Silence Blog

Dicer-1 rules!

It has been more than a decade since Dicer was identified as the siRNA and miRNA-generating enzyme. Dicer proteins exist in nearly all eukaryotes and are well know for their ability to convert long, double-stranded RNA, or miRNA precursors (pre-miRNAs) into 21-23 nt long duplexes. Therefore, Dicer has been dubbed a ‘molecular ruler.’

In Drosophila melanogaster there are two Dicer proteins. Dicer-2—along with its partner R2D2—processes long, double-stranded RNA; whereas Dicer-1 cleaves miRNA precursors—stem-loop RNAs ∼70 nt long. What restricts Dicer-1 to processes primarily pre-miRNA into and not long double-stranded RNA?

Using classical biochemistry, Tsutsumi et al. (doi:10.1038/nsmb.2125) now show that fly Dicer-1 recognizes the single-stranded terminal loop of pre-miRNA through the Dicer-1 helicase domain, sensing terminal loop size and measuring the distance from the 3ʹ overhang to the terminal loop. Stem-loop RNAs whose stems are longer than those of canonical pre-miRNAs are poor Dicer-1 substrates. Consequently, fly Dicer-1 fails to process long, double-stranded RNAs. By measuring the distance from the 3´ end to the loop, Dicer-1 indeed earns the title ‘Molecular ruler.’ 

Carlos Fabián Flores-Jasso, Ph.D.

Posted by Elizabeth Bal at 17:13    Comments (0)

Yijun Qi joins Silence as co-Editor-in-Chief

We are delighted to announce the appointment of Yijun Qi to co-Editor-in-Chief of Silence, alongside Phillip Zamore.

Yijun is currently an Associate Investigator at the National Institute of Biological Sciences in Beijing, and his research focuses on dissecting the mechanisms of RNAi pathways and the biological functions of small RNAs in plants.

His lab has discovered miRNAs in single-celled green algae, identified a novel class of long miRNAs that direct DNA methylation, and provided insights into how small RNAs are sorted into plant Argonaute complexes.

Posted by Elizabeth Bal at 09:39    Comments (0)

HOT paper: A genome-scale shRNA resource for transgenic RNAi in Drosophila

Currently, transgenic flies expressing long inverted repeat transcripts that generate double-stranded RNA are available for nearly every Drosophila gene, allowing genome-wide RNAi screening. However, long double-stranded RNA does not efficiently trigger RNAi in the ovary germline. To achieve effective RNAi in the female germline, Ni et al. (Ni et al., Nature Methods 8, 405-407, 2011) systematically tested the efficiency of artificial microRNA precursors (shRNAs) to silence specific genes in the germline. The result of their studies was Valium22, a vector that combines optimized germline-specific expression promoter sequences with a modified backbone from pre-miR-1. They evaluated this construct by generating shRNA transgenic flies targeting four piRNA pathway genes. The RNAi phenotypes from each Valium22 line phenocopied the corresponding null mutants. Their results suggest that Valium22 will become the vector of choice for RNAi in the Drosophila germline. The authors are now generating a genome-wide shRNA collection of fly stocks targeting all 14,208 annotated Drosophila protein-coding genes.

Zhao Zhang

Posted by Elizabeth Bal at 10:37    Comments (0)

HOT paper: Structural basis for site-specific ribose methylation by box C/D RNA protein complexes

In a recent issue of Nature, Lin et al. {Lin et al., 2011, Nature, 469, 559-63}  reported the structure of box C/D ribonucleoprotein (RNP) in complex with its substrate, highlighting the mechanism behind site-specific ribose methylation. Box C/D RNP and H/ACA RNP belong to the small nucleolar RNP (snoRNP) family that methylates and pseudouridylates pre-rRNA respectively. In addition, the founding member of the family, U3 snoRNP, cleaves and liberates rRNA for ribosome assembly {Maxwell and Fournier, 1995, Annu Rev Biochem, 64, 897-934}. Intriguingly, the study by Lin et al. reveals that box C/D methylation bears some resemblance to RNA interference (RNAi), where sequence specific base pairing between RNA guide and substrate acts as the molecular ruler: the site of modification by C/D RNP occurs 5 base pairs upstream of the box D element. The first nucleotide of the guide RNA immediately upstream of the box D element is unpaired and subsequent nucleotides (especially base 1 to 5) interact extensively with the protein {Lin et al., 2011, Nature, 469, 559-63} much like the seed region of the guide in Argonaute {Parker et al., 2004, EMBO J, 23, 4727-37; Ma et al., 2005, Nature, 434, 666-70}, suggesting that this region of the guide sequence is rigid and sensitive to perturbation. Indeed, disrupting base pairing between the RNA-guide and substrate abrogates modification {Cavaille et al., 1996, Nature, 383, 732-5}. Furthermore, superimposition of the C/D RNP in the free state with the substrate-bound RNP illustrates a conformational change much like Argonaute when it undergoes a transition into the active form {Wang et al., 2009, Nature, 461, 754-61}.

Given that the degree of pairing to the RNA substrate affects restructuring of Argonaute, it will be interesting to see if the same applies to C/D RNP. Amazingly, the structure underscores the uniqueness of the functional unit of C/D RNP as a highly coordinated arrangement and positioning of RNA and proteins to ensure fidelity of site-specific modification. This is crucial in light of the possibility that 2 separate modifications can occur concurrently and independently on the same bipartite molecule {Lin et al., 2011, Nature, 469, 559-63}. We can only anticipate the structures of other snRNPs to consolidate the arsenal of RNA-centric biological machineries and the myriad of surprises it may bring. 

LiangMeng Wee

Posted by Elizabeth Bal at 14:14    Comments (0)

HOT paper: Many X-linked microRNAs escape meiotic sex chromosome inactivation

During meiotic prophase, homologous chromosomes must pair (synapse) in order to be separated appropriately during the first meiotic division. It is believed that meiotic sex chromosome inactivation (MSCI) evolves to mask the sex chromosome asynapsis for the heterogametic sex with distinctive heteromorphic sex chromosomes. It is has been shown that during pachytene stage, almost all the protein coding genes on the sex chromosomes are silenced. However, a study from the Yan lab (Song et al.) has found that many X-linked miRNAs are transcribed by Pol II at the pachytene stage, suggesting that mechanisms of MSCI may be different from conventional heterochromatin silencing, and the miRNA on sex chromosomes may undergo distinct regulation. It would be interesting to check the expression of  X-linked miRNAs in meiotic mutants in which MSCI is disrupted, which would indicate whether the transcription of these miRNAs is dependent on MSCI.

Xin Chenglin Li

Posted by Elizabeth Bal at 14:12    Comments (0)

Regulation of ARGONAUTE1 in Arabidopsis thaliana

In an article recently published in Silence, Earley et al. describe how loss-of-function mutations in the F-box-gene FBW2 serve to increase the protein levels of ARGONAUTE1 (AGO1), a core component of the RNA-induced silencing complex in Arabidopsis thaliana.

Conversely, over-expression of the endogenous F-box protein leads to a decrease in the abundance of AGO1 protein levels, but not AGO1 mRNA. Together, these results indicate the novel role of FBW2 as a negative regulator of AGO1 protein levels, contributing yet another layer of complexity to the mechanisms involved in AGO1 homeostasis.

Although FBW2 mutants possess no obvious morphological phenotype, they do display a reduced sensitivity to the plant growth regulator abscisic acid (ABA), suggesting that FBW2 may also play a role in hormone response pathways. The authors predict that the potential involvement of FBW2 in such regulatory pathways will prove to be an interesting subject for future research.

Posted by Elizabeth Bal at 14:33    Comments (0)

Olivier Voinnet joins Editorial Board

David Baulcombe and Phillip Zamore are very happy to announce the addition of Olivier Voinnet to Silence as Senior Editor.

Currently a senior researcher at the CNRS Institute of Plant Molecular Biology in Strasbourg, Voinnet  gained his PhD in David Baulcome’s lab in 2001, before establishing his own lab in 2002.

In 2009, he was awarded an EMBO Gold Medal for his pioneering work on the mechanisms and roles of RNA silencing in plants.

Posted by Emma Pettengale at 14:21    Comments (0)

Mini-review published highlighting new antisense technologies paper in Silence

In an article published in Silence today, Zheng et al describe how dextran was conjugated with 2'-O-methyl oligoribonucleotides to develop fluorescently labeled antisense reagents which were targeted to block specific miRNAs.  These antisense oligonucleotides were introduced into the germ line of adult hermaphrodite C. elegans, and passed onto the worms progeny, where they were successfully shown to have efficiently and specifically inhibited Lin-4 miRNA in several different tissues.

The article is further discussed in a mini-review by Slack et al, published in Journal of Biology.

The authors anticipate that this new class of antisense oligonucleotide will offer scientists a new experimental approach complimentary to the mutational strategy currently used for the study of miRNA function in vivo.

Posted by Emma Pettengale at 10:49    Comments (0)

HOT paper: Mature and functional viral miRNAs transcribed from novel RNA polymerase III promoters

Viruses take advantage of the host cell machinery to replicate their genome and produce more viral particles. Viruses are known to evolve rapidly and find novel ways to take advantage of existing cellular machinery to propagate. For example, Hepatitis C virus (HCV) utilizes signaling pathways such as epidermal growth factor-receptor pathway, the PI3K/Akt cascade or Src kinase-dependent pathways (Bode et al. 2009).

The miRNA pathway is no exception to this phenomenon. Previously, viruses were demonstrated to have miRNA genes (Pfeffer et al. 2005, 2004; Gottwein and Cullen 2008; Cullen 2009).

In this paper, Diebel KW. and colleagues reported the existence of Murid herpesvirus 4 (MUHV-4) microRNAs  in lytically infected cells and infected tissues samples ex vivo. For the first time, they could detect gHV68 pri-miRNAs, processed intermediates and mature miRNAs existing in lytically infected cells. Authors not only report the existence of these miRNAs and their precursors in lytically infected cells, but they also confirm that these miRNAs are  functional by using a luciferase reporter system in the infected cells.

Unlike the previously reported mature miRNAs from Epstein-Barr virus (EBV) and Kaposi’s sarcoma- associated herpesvirus (KSHV), that are processed from  RNA-pol II transcribed pri-miRNAs, the precursors of the miRNAs from MUHV-4  seem to be transcribed by RNA pol III.   In fact, MuvHV-4 is the only herpesvirus examined to date that has been predicted to generate its pri-miRNA transcript through RNA polymerase III (pol III) transcription.

Most of the human miRNAs are transcribed by RNA pol II; and some miRNAs genes that are interspersed among Alu repeats require RNA pol III for transcription(Borchert et al. 2006).  The transcription of these miRNA precursors, but not control pol-II dependent transcripts, are -amanitin sensitive, suggesting that they don't require RNA pol II for transcription.  Furthermore, the specific deletion of the RNA polymerase III promoter elements of MuvHV-4 result in the complete loss of miRNA detection.  Some future experiments such as ChIPs using antibodies specific for RNA pol III might also be done to  address direct interaction of RNA pol III with the RNA polymerase III promoter elements of MuvHV-4.

In summary, the authors  propose the existence of novel viral transcripts that are transcribed by RNA pol III  and  are processed by cellular RNAse III enzymes to produce functional mature viral miRNAs.  Further sequencing and functional studies with infected cells might reveal more virally expressed miRNAs.

A deeper understanding of viral miRNAs may provide the basis for novel therapies to combat viruses (Bode et al. 2009).

Elif Sarinay
 
References:
 
1) Bode JG, Brenndorfer ED, Karthe J, Haussinger D. Interplay between host cell and hepatitis C virus in regulating viral replication. Biol Chem. 2009 Oct;390(10):1013-32.

2) Pfeffer S, et al. Identification of microRNAs of the herpesvirus family. Nat Methods. 2005 Apr;2(4):269-76. Epub 2005 Feb 16.

3) Gottwein E, Cullen BR. Viral and cellular microRNAs as determinants of viral pathogenesis and immunity. Cell Host Microbe. 2008 Jun 12;3(6):375-87.

4) Umbach JL, Cullen BR. The role of RNAi and microRNAs in animal virus replication and antiviral immunity. Genes Dev. 2009 May 15;23(10):1151-64.

5) Kevin W. Diebel, Anna L. Smith and Linda F. van Dyk. Mature and functional viral miRNAs transcribed from novel RNA  polymerase III promoters. RNA 2010 16(1):170-85

6) Borchert et al. RNA polymerase transcribes human microRNAs. Nature Structural & Molecular Biology 2006 13(12):1097-101.

Posted by Elif Sarinay at 06:43    Comments (0)

HOT paper: Loqs and R2D2 act sequentially in the siRNA pathway in Drosophila

Two small RNA pathways, namely the small-interfering RNA (siRNA) and microRNA (miRNA) pathway, act ubiquitously in Drosophila somatic tissues (1).  These two pathways differ in the factors required for the biogenesis and function of small RNAs: Dicer-1 (Dcr-1) and its double-stranded RNA (dsRNA)-binding partner, Loquacious (Loqs), generate miRNAs, whereas siRNA-formation requires Dicer-2 (Dcr-2) and the dsRNA-binding protein R2D2 (2,3,4,5,6).

Strikingly, Loqs was also reported to affect siRNA-mediated silencing by an artificially introduced inverted-repeat (IR) transgene targeting the white gene (3). Following the discovery of endogenous siRNAs, the restriction of Loqs to the miRNA pathway was again challenged: Loqs was required for generation of siRNAs derived from endogenous hairpin RNAs, termed structured loci (7,8). However, these hairpins were only partially base-paired (similar to the shorter pre-miRNAs), in contrast to the completely base-paired white-IR transgene (9). It was proposed that Dcr-2 partners with Loqs to generate siRNAs from structured loci, with no dependence on R2D2 (7,8).

Marques et al., in their recent Nature Structural and Molecular Biology paper, followed up on these controversial observations by detailed analysis of the involvement of either R2D2 or Loqs on biogenesis and function of siRNAs derived from different sources (10). High-depth sequencing from loqs mutants confirmed, that the production of miRNAs as well as siRNAs derived from an inverted-repeat transgene requires Loqs. R2D2, however, was only required for normal accumulation of siRNAs, but not miRNAs. They also observed a stronger dependence on Loqs relative to R2D2 for in vitro and in vivo processing of long dsRNA precursors. Moreover, r2d2 mutants failed to assemble a siRNA duplex into an Argonaute complex in vitro, however complex assembly was functional though severely deprived in loqs mutants. Therefore, the authors suggest that the two dsRNA binding proteins act sequentially in the RNAi pathway: Loqs is required along with Dcr-2 for producing siRNAs from dsRNA, whereas R2D2 partners with Dcr-2 to load siRNA duplexes into Ago2.

To further corroborate their conclusion, they show similar results for endogenous siRNAs. Interestingly, they report a dependency for Loqs not only for structured loci-derived siRNAs, confirming previous results (7,8), but also transposons-derived siRNAs. Remarkably, silencing of target mRNAs by endogenous siRNAs is dependent on R2D2, in contrast to previous results observed in S2 cells (7).

Although the disruption of the siRNA pathway in loqs mutants is cogent, the final approval of a hierarchical role for Loqs and R2D2 requires further analysis of Dcr-2-mediated processing using kinetic rather than endpoint assays, possibly in a minimal, reconstituted system. Finally, the effect of loqs mutation on the siRNA pathway could be an indirect effect, even more so, since a physical association between Loqs and Dcr-2 could not be shown.

Megha Ghildiyal and Stefan L. Ameres

References:

1.   Ghildiyal, M. & Zamore, P. D. Small silencing RNAs: an expanding universe. Nat Rev Genet 10, 94-108 (2009).

2.   Lee, Y. S. et al. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69-81 (2004).

3.   Förstemann, K. et al. Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol 3, e236 (2005).

4.   Saito, K., Ishizuka, A., Siomi, H. & Siomi, M. C. Processing of pre-microRNAs by the Dicer-1-Loquacious complex in Drosophila cells. PLoS Biol 3, e235 (2005).

5.   Jiang, F. et al. Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila. Genes Dev 19, 1674-1679 (2005).

6.   Liu, Q. et al. R2D2, a Bridge Between the Initiation and Effector Steps of the Drosophila RNAi Pathway. Science 301, 1921-1925 (2003).

7.   Czech, B. et al. An endogenous small interfering RNA pathway in Drosophila. Nature 453, 798-802 (2008).

8.   Okamura, K. et al. The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 453, 803-806 (2008).

9.   Lee, Y. S. & Carthew, R. W. Making a better RNAi vector for Drosophila: use of intron spacers. Methods 30, 322-329 (2003).

10.   Marques, J. T. et al. Loqs and R2D2 act sequentially in the siRNA pathway in Drosophila. Nature Structural & Molecular Biology (2009).

Posted by Stefan Ameres at 10:30    Comments (0)

Welcome to the Silence blog

Welcome to the Silence blog.  We hope this site will become the forum for the exchange of ideas within the RNA silencing community.

We welcome short pieces on interesting topics such as recent research papers, new methodologies, policy directions, funding opportunities and upcoming meetings. Blogs on all aspects of genetic and epigenetic control mediated by RNA are welcome.  Bloggers for this site come from a wide range of backgrounds, and you are encouraged to post comments or continue discussions through the site, enabling the free and rapid flow of information, thereby promoting debate.

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We look forward to reading your contributions!

Posted by Emma Pettengale at 10:30    Comments (0)