loci in order to achieve improvement in the crop.
 Phenotypic variation in relatives of tomato. Some of this variation may be due to the effects of siRNAs (picture from Tanksley and McCouch Science 277 (1997) 1063-1066) |