Gene Silencing by Small RNAs
The Keystone Symposia on Gene Silencing by Small RNAs was held February 7-12th in Vancouver, British Columbia. Our guest correspondent for this event was locally based Athanasios Zovoilis from the Genome Sciences Center who was able to avoid all of Vancouver’s natural wonders long enough to share his report with us. Check out some of the meeting’s highlights:
As the name implies, the meeting focused on gene silencing from the prospective of small RNAs. The conference program covered practically the entire range of gene silencing, from prokaryotes to eukaryotes, from plants to mammals, from structure to function. Perhaps this was the main benefit for conference participants, since they could track so many different organisms at the same time and follow the most recent advances in the field to get a more holistic view of the conserved mechanisms that govern gene silencing via small RNAs.
Besides hearing the latest research from highly respected speakers (some of which have practically set the foundations of this new field), the conference also offered a great chance for discussion between attendees during and after the sessions.
The main conclusion… there is still a lot to be found out there. Most speakers and participants agree that what we see at the moment may be just the tip of the iceberg regarding the potential of small RNAs to control cellular functions.
Inevitably, the conference included a lot about miRNAs, crRNAs and piRNAs, since at the moment they comprise some from the most intensively and widely tested small RNAs. However, a significant number of talks, especially those that were invited short talks from already presented posters, extended to totally new and maybe unexpected functions of the RNA silencing machinery like alternative splicing.
In addition, the presented posters included a lot of currently unpublished work that definitely will make the headlines of many good publications in near future. Some posters that triggered a lot of discussion included evidence that:
- Dicer might be dispensable for asymmetric RISC loading in mammals
- Small RNAs do play a role in DNA break repair
- piRNA may mediate transgenerational inheritance of an acquired trait as well as small RNAs in C.elegans may also play a role in transgenerational epigenetic inheritance. (This point really triggered a lot of discussion among the participants.)
A portion of these talks are described in detail below:
CRISPR-derived RNAs in Immune Response
Jennifer Doudna, UC Berkeley
In her talk, Dr Doudna focused on small CRISPR (clustered regularly interspaced short palindromic repeats)-derived RNAs (crRNAs). crRNAs based immune systems in prokaryotes protect their genomes from invading foreign genetic elements and Doudna’s main question was how this system avoids self targeting. It seems that a short conserved sequence (PAM) plays an important role in this together with members of the Cascade (CRISPR-associated complex).
Doudna presented an experiment testing the importance of one of these members, CasA for DNA binding and its role in self vs non-self recognition. She found that DNA binding occurs immediately upon CasA incorporation, which in turn, raised the question: why is CasA required for dsDNA binding? Elucidation of CasA crystal structure suggested that CasA is sitting at the base of the complex and near the location of the seed. By testing cascade assembly with CasA mutants for loop L1 of CasA she showed that CasA L1 interacts with the target strand of the conserved sequence PAM, which is critical for CRISPR system.
Human Nuclear Argonaute Couples Chromatin-mediated Silencing to Regulation of Alternative Splicing.
Annick Harel-Bellan, CEA
Dr Bellan presented an interesting correlation between RNAi processes and alternative splicing. Based on previous work, in which she had shown that alternative exons of the CD44 gene are distinguished by H3K9 tri-methylation marks, now she provided a first characterization of the mechanism underlying this marking. She presented data that revealed association of HP1γ and members of the spliceosome with AGO1 and AGO2 in this process. In addition, RNAs that map to exon extremities were mentioned also to be involved. The process is DICER and HP1γ dependent and experiments were able to show that this machinery (termed RNAi-SPLITS) can affect H3K9 trimethylation, alternative splicing of CD44 and PNA Pol II elongation rate.
Regulation of MicroRNA
Narry Kim, Seoul National University
Dr Kim described her group’s current work regarding the role of mono-uridylation (Mono-U) of precursor miRNA on further processing towards mature miRNA. As she mentioned, in contrast to Poly-uridylation that is well studied, it is currently unknown which the impact of Mono-U is on final miRNA levels, and for this reason she focused on let-7 as an example. In this case, mono-U of the one 3’ overhang nucleotides of the pre-miRNA promotes Dicer processing of pre-let7.
In order to reveal which enzyme is involved in this process she tested various members of the human TUTases family through immuno-purification and in vitro uridylation assays. TUTases 2,4 and 7 (TUT 7 and TUT4 are specific for UTP) were shown to be good candidates for mono-U, since the can cause mono-U of pre-let7a-1 and promote let-7 biogenesis. From her presented data it seemed that TUT7 was the enzyme primarily doing this job. Subsequently, it was shown that let7 mono-U was TUT-2/4/7 dependent also in vivo. Other examples included miR-105. Based on these, the speaker concluded that we are in front of a new type of modification with functional relevance in miRNA biogenesis, mono-uridylation.
Silencing Factor Links Heterochromatin Transcription to RNA Degradation
Marc Bühler, FMI
Dr Bühler announced the identification of an already well known silencing factor with RNA degradation at heterochromatin regions. This is Swi6(HP1). Bühler tested heterochromatin regions other than centromers. Firstly, he showed that RNA transcribed from these regions (which has already been shown by other studies to apply even at these regions) is not translated into proteins, even when the exosome is blocked, mainly due to binding via HP1. Swi6 was shown to bind the RNA via the hinge region. He showed that Swi6-RNA binding is dispensable for the chromosome structural integrity.
Through the chromodomain the protein binds also chromatin and Bühler showed that the amount of Swi6 bound to H3K9 me is reduced as the RNA binding increases. To this end, he proposed a model, in which RNA transcribed by heterochromatin regions is eliminated through binding via HP1 and change in HP1 structural conformation, release of HP1 from chromatin (H3K9me) and transfer to exosome for degradation.
Systems Approaches to miRNAs
David Bartel, MIT
Dr Bartel started his talk by setting the question why and how miR* miRNAs get discriminated from the respective miRNAs. To answer this question he performed experiments over-expressing C. elegans miRNAs (pri-miRNAs) in human cells, that normally do not get further processed (the situation is much better when trying to do this with Drosophila miRNA primary transcripts). Based on these findings he questioned whether there are other determinants at the regions flanking processing sites by Drosha and related enzymes that affect primary transcript processing.
He described a strategy to elucidate such a sequence determinant by employing functional and non functional pri-miRNA variants with their ends closed to a circular conformation and subsequent massive parallel sequencing (splint ligation paired end sequencing). Based on this he identified a new CNNC motif at the upstream flanking region important for processing of many miRNAs. This motif was found to be present also in Drosophila but not C.elegans miRNA transcripts, which can explain the reason why only the first could be also successfully processed in human cells.
piRNA Pathway in Flies and Mice
Alexei Aravin, California Institute of Technology
Dr Aravin focused on the basic characteristics that define a piRNA and on a number of critical questions about their nature raised by their incredible heterogeneity such as what makes a piRNA a piRNA, whether we can create artificial piRNAs, and under which nucleotide sequence conditions. Using the piRNAs derived from traffic jam 3’UTR as a basis and inserting a GFP sequence into the cassette, he showed that active artificial piRNAs of any sequence can be produced if heterogenous sequence is inserted into a piRNA cluster. Then he questioned whether we could create transgenic piRNAs in a new genomic locus. To this end he showed that both local and long-range sequence environments define the generation of individual piRNAs from precursor transcripts.
Findings regarding gene silencing mechanisms in prokaryotes and plants may be very useful in other model systems although not always applicable. But even if they are different between organisms (as in the case presented by Dr. Bartel) they can help to identify new aspects of their regulation. In addition, the potential of small RNAs seems to extend to other aspects of gene expression regulation, such as alternative splicing, and players of the RNA silencing machinery seem to share a much wider range of functions than previously believed.
**EpiGenie would like to thank Dr. Athanasios Zovoilis from the Genome Sciences Center in Vancouver, Canada for providing this conference coverage.