Papers by Wolfgang Nellen
Nucleic Acids Research, 2010
The centromeric histone H3 variant (CenH3) serves to target the kinetochore to the centromeres an... more The centromeric histone H3 variant (CenH3) serves to target the kinetochore to the centromeres and thus ensures correct chromosome segregation during mitosis and meiosis. The Dictyostelium H3-like variant H3v1 was identified as the CenH3 ortholog. Dictyostelium CenH3 has an extended N-terminal domain with no similarity to any other known proteins and a histone fold domain at its C-terminus. Within the histone fold, a-helix 2 (a2) and an extended loop 1 (L1) have been shown to be required for targeting CenH3 to centromeres. Compared to other known and putative CenH3 histones, Dictyostelium CenH3 has a shorter L1, suggesting that the extension is not an obligatory feature. Through ChIP analysis and fluorescence microscopy of live and fixed cells, we provide here the first survey of centromere structure in amoebozoa. The six telocentric centromeres were found to mostly consist of all the DIRS-1 elements and to associate with H3K9me3. During interphase, the centromeres remain attached to the centrosome forming a single CenH3-containing cluster. Loading of Dictyostelium CenH3 onto centromeres occurs at the G2/prophase transition, in contrast to the anaphase/telophase loading of CenH3 observed in metazoans. This suggests that loading during G2/ prophase is the ancestral eukaryotic mechanism and that anaphase/telophase loading of CenH3 has evolved more recently after the amoebozoa diverged from the animal linage.
Schematic representation of dsRBD containing proteins in <i>D</i>. <i>discoideum</i>
<p><b>Dhx9</b> (1472 aa: dsRBD (1) [365–440], dsRBD (2) [532–607], DEXDc [715–9... more <p><b>Dhx9</b> (1472 aa: dsRBD (1) [365–440], dsRBD (2) [532–607], DEXDc [715–903], HELICc [963–1069], HA2 [1132–1243]), <b>HelF</b> (837 aa: dsRBD [2–76], DEXDc [228–431], HELICc [608–687]), <b>RbdA</b> (297 aa: dsRBD [4–70]), <b>RbdB</b> (733 aa: dsRBD [9–75]), P-rich [510–584]. Numbers in brackets indicate the position of protein domains in the amino acid (aa) sequence predicted by SMART [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006057#pgen.1006057.ref045" target="_blank">45</a>]. DsRBD (<i>double stranded</i> RNA <i>binding domain</i>), DEXDc (<i>Dead-like Helicases superfamily domain</i>), HA2 (<i>Helicase associated domain</i> 2), P-rich site (Proline rich site). Domains are drawn to scale.</p
dsRBD containing proteins in <i>D</i>. <i>discoideum</i> (AX4)
RNA Biology, 2016
A group of homologous nucleic acid modification enzymes called Dnmt2, Trdmt1, Pmt1, DnmA, and Ehm... more A group of homologous nucleic acid modification enzymes called Dnmt2, Trdmt1, Pmt1, DnmA, and Ehmet in different model organisms catalyze the transfer of a methyl group from the cofactor S-adenosyLmethionine (SAM) to the carbon-5 of cytosine residues. Originally considered as DNA MTases, these enzymes were shown to be tRNA methyltransferases about a decade ago. Between the presumed involvement in DNA modification-related epigenetics, and the recent foray into the RNA modification field, significant progress has characterized Dnmt2-related research. Here, we review this progress in its diverse facets including molecular evolution, structural biology, biochemistry, chemical biology, cell biology and epigenetics.
PLOS Genetics, 2016
We identified the dsRNA binding protein RbdB as an essential component in miRNA processing in Dic... more We identified the dsRNA binding protein RbdB as an essential component in miRNA processing in Dictyostelium discoideum. RbdB is a nuclear protein that accumulates, together with Dicer B, in nucleolar foci reminiscent of plant dicing bodies. Disruption of rbdB results in loss of miRNAs and accumulation of primary miRNAs. The phenotype can be rescued by ectopic expression of RbdB thus allowing for a detailed analysis of domain function. The lack of cytoplasmic dsRBD proteins involved in miRNA processing, suggests that both processing steps take place in the nucleus thus resembling the plant pathway. However, we also find features e.g. in the domain structure of Dicer which suggest similarities to animals. Reduction of miRNAs in the rbdB-strain and their increase in the Argonaute A knock out allowed the definition of new miRNAs one of which appears to belong to a new non-canonical class.
The Journal of biological chemistry, Jan 19, 2014
The retrotransposon DIRS-1 is the most abundant retroelement in Dictyostelium discoideum and cons... more The retrotransposon DIRS-1 is the most abundant retroelement in Dictyostelium discoideum and constitutes the pericentromeric heterochromatin of the six chromosomes in D. discoideum. The vast majority of cellular siRNAs is derived from DIRS-1, suggesting that the element is controlled by RNAi-related mechanisms. We investigated the role of two of the five Argonaute proteins of D. discoideum, AgnA and AgnB, in DIRS-1 silencing. Deletion of agnA resulted in the accumulation of DIRS-1 transcripts, the expression of DIRS-1-encoded proteins, and the loss of most DIRS-1-derived secondary siRNAs. Simultaneously, extrachromosomal single-stranded DIRS-1 DNA accumulated in the cytoplasm of agnA- strains. These DNA molecules appear to be products of reverse transcription and thus could represent intermediate structures before transposition. We further show that transitivity of endogenous siRNAs is impaired in agnA- strains. The deletion of agnB alone had no strong effect on DIRS-1 transposon re...
Nucleic Acids Research, 2013
Although the DNA methyltransferase 2 family is highly conserved during evolution and recent repor... more Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNA Asp(GUC) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified tRNA Glu(CUC/UUC) and tRNA Gly(GCC) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.
Moment Mal!
Biologie in unserer Zeit, 2016
Detection of siRNAs based on the bidirectional RNA expression driven by the left and right DIRS-1 ITRs
Analysis of truncated RbdB variants
<p>A: Schematic representation of RbdB and truncated protein variants. In RbdB Δ504–733 GFP... more <p>A: Schematic representation of RbdB and truncated protein variants. In RbdB Δ504–733 GFP, 230 amino acids were deleted from the C-terminus. RbdB Δ504–612 GFP lacks the Prich-site. B: left: Both truncated RbdB-GFP versions were expressed in the knockout background and visualized by fluorescence microscopy. RbdB Δ504–612 GFP showed the same distribution as RbdB GFP. In contrast, RbdB Δ504–733 GFP was not detectably by fluorescence microscopy. right: Western Blot showing expression of RbdB Δ504–733 GFP (84 kDa), RbdB Δ504–612 (98 kDa) and of RbdB GFP (109 kDa). Note that all proteins run at higher molecular levels than calculated. SevA (40 kDa) is shown as loading control. C: left: Northern Blot analysis of rbdB- strains expressing RbdB GFP [R1], RbdB Δ504–733 GFP [R2], RbdB Δ504–612 GFP [R3] on miRNAs. 12 μg total RNA were loaded per lane. As a control, RNA from the AX2 wt and from an rbdB- strain was used. The mature miRNAs ddi-miR-1176 and ddi-miR-1177 were detected by <sup>32</sup>P labelled probes as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006057#pgen.1006057.g003" target="_blank">Fig 3A</a>. Hybridisation to snoRNA DdR6 was used as a loading control. Right: miRNA signals (ddi-miR-1176 left, ddi-miR-1177 right) were quantified relative to DdR6 from different Northern Blots and normalized to the AX2 wt. R1-R3: rbdB- mutants were transformed with pDM323 RbdB Δ504–733 GFP (R1), pDM323 RbdB Δ504–612 GFP (R2) and with pDM323 RbdB GFP (R3). According to paired t-test, no significant difference was seen in miRNA accumulation between the wild type and the mutants.</p
Identification of new miRNAs
Co-IP of RbdB Δ 504–612 GFP and DrnB 3xHA
Model for gene silencing by natural and transgene derived siRNAs
<p>(A) The endogenous DIRS–1 sequences are transcribed in both directions and generate dsRN... more <p>(A) The endogenous DIRS–1 sequences are transcribed in both directions and generate dsRNAs (grey) which are cleaved by Dicer to ~21 nt siRNAs. siRNAs recruit RrpC in a AgnA-dependent manner. The resulting dsRNA which may be synthesised primer dependent or primer independent in 5’ and 3’ direction is processed to secondary siRNAs. Primer independent siRNAs carry a 5’-triphosphate. Argonaute proteins (probably AgnA) silence DIRS–1 by mRNA destabilisation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131271#pone.0131271.ref010" target="_blank">10</a>]. (B) A transgene fusion of GFP and a DIRS-1 ORF is targeted by endogenous DIRS-1 siRNAs <i>in trans</i>. These may be primary or secondary siRNAs. They recruit the RdRP RrpC, which produces primer dependent and primer independent secondary siRNAs in 5’ and 3’ direction. Consequently, siRNAs matching adjacent sequences (here GFP) are generated. Silencing occurs on the level of mRNA destabilisation. DIRS–1 sequences are colour coded grey, GFP sequences green. Secondary siRNAs have a 5’-triphosphate or a monophosphate when they are Dicer products from longer dsRNAs or primer dependent. (C) siRNAs are generated from a bidirectionally transcribed trigger transgene. They can target the trigger transcripts <i>in cis</i> and complementary sequences on mRNAs of the corresponding endogene. They produce secondary siRNAs either from the trigger only or from both the target and the trigger transcript. If transgene derived siRNAs can only recruit RdRPs <i>in cis</i>, i.e. on the transgene itself, this could explain the lack of spreading along the mRNA of the endogene. Trigger sequences are colour coded grey, flanking sequences of the corresponding endogene blue.</p
RNAi and epigenetic regulation of Dictyostelium retrotransposon
GBM Annual Fall meeting Berlin/Potsdam 2005, 2005
Nucleic Acids Research, 2005
We have identified a DNA methyltransferase of the Dnmt2 family in Dictyostelium that was denomina... more We have identified a DNA methyltransferase of the Dnmt2 family in Dictyostelium that was denominated DnmA. Expression of the dnmA gene is downregulated during the developmental cycle. Overall DNA methylation in Dictyostelium is $0.2% of the cytosine residues, which indicates its restriction to a limited set of genomic loci. Bisulfite sequencing of specific sites revealed that DnmA is responsible for methylation of mostly asymmetric C-residues in the retrotransposons DIRS-1 and Skipper. Disruption of the gene resulted in a loss of methylation and in increased transcription and mobilization of Skipper. Skipper transcription was also upregulated in strains that had genes encoding components of the RNA interference pathway disrupted. In contrast, DIRS-1 expression was not affected by a loss of DnmA but was strongly increased in strains that had the RNAdirected RNA polymerase gene rrpC disrupted. A large number of siRNAs were found that corresponded to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA modification. No siRNAs corresponding to the standard Skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene silencing in Dictyostelium but that different, partially overlapping mechanisms control transposon silencing.
Nucleic Acids Research, 2007
Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we repor... more Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 18-26 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase.
Biologie in Unserer Zeit, Nov 8, 2021
Epigenetisches Genesilencing: RNA Interferenz und Antisense RNA
Experientia, 1995
Over the past ten years, powerful molecular genetic techniques have been developed to analyze gen... more Over the past ten years, powerful molecular genetic techniques have been developed to analyze gene function in Dictyostelium. DNA-mediated transformation using a variety of selections and vectors has allowed the introduction of wild-type or modified genes that are under various forms of transcriptional control. Homologous recombination is efficient and can be used to modify the genome in precise ways. In addition, it is now possible to clone genes based on their mutant phenotype alone, either by insertional mutagenesis, or by screening antisense expression cDNA libraries. Finally, a nearly complete physical map of the genome is available and so genes are easily mapped by physical techniques. We discuss many of these advances within the context of major research problems presently under study.
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Papers by Wolfgang Nellen