Read more]]> 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 domain of Shutdown can potentially interact with Hsp90, a chaperone protein required to load siRNAs into Argonaute proteins, the Brennecke group mutated an amino acid in Shutdown that is predicted to mediate Hsp90 binding; this mutant does not support piRNA production. Thus, Shutdown may act with Hsp90 to facilitate loading of piRNAs into PIWI proteins.

The Pillai group, working in mice, examined the shutdown homolog FK-506 binding protein 6, fkbp6. As in Drosophila, fkbp6 mutant mice suffered transposon over expression, but in the testes, where piRNAs play their most important role in mammals. Interestingly, in newborn mice, fkbp6 is required for production of Miwi2-, but not Mili-associated piRNAs. Supporting the idea that Shutdown/FKBP6 may collaborate with Hsp90 in piRNA loading, Hsp90 and FKBP6 co-immunoprecipitated from mouse testes extracts; the association required the FKBP6 C-terminal TPR domain.

Thus, the requirement for chaperones to load piRNAs into Piwi proteins appears to be widely conserved in the transposon silencing piRNA pathway.

—Zhao Zhang, University of Massachusetts Medical School

*Preall, J. B., Czech, B., Guzzardo, P. M., Muerdter, F., Hannon, G. J. (2012). shutdown is a component of the Drosophila piRNA biogenesis machinery. RNA 18:1446-57; Olivieri, D., Senti, K. A., Subramanian, S., Sachidanandam, R., Brennecke, J. (2012). The Cochaperone Shutdown Defines a Group of Biogenesis Factors Essential for All piRNA Populations in Drosophila. Mol Cell, in press; Xiol, J. et al. (2012). A Role for Fkbp6 and the Chaperone Machinery in piRNA Amplification and Transposon Silencing. Mol Cell, in press.

Read more]]> 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 the dye-labeled nucleic acid contents to be imaged by Total Internal Reflection Fluorescence (TIRF) microscopy. With one of the strands tagged with Cy3 (donor) and the other with Cy5 (acceptor), annealing between the two strands brings the dyes together, producing a high FRET state; their dissociation produces a low FRET state. DNA or RNA strands with less than 7 nt of contiguous base pairing displayed an association rate 2 orders of magnitude smaller than those with seven or more base pairs. A minimum of 7 nt is therefore required for a duplex to form quickly.

The number seven immediately brings to mind the seven-to-eight-nucleotide “seed sequence” of miRNAs and siRNAs. The seed sequence, spanning nucleotides 2 to 8 of a small silencing RNA guide, is created when the RNA guide binds an Argonaute protein; the seed sequence provides nearly all the binding specificity of the Argonaute: small RNA silencing complex. Remarkably, studies of the bacterial CRISPR system also point to a seven-nucleotide seed that allows the machinery to bind foreign nucleic acids (Bhaya et al., 2011). Thus, regulation by Argonaute-bound small RNAs or the CRISPR machinery may have evolved to harness the natural biophysical properties of nucleic acid pairing: the minimal 7 nt pairing rule allows them to rapidly lock in on their targets.

—Liang-Meng Wee, University of Massachusetts Medical School

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 ...

Read more]]> 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 find that Dicer expression is decreased in the retinal pigmented epithelial cells in patients with geographic atrophy, which allows the accumulation of long dsRNA Alu elements. These repetitive Alu elements produce inflammatory changes leading to the loss of the retinal pigmented epithelium and producing the disease phenotype. The authors were able to recapitulate geographic atrophy in a mouse model by making a tissue-specific knockout of Dicer in the RPE cells. This study highlights a role for Dicer in suppressing repetitive elements beyond the more well studied role of Dicer in producing siRNAs and miRNAs that function as guides for RISC.

Keith Boundy

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 ...

Read more]]> 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 best maintained the GC content and length distribution of the library throughout 40 cycles of PCR, while Phusion polymerase in HF buffer introduced dramatic bias in both parameters relative to the original library.  Neanderthal DNA is a limited resource and contains a high level of GC-rich microbial contamination.  Of the ten polymerase-buffer systems tested for ancient DNA library preparation, AccuPrime Pfx produced the highest levels of endogenous sequences, while Phusion in HF buffer  preferentially amplifies the GC-rich microbial templates. 

PCR polymerases are a principal source of GC content and length bias in high throughput library preparation. Other uninvestigated  biases, such as thermocycling parameters, likely exist but optimizing the polymerase-buffer system can contribute to generating a library that accurately represents the starting material.

Desiree Boltz

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.

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 ...

Read more]]> 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.

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.

Read more]]> 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

Read more]]> 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

Read more]]> 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