Shutdown rules in germline transposon silencing and piRNA production

samrose on September 20, 2012 at 3:46 pm - 0 Comments

In animal gonads, the piRNAs, bound to PIWI-clade Argonaute proteins, silence transposons and maintain genomic integrity. Failure of this pathway triggers transposon activation and induces DNA damage, resulting in sterility. Now, the Hannon, Pillai, and Brennecke labs* describe a new gene in the piRNA pathway, shutdown, which encodes an evolutionarily conserved FKBP-family co-chaperone protein. shutdown was originally discovered in a large-scale Drosophila genetic screen for female sterility (Schupbach and Wieschaus, 1991). Working in flies, the Hannon and Brennecke labs report that mutation of shutdown in fruit fly, like other piRNA pathway genes, leads to transposon de-silencing in both the somatic and germline cells of the ovary. Small RNA sequencing reveals that shutdown mutants block piRNA production. The C-terminal TPR …

Measuring nucleic acid base pairing using FRET: significance of the number 7

Andrea Melendez on August 31, 2012 at 1:34 pm - 0 Comments

Nucleic acid base pairing occurs during DNA repair (San Filippo et al., 2008), splicing (Black, 2003), ribosomal RNA modification (Weinstein and Steitz, 1999) and gene regulation by small silencing RNAs (Ghildiyal and Zamore, 2009). Recent work by Ha and colleagues (Cisse et al., 2012) on the association and dissociation of complementary nucleic acids provides new insight into how short regions of nucleic acid complementarity mediate these biological functions. Ha and coworkers measured the annealing and melting of complementary oligonucleotides using fluorescence resonance energy transfer (FRET). Unlike classical FRET studies, these authors enclosed a single pair of complementary nucleic acid molecules in liposomes of defined volume. The liposomes were then adhered to a glass slide, allowing …

DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration

Elizabeth Bal on May 22, 2012 at 11:13 am - 0 Comments

Dicer has long been known for its ability to produce siRNAs and miRNAs, the guides which drive RNA silencing, from long, double-stranded RNA or miRNA precursors (pre-miRNAs). Kaneko et al. now report a fascinating role for mammalian Dicer, separate from its function in RNA silencing.

In a series of elegant experiments, the authors show a role for mammalian Dicer in preventing the inflammatory changes of geographic atrophy or “dry” age related macular degeneration (AMD) by degrading Alu dsRNA in the retinal pigmented epithelial (RPE) cells of both mice and humans. Interestingly, this effect was due to the endonuclease activity of Dicer alone
degrading dsRNA, independent of siRNA loading in the RNA-induced silencing complex (RISC).

They …

Length and GC-biases during sequencing library amplification

Elizabeth Bal on May 18, 2012 at 11:21 am - 1 Comment

High throughput sequencing has broadened our understanding of countless biological processes and led to scientific advancements in clinical therapeutics. With massively parallel sequencing becoming a staple in laboratories worldwide, we need to understand the limitations and biases of the technology. PCR amplification is an essential step in the preparation of a library for high throughput sequencing. However, biases introduced by PCR, reflecting the GC content and length of the template, can have detrimental effects on library generation.

Dabney and Meyer have now compared ten commercially available PCR polymerase-buffer systems (Dabney and Meyer, Biotechniques 52:87-94, 2012) to determine the biases they introduce in sequencing both modern and ancient DNA. For modern DNA library preparation, Herculase II Fusion polymerase …

Call for papers – ‘RNA: Methods and Protocols’ series

Elizabeth Bal on April 3, 2012 at 10:22 am - 0 Comments

Silence is inviting submissions for a new article series on methods and protocols in analyzing RNA.

This special collection of articles will provide novel insights into the latest methods within the field of RNA, addressing questions of general interest to the RNA community, as well as predictions on future uses, modifications and improvements that would advance existing methods beyond their current use.

To submit your manuscript, please use our online submission system and indicate in your covering letter that you would like it to be considered for the series, or alternatively, send a pre-submission enquiry to editorial@silencejournal.com.

Dicer-1 rules!

Elizabeth Bal on September 19, 2011 at 5:13 am - 0 Comments

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 …

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

Elizabeth Bal on June 17, 2011 at 9:39 am - 0 Comments

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.

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

Elizabeth Bal on May 17, 2011 at 10:37 am - 0 Comments

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. …

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

Elizabeth Bal on April 26, 2011 at 2:14 am - 0 Comments

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 …

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

Elizabeth Bal on April 21, 2011 at 2:12 am - 0 Comments

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 …