Trans-kingdom cross-talk:small RNAs on the move

This review focuses on the mobility of small RNA (sRNA) molecules from the perspective of trans-kingdom gene silencing. Mobility of sRNA molecules within organisms is a well-known phenomenon, facilitating gene silencing between cells and tissues. sRNA signals are also transmitted between organisms of the same species and of different species. Remarkably, in recent years many examples of RNA-signal exchange have been described to occur between organisms of different kingdoms. These examples are predominantly found in interactions between hosts and their pathogens, parasites, and symbionts. However, they may only represent the tip of the iceberg, since the emerging picture suggests that organisms in biological niches commonly exchange RNA-silencing signals. In this case, we need to take this into account fully to understand how a given biological equilibrium is obtained. Despite many observations of trans-kingdom RNA signal transfer, several mechanistic aspects of these signals remain unknown. Such RNA signal transfer is already being exploited for practical purposes, though. Pathogen genes can be silenced by plant-produced sRNAs designed to affect these genes. This is also known as Host-Induced Genes Silencing (HIGS), and it has the potential to become an important disease-control method in the future

DNA Methylation and Normal Chromosome Behavior in Neurospora Depend on Five Components of a Histone Methyltransferase Complex, DCDC

Methylation of DNA and of Lysine 9 on histone H3 (H3K9) is associated with gene silencing in many animals, plants, and fungi. In Neurospora crassa, methylation of H3K9 by DIM-5 directs cytosine methylation by recruiting a complex containing Heterochromatin Protein-1 (HP1) and the DIM-2 DNA methyltransferase. We report genetic, proteomic, and biochemical investigations into how DIM-5 is controlled. These studies revealed DCDC, a previously unknown protein complex including DIM-5, DIM-7, DIM-9, CUL4, and DDB1. Components of DCDC are required for H3K9me3, proper chromosome segregation, and DNA methylation. DCDC-defective strains, but not HP1-defective strains, are hypersensitive to MMS, revealing an HP1-independent function of H3K9 methylation. In addition to DDB1, DIM-7, and the WD40 domain protein DIM-9, other presumptive DCAFs (DDB1/CUL4 associated factors) co-purified with CUL4, suggesting that CUL4/DDB1 forms multiple complexes with distinct functions. This conclusion was supported by results of drug sensitivity tests. CUL4, DDB1, and DIM-9 are not required for localization of DIM-5 to incipient heterochromatin domains, indicating that recruitment of DIM-5 to chromatin is not sufficient to direct H3K9me3. DIM-7 is required for DIM-5 localization and mediates interaction of DIM-5 with DDB1/CUL4 through DIM-9. These data support a two-step mechanism for H3K9 methylation in Neurospora

The <it>SLEEPER</it> genes: a transposase-derived angiosperm-specific gene family

<p>Abstract</p> <p>Background</p> <p><it>DAYSLEEPER</it> encodes a domesticated transposase from the hAT-superfamily, which is essential for development in <it>Arabidopsis thaliana</it>. Little is known about the presence of <it>DAYSLEEPER</it> orthologs in other species, or how and when it was domesticated. We studied the presence of <it>DAYSLEEPER</it> orthologs in plants and propose a model for the domestication of the ancestral <it>DAYSLEEPER</it> gene in angiosperms.</p> <p>Results</p> <p>Using specific BLAST searches in genomic and EST libraries, we found that <it>DAYSLEEPER</it>-like genes (hereafter called <it>SLEEPER</it> genes) are unique to angiosperms. Basal angiosperms as well as grasses (Poaceae) and dicotyledonous plants possess such putative orthologous genes, but <it>SLEEPER</it>-family genes were not found in gymnosperms, mosses and algae. Most species contain more than one <it>SLEEPER</it> gene. All <it>SLEEPER</it>s contain a C₂H₂ type BED-zinc finger domain and a hATC dimerization domain. We designated 3 motifs, partly overlapping the BED-zinc finger and dimerization domain, which are hallmark features in the <it>SLEEPER</it> family. Although <it>SLEEPER</it> genes are structurally conserved between species, constructs with <it>SLEEPER</it> genes from grapevine and rice did not complement the <it>daysleeper</it> phenotype in Arabidopsis, when expressed under control of the <it>DAYSLEEPER</it> promoter. However these constructs did cause a dominant phenotype when expressed in Arabidopsis. Rice plant lines with an insertion in the <it>RICESLEEPER</it>1 or 2 locus displayed phenotypic abnormalities, indicating that these genes are functional and important for normal development in rice. We suggest a model in which we hypothesize that an ancestral hAT transposase was retrocopied and stably integrated in the genome during early angiosperm evolution. Evidence is also presented for more recent retroposition events of <it>SLEEPER</it> genes, such as an event in the rice genome, which gave rise to the <it>RICESLEEPER</it>1 and 2 genes.</p> <p>Conclusions</p> <p>We propose the ancestral <it>SLEEPER</it> gene was formed after a process of retro-transposition during the evolution of the first angiosperms. It may have acquired an important function early on, as mutation of two <it>SLEEPER</it> genes in rice, like the <it>daysleeper</it> mutant in <it>A. thaliana</it> gave a developmental phenotype indicative of their importance for normal plant development.</p

A generalized overview of RNA transfer from one cell (blue) to another (red).

<p>sRNA is produced by Dicing of larger dsRNA molecules in the transmitting cell. On the left, non-vesicular dsRNA and sRNA are secreted by unknown mechanisms. Uptake of this RNA is depicted in a manner that resembles SID-1/SID-2 mediated uptake <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004602#pgen.1004602-McEwan1" target="_blank">[39]</a>. DsRNA is bound by a receptor and internalized, after which it is taken up into the cytosol by a transmembrane channel, such as SID-1. In the middle, transfer of sRNAs through MVB-mediated exosomes is depicted. A model for loading of sRNA into intraluminal vesicles of MVBs (<b>MVB</b>) is suggested <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004602#pgen.1004602-VillarroyaBeltri1" target="_blank">[49]</a>. These vesicles are released in the intercellular space as exosomes after fusion of MVBs with the plasma membrane (<b>PM</b>). Exosomes are taken up by endocytosis into the receiving cell. It is unknown how sRNA is released into the cytosol, but one could envisage a fusogenic protein (<b>F</b>) to be involved, which facilitates fusion of the endosomal and exosomal membranes. On the right, transfer of sRNA in shedding vesicles (<b>SV</b>), which are generated directly from the PM, is depicted. How RNA is loaded into SV is unknown. The recipient cell takes up the sRNA after fusion of the SV with the PM in a process that requires fusogenic proteins. SVs might be taken up in an endocytosis-dependent manner and exosomes might be taken up in a membrane fusion event. In the cytosol of the recipient cell, the sRNA is recognized by the RNAi machinery and triggers gene silencing, either through post-transcriptional gene silencing (<b>PTGS</b>) or transcriptional gene silencing (<b>TGS</b>). During PTGS, amplification of the sRNA signal is provided by RNA-dependent RNA polymerases (<b>RdRP</b>), which give rise to secondary sRNAs that can target the same or other transcripts.</p

Overview of different situations in which sRNA transfer occurs.

<p><b>A,</b><i>Botrytis cinerea</i> can transfer Bc-siRNA to its host. This process has been shown to be dependent on AGO1 in the host, <i>Arabidopsis thaliana</i> and on both Dcl1 and 2 in <i>Botrytis cinerea</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004602#pgen.1004602-Weiberg1" target="_blank">[23]</a>. <b>B,</b> Human miRNAs can be translocated to the malaria-parasite, <i>P. falciparum</i>, where they interfere with translation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004602#pgen.1004602-LaMonte1" target="_blank">[16]</a>. <b>C,</b> The nematode <i>C. elegans</i> has been shown to take up <i>E. coli</i>-produced ncRNAs that subsequently influence their foraging behavior. This is dependent on the <i>C. elegans</i> protein RDE-2, that is essential for RNAi <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004602#pgen.1004602-Liu1" target="_blank">[17]</a>. <b>D,</b> The Chagas disease-causing parasite, <i>T cruzi</i>, produces tRNA-derived sRNAs (tsRNAs) that are exported from the cell in vesicles. These vesicles are shown to increase infectability of host cells, suggesting this might be caused by the tsRNAs. This has not been shown directly though <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004602#pgen.1004602-GarciaSilva1" target="_blank">[14]</a>. <b>E,</b> The expression of sRNA-generating constructs to silence genes in pathogens, or other closely associated species, has now been demonstrated for many species combinations. This process is suggested to be dependent on Dcl1, since Dcl2, 3, and 4 seem to be dispensable to induce silencing by an Arabidopsis-expressed hairpin in the insect, <i>Helicoverpa armigera</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004602#pgen.1004602-Mao1" target="_blank">[24]</a>.</p