Keystone Symposia RNA Silencing – Mechanism, Biology and Application
The snow may have been light, but there was a blizzard of non-coding RNA wisdom dumping throughout last month’s Keystone RNA Silencing: Mechanism, Biology, and Application Conference January 14th-19th. EpiGenie guest correspondent, David Cummins made the trip up from Australia, where he’s a postdoc working with RNAi technologies at the CSIRO.
The Making of a MicroRNA
V. Narry Kim, Seoul National University, South Korea
Narry Kim talked about the role of uridylation and its effect on Dicer processing. The example discussed was the role of Lin28 in downregualting let-7 miRNA by inducing uridylation of let-7 precursor (pre-let-7). TUTase4 (TUT4) was identified as the enzyme responsible for uridylyl transferase. TUT4 can execute mono-uridylation as well as oligo-uridylation. Oligo-uridylation results in the inhibition of Dicer processing, however, little was known about the effect of mono-uridylation on Dicer processing. Narry Kim went on to demonstrate that the presence of one, two or three U’s at the 3’ end of the miRNA, resulted in the same Dicer product, suggesting that Dicer processing can not only be 3’ end specific, but also 5’ end specific. Narry Kim also showed that it is the presence of a 5’ phosphate group in which Dicer recognises for 5’ end processing. If a 5’ end phosphate is not present, dicer processing returns to 3’ dicing.
Dicer and Beyond: Mechanisms of Regulatory RNA Processing and Targeting
Jennifer A Doudna, University of California, Berkeley, USA
Jennifer Doudna discussed in detail, structural insights into RNA processing by the human RISC loading complex. Using single-particle EM analysis, the structure of the human Dicer was shown to form an L-shape. At the N-terminal, the DExH/D domain interacts with the RNA binding protein (TRBP), whereas the C-terminal catalytic domains are proximal to AGO2 within the RISC loading complex (RLC). Her analysis indicates that TRBP is flexibly bound to the Dicer DExH/D domain. The location and flexibility of TRBP, allows access to the PAZ domain of AGO2 in her model, suggesting a role for TRBP in bridging the steps between release of the siRNA by Dicer and loading of the duplex into AGO2. Binding by TRBP may allow the siRNA intermediate to stay associated with the RLC after release from Dicer and may also help in orientation of the siRNA for AGO2 loading. Furthermore, it may be predicted that TRBP is important for proper strand selection during RISC loading, much like the roles of Dicer-2 and R2D2 (a TRBP homolog) in Drosophila.
Herpesvirus saimiri U RNAs Affect Expression of Cellular micoRNAs in latently Infected T cells.
Demian E. Cazalla, Yale University, USA
Demian discussed the role of Herpisvirus saimiri (HSV) Sm-class small nuclear RNAs, named HSURs for Herpesvirus saimiri U RNAs. These are the most abundantly expressed gene products in HSV transformed cells. HSUR 1 and 2 are the most conserved of all HSURs and are shown to associate with host miRNAs in virally transformed cells. Demian present data indicating the HSUR-1 binds directly to miRNAs through predicted complementary sequences and this is in a ARE (AU rich region) dependent manner. In addition, HSURs 1 and 2 affect the levels of miR-27 (both miR-27a and miR-27b) and its targets in virally transformed cells.
Highlights from Small RNA Therapeutic Talks
Premlata Shanker, Yale School of Medicine
Dr. Shanker was unable to present, instead Priti Kumar spoke on her behalf. This talk was about discovering a new delivery method for siRNAs and treatment of HIV infection. A scFvCD7-9R/siRNA complex targeting the cellular receptor CCR or HIV targets Vif and Tat were development. It was shown these complexes had no toxicity and did not induce the IFN or TLR response. Significantly, the antiviral scFvCD7-9R/siRNA complexes could suppress an established HIV infection in mice.
Daniel G. Anderson, MIT
Delivery, Delivery, Delivery. This was the message from Daniel. Using high throughput development of biomaterials, Daniel has developed and tested a wide range of products for siRNA delivery for the treatment of various cancers. He showed using a current nanoparticle formulation targeting the Claudin 3 (CLND3) in ovarian cancer, tumour growth was suppressed. Daniel also gave another example of targeting kinesin spindle protein for liver cancer. A next generation library has also been developed to improve the potency of the current nanoparticles. Results to date have shown a 2-3 order of magnitude in siRNA delivery efficiency in mice.
Small RNAs are Mobile and Trigger DNA Methylation in Distant Tissue
Attila Molnar, University of Cambridge, UK
One of several plant talks during the conference meeting. Attila Molnar discussed that RNAi silencing is not cell-autonomous in flowering plants. Initial studies showed that a plant expressing small RNAs targeting GFP, when grafted to a GFP expressing plant resulted in the destruction of the GFP signal. This lead Molnar to identify if mobile silencing elements exist in plants. Through a range of grafting experiment using Arabidopsis thaliana mutants lacking Dicers 2, 3 and 4, Molnar was able to swap the shoot and roots of mutant and wild type plants lacking these key genes. He identified small RNAs in both the shoot of Dicer knockout grated to wt roots, in addition to small RNAs in the roots of dicer knockouts grafted to WT shoots, indicating that these small RNAs must have originated form the WT part of the plant. Molnar went on to examine the content of the small RNAs. It was observed that the majority of small RNAs were 24nt products, originating form Dicer-3. However, in addition to 24nt siRNAs, both 2 nt and 23nt siRNAs are also mobile. Therefore mobility is not an exclusive feature of one size class of small RNAs. In conclusion, Molnar also stated that these small mobile RNAs can direct methylation in distant tissues, via long distance epigenetic signalling.
MicroRNAs and Stem Cells
Elaine Fuchs, Howard Hughes Medical Institute, Rockefeller University, USA
Elaine discussed the role of microRNAs in stem cells, using skin as a model. Elaine demonstatated that stem cells isolated from mice hair follicles resulted in hair growth on hairless mice following injection of the stem cells. Elain asked the question if microRNAs were involved in this process. MicroRNAs were sequenced from the epidermis and the hair follicle and shown to be differentially expressed. In situ hybridization was also used to determine the location of several miRNAs. In the case of miR-203, its expression pattern varied depending on the developmental stage of the skin. Another microRNA, miR-205 is shown to be highly expressed in normal skin, yet severely downregulated in human epithelial tumours. An additional experiment using mice that are defective for DGCR8 within their skin, demonstrated that miRNAs are important for hair follicle development. Elaine’s research has implications for skin disorders and cancers.