Wolfgang Nellen

What makes an mRNA anti-sensi-itive?

Antisense RNA has been used for some time as a versatile tool for silencing gene expression. There is ample evidence for gene regulation by endogenous antisense transcripts in prokaryotes and increasing insight into the molecular mechanisms underlying such regulation. The introduction of antisense gene constructs into eukaryotes has now become routine but the mechanisms by which gene expression is inhibited are barely understood. In recent years, several examples of endogenous eukaryotic antisense transcripts have been discovered, some of which probably serve regulatory functions. Here we will discuss a model to explain mechanisms of antisense-mediated gene silencing.

Human DNMT2 methylates tRNAAsp molecules using a DNA methyltransferase-like catalytic mechanism

Although their amino acid sequences and structure closely resemble DNA methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to function as RNA methyltransferases transferring a methyl group to the C5 position of C38 in tRNA(Asp). We observe that human DNMT2 methylates tRNA isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium discoideum. RNA extracted from wild type D. melanogaster was methylated to a lower degree, but in the case of Dictyostelium, there was no difference in the methylation of RNA isolated from wild-type and Dnmt2 knock-out strains. Methylation of in vitro transcribed tRNA(Asp) confirms it to be a target of DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA methyltransferase-like mechanism, because similar residues from motifs IV, VI, and VIII are involved in catalysis as identified in DNA methyltransferases. In addition, exchange of C292, which is located in a CFT motif conserved among Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2 represents the first example of an RNA methyltransferase using a DNA methyltransferase type of mechanism.

Differential antisense transcription from the Dictyostelium EB4 gene locus: Implications on antisense-mediated regulation of mRNA stability

The 2.2 kb mRNA of the Dictyostelium discoideum prespore gene EB4-PSV is constitutively transcribed during growth and development, but the message is only accumulated when cells form aggregates and establish the prespore-prestalk pattern. Disruption of the pattern by mechanical disaggregation results in a rapid loss of the mRNA, while transcription remains nearly unchanged. In early development and after disaggregation, when the mRNA is unstable, a 1.8 kb antisense transcript originating from the same gene locus is detected. This RNA has apparently no coding capacity and is transcriptionally regulated by a promoter located within the translated region of the gene. Excess transcription of antisense RNA in vegetative cells and after disaggregation suggests its involvement in the control of mRNA stability. In agreement with this assumption, the inhibition of RNA synthesis during disaggregation prevents destabilization of the mRNA. This stability regulation of an endogenous mRNA is reminiscent of the loss of specific mRNAs in cells transformed with antisense constructs.

Formation of nucleoprotein filaments by mammalian DNA methyltransferase Dnmt3a in complex with regulator Dnmt3L

The C-terminal domains of Dnmt3a and Dnmt3L form elongated heterotetramers (3L-3a-3a-3L). Analytical ultracentrifugation confirmed the Dnmt3a-C/3L-C complex exists as a 2:2 heterotetramer in solution. The 3a–3a interface is the DNA-binding site, while both interfaces are essential for AdoMet binding and catalytic activity. Hairpin bisulfite analysis shows correlated methylation of two CG sites in a distance of ∼8-10 bp in the opposite DNA strands, which corresponds to the geometry of the two active sites in one Dnmt3a-C/3L-C tetramer. Correlated methylation was also observed for two CG sites at similar distances in the same DNA strand, which can be attributed to the binding of two tetramers next to each other. DNA-binding experiments show that Dnmt3a-C/3L-C complexes multimerize on the DNA. Scanning force microscopy demonstrates filament formation rather than binding of single tetramers and shows that protein–DNA filament formation leads to a 1.5-fold shortening of the DNA length.

Oligomerization and Binding of the Dnmt3a DNA Methyltransferase to Parallel DNA Molecules: HETEROCHROMATIC LOCALIZATION AND ROLE OF Dnmt3L

Structural studies showed that Dnmt3a has two interfaces for protein-protein interaction in the heterotetrameric Dnmt3a/3L C-terminal domain complex: the RD interface (mediating the Dnmt3a-3a contact) and the FF interface (mediating the Dnmt3a-3L contact). Here, we demonstrate that Dnmt3a-C forms dimers via the FF interface as well, which further oligomerize via their RD interfaces. Each RD interface of the Dnmt3a-C oligomer creates an independent DNA binding site, which allows for binding of separate DNA molecules oriented in parallel. Because Dnmt3L does not have an RD interface, it prevents Dnmt3a oligomerization and binding of more than one DNA molecule. Both interfaces of Dnmt3a are necessary for the heterochromatic localization of the enzyme in cells. Overexpression of Dnmt3L in cells leads to the release of Dnmt3a from heterochromatic regions, which may increase its activity for methylation of euchromatic targets like the differentially methylated regions involved in imprinting.

Determination of preferential binding sites for anti-dsRNA antibodies on double-stranded RNA by scanning force microscopy

The monoclonal anti-dsRNA antibody J2 binds double-stranded RNAs (dsRNA) in an apparently sequence-nonspecific way. The mAb only recognizes antigens with double-stranded regions of at least 40 bp and its affinity to poly(A) poly(U) and to dsRNAs with mixed base pair composition is about tenfold higher than to poly(I) poly(C). Because no specific binding site could be determined, the number, the exact dimensions, and other distinct features of the binding sites on a given antigen are difficult to evaluate by biochemical methods. We therefore employed scanning force microscopy (SFM) as a method to analyze antibody-dsRNA interaction and protein-RNA binding in general. Several in vitro-synthesized dsRNA substrates, generated from the Dictyostelium PSV-A gene, were used. In addition to the expected sequence-nonspecific binding, imaging of the complexes indicated preferential binding of antibodies to the ends of dsRNA molecules as well as to certain internal sites. Analysis of 2,000 bound antibodies suggested that the consensus sequence of a preferential internal binding site is A2N9A3N9A2, thus presenting A residues on one face of the helix. The site was verified by site-directed mutagenesis, which abolished preferential binding to this region. The data demonstrate that SFM can be efficiently used to identify and characterize binding sites for proteins with no or incomplete sequence specificity. This is especially the case for many proteins involved in RNA metabolism.

Regulation of the Discoidin I gamma gene in Dictyostelium discoideum: identification of individual promoter elements mediating induction of transcription and repression by cyclic AMP.

We dissected the promoter of the developmentally induced and cyclic AMP-repressed discoidin I gamma gene and identified a sequence element essential for developmental induction. Transfer of the element to an inactive heterologous promoter demonstrated that this sequence is sufficient to confer expression in axenically growing cells and to induce gene activity in development after growth on bacteria. A 16-base-pair sequence within this element was shown to be sufficient for induction in the discoidin promoter context and was used to reactivate different truncated promoter constructs. This led to the localization of an element necessary for down regulation of gene expression by extracellular cyclic AMP.

A developmentally regulated membrane protein gene in Dictyostelium discoideum is also induced by heat shock and cold shock

We have analyzed the expression of the Dictyostelium gene P8A7 which had been isolated as a cDNA clone from an early developmentally regulated gene. The single genomic copy generated two mRNAs which were subject to different control mechanisms: while one mRNA (P8A7S) was regulated like the cell-type-nonspecific late genes, the other one (P8A7L) was induced during development, when cells were allowed to attach to a substrate, and when cells were subjected to stress, such as heat shock and cadmium. Interestingly the same induction was also observed with cold shock. RNA processing was inhibited by heat and cold shock, leading to nuclear accumulation of a precursor. The translated region of the cDNA was common to both mRNAs and encoded an unusually hydrophobic peptide with the characteristics of a membrane protein.

Dynamics of a novel centromeric histone variant CenH3 reveals the evolutionary ancestral timing of centromere biogenesis

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, α-helix 2 (α2) 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.

In vitro and in vivo action of antisense RNA

The transient or permanent expression of antisense RNA represents one option to apply antisense techniques in biotechnology and medical research. Despite the increasing importance and use of antisense nucleic acids as well as their significant antisense-specific phenotypic effects in vivo, there is an obvious lack of explanation for the mechanism of their action. By studying naturally occurring antisense RNA and analyzing their mechanism of action we attempt to learn more about the design, the use, and the critical parameters of artificial antisense RNA. Attempts to derive models from biochemical and structural studies for the interactions between antisense RNAs and their targets will be discussed.