How RNA dependent RNA polymerases control retrotransposons and thereby warrant genomic integrity


The integrity of eukaryotic genomes is under constant threat from external sources like UV radiation, viruses or mutagenic chemicals, and internally from mobile genetic elements. Lack of counter measures and control mechanisms can lead to a plethora of undesired outcomes, and, in consequence the development of serious diseases, including cancer. In this project, we wish to study how the class of RNA template dependent RNA polymerases (RdRPs) acts in the important control of retrotransposons. RdRPs form 3’-5‘ phosphodiester bonds between ribonucleotides, and their presence is almost ubiquitous from viruses to higher eukaryotes. In viruses, RdRPs are responsible for viral genome replication and in cellular organisms they mediate gene silencing by RNA interference, DNA methylation and heterochromatin formation. In the model organism Dictyostelium discoideum, the three RdRPs RrpA-C are encoded, and RrpA and RrpC have been reported by the host laboratory to be important for silencing of the retrotransposons DIRS-1 and Skipper. These mobile genetic elements form the centromeric sequences, thus they must be kept in a heterochromatic state as otherwise separation of duplicated chromosomes would be impeded with potentially fatal consequences. How this is achieved biochemically is poorly understood. Hence, we plan to determine the enzymatic activity, kinetic and thermodynamic parameters of RrpA and RrpC. They will be purified by Streptavidin affinity purification from strep-tagged over expression clones in Dictyostelium discoideum, and used in in vitro reactions with retrotransposon RNA substrates. Thermodanymic properties of these RdRPs will be analyzed by isothermal titration calorimetry. The importance of five phosphorylation sites in RrpC will be determined by the exchanging amino acids by site directed mutagenesis and assaying the activity of the mutant enzymes. We also plan to investigate the poorly understood mechanism of DIRS1 retrotransposition that can be observed in rrpC gene deletion strains by Northern blotting and RNA FISH. Furthermore, we will examine and compare the siRNA populations in the wild type and RdRP mutants by deep RNA sequencing. This work will determine the biochemical properties of RdRPs, and mechanistic details of retrotransposition and retrotransposon silencing. Importantly, we expect to gain novel insights into the role of RdRPs controlling retrotransposons, and thus the maintenance of genomic integrity.

Key words: Dictyostelium discoideum, RdRPs, RNA FISH, DIRS-1, genomic integrity


Cellular functions of the RdRPs have been analyzed by generating deletion strains of rrp gene using a convenient one-step cloning protocol for gene deletion vectors (1), both individually, and also in all possible combinations. The fact that even the triple knockout strain (rrpA¾:rrpB¾:rrpC¾) could be obtained, indicated that this gene family is not essential in the amoeba (2).

Figure 1: Currently discussed mechanisms of RNA-dependent RNA Polymerases (RdRPs). (A) Primary siRNAs are reaction products of the RNase III enzyme Dicer that can process various substrates with stretches of double-stranded RNA. (B) One of the siRNA strands could interact with an RdRP, which might be part of a multi-protein complex as shown. The siRNA strand bound to this RdRP would act on a cognate mRNA as guide (D) or primer (E). In the latter case, the resulting long RNA double strand might be a Dicer substrate, which would generate secondary siRNAs that are chemically identical to the primary siRNAs. In the guiding mode, secondary siRNAs would be de novo synthesized and are characterized by a 5’-triphosphate. In a primer-independent mode, the RdRP would recognise hitherto unknown features in the RNA target that render it into a suitable substrate.


To analyze the activity of the RdRPs, a lacZ reporter system was developed (2), in which a colorimetric enzyme assay was used to assess the silencing effect of RNA constructs against the lacZ. This allowed to determine that RNAi regulation takes place in D. discoideum predominantly on the translational level and the low levels of β-gal siRNAs were detected in antisense RNA expressing rrpC knock out strains only, but in contrast to this, and at significantly higher levels, all hairpin RNA expressing strains featured β-gal siRNAs. Distribution of the silencing signal to mRNA sequences 5′ of the original hairpin trigger was observed in all strains, except when the rrpC gene was deleted or that of the dicer-related nuclease DrnB (2). This study thus answered several important questions on RNA silencing in D. discoideum: transitivity of an RNA silencing signal exists in the amoeba, and it requires, next to DrnB, the RdRP RrpC. Further, RrpC is involved small RNA generation in both, antisense RNA-induced silencing and hairpin RNA-induced RNAi, but these two mechanisms appear to overlap only partially.


Earlier, the host group observed that RrpC is involved in the regulation of the Dictyostelium intermediate repeat sequence 1 (DIRS-1), (3). DIRS-1 is the first member of a poorly characterized class of long terminal repeat (LTR) retrotransposons featuring inverted instead of direct terminal repeats, lacking an aspartic protease domain and using a tyrosine recombinase instead of a DDE-type integrase protein for integration. In the genome of amoeba the occurrence of ~40 intact copies and ~200–300 DIRS-1 fragments, making it the most frequently occurring LTR retrotransposon D. discoideum (4). In D. discoideum DIRS-1 sequences accumulate at one end of each chromosome, and these clusters represent the centromeric region of each chromosome (5-6). Genome integrity is severely affected due to the uncontrolled amplification of DIRS-1 elements and its integration into centromeric regions. DIRS-1 is transcribed as a 4.5-kb-long messenger RNA (mRNA) with three overlapping open reading frames (ORFs; Fig. 2), (7). The left LTR possesses promoter activity to drive the transcription of the DIRS-1 mRNA (8). These DIRS-1 mRNA transcripts were found to accumulate during D. discoideum development and contain parts of the two LTRs (8). Heat shock or other stress conditions can trigger the expression of a 900-nt-long antisense transcript “E1” (Fig. 2.) additionally (9-10).



Figure 2. Schematic illustration of the DIRS-1 retrotransposon with the left and right inverted long terminal repeats. The three open reading frames (ORFs), encoding the GAG protein (ORF I), the tyrosine recombinase (ORF II) and reverse transcriptase/RNase H domain and a methyltransferase (ORF III) are displayed collectively with the 900-nt E1 antisense transcript.


In a recently published study (11), the host lab has detailed the regulation of DIRS-1 by RrpC. In either deletion strains of RrpC, full-length and shorter DIRS-1 messenger RNAs are strongly enriched. In DIRS-1 antisense orientation a novel long non-coding RNA (approx. 4000 nts) was discovered and to discriminate it from the 900-nt E1 antisense transcript that is produced under stress conditions it was termed as lasDIRS-1 (9-10). The right LTR of DIRS-1 (Fig. 2) serves as promoter of lasDIRS-1. Using a newly established protocol for RNA-FISH in D. discoideum (12), DIRS-1 sense and antisense transcripts were shown to accumulate in nuclei of the rrpC strain, but not the wild-type.

Concurrent with the accumulation of long transcripts in rrpC strains, the vast majority of small (21 mer) DIRS-1 RNAs vanished as shown by Northern blotting. In the AX2 wild-type strain RNASeq revealed an asymmetric distribution of the DIRS-1 small RNAs, both along DIRS-1 and with respect to sense and antisense orientation (Fig. 3). If these small RNAs were products by one of the two Dicer proteins in the amoeba, one should expect a symmetric distribution in sense and antisense orientation. The observation that this is not the case suggests that they are direct RdRP products, likely formed either in the primer-independent or in the guide mode. This interpretation was beautifully confirmed by a deep sequencing analysis of the rrpC strain, in which the vast majority of the siRNAs had disappeared (Fig. 3), identifying RrpC as the main RdRP acting in the production of DIRS-1 siRNAs.

Figure 3. Distribution of DIRS-1-specific small RNAs. (A) Schematic representation of the DIRS-1 sequence (for details Fig. 5). (B) Small RNA reads from the wild-type AX2 strain are displayed separately for sense and antisense orientations, and together in (C). (D) shows the small RNA reads in the rrpC strain. All reads are mapped onto the DIRS-1 sequence in (A). Reads are displayed in the same scale, ranging from 0 to 400.000 reads and the signal height reflects the number of small RNAs. Regions with particularly visible deviations between the AX2 and rrpC strains are boxed (I, II, III, IV).



To address the mechanism by which the RrpC-dependent DIRS-1 siRNAs silence DIRS-1, the host group made use of GFP-fusions of the three ORFs of DIRS-1 (Fig. 4) and transformed them as extrachromosomal vectors in the AX2 wild-type and the rrpC strain. Expression of the two ORFs (ORF II & ORF III) was not detectable in the AX2 wild-type, while the expression of ORF I-GFP fusion protein was readily expressed in both strains, as monitored by Western blotting (Fig. 4A, B). This indicates that the RrpC-dependent siRNAs that are present in the AX2 wild-type strain are essential and adequate to silence post-transcriptionally ORF II and ORF III, but not ORF I of the retrotransposon, even though siRNAs in the 3′ half of ORF I are mostly antisense to the coding transcript. With the β-Galactosidase reporter system, the host group had shown that RrpC is essential for the appearance of small RNAs 5′ of the original silencing signal with respect to the target RNA.


Figure 4. Overexpression of DIRS-1 ORFs. C-terminal GFP fusions in the AX2 wild-type and in the rrpC strain shown by DIRS-1 ORF I (A) and ORF II or ORF III (B). The expression of the fusion proteins was monitored by western blotting using a GFP specific antibody. (C) Small GFP RNAs from both AX2 wild type and rrpC strains were analyzed by northern blot using a strand-specific radioactively labeled probe. The sizes of a radioactively labeled RNA markers are shown to the left. Ethidium bromide-stained rRNA served as loading control, and northern blots using a sno6 riboprobe acted to monitor transfer efficiency. (D) GFP only expression in AX2 wild-type cells. (E) Small GFP containing RNAs expression in both the above mention strains. In (A, B and D), severin or discoidin were used as loading controls, and the sizes of a protein marker are shown. M in (C and E) denotes small RNA size markers.





This phenomenon is usually referred to as transitive silencing, in which the target RNA is thought to serve as the template, from which an RdRP synthesize a complementary RNA strand. Due to the directionality of all nucleic acid polymerases, such a secondary silencing signal is expected to localize 5′ of the original trigger. However, in C. elegans Pak and Fire reported that a subset of secondary RNAs could also localize 3′ of the original trigger an observation that otherwise is mainly seen in the plant kingdom. Northern blot analyses was performed to identify small RNAs derived from GFP, to check whether 3′ spreading might also be observed in the case of the DIRS-1 ORF-GFP fusion constructs,. Significant amounts of small GFP-specific sense RNAs were detected in the ORF II-GFP and ORF III-GFP expressing cells in the AX2 wild-type background, but not in the rrpC strain (Fig. 4C). GFP expression construct alone was transformed into the AX2 wild-type cells, to test that GFP itself does not cause the generation of siRNAs, which resulted in GFP expressing cells (Fig. 4D) and did not cause generation of small GFP RNAs as monitored by northern blotting (Fig. 4E). To serve as a control for the functionality of the blot, the inclusion of RNA from the C-terminal GFP fusions of the DIRS-1 ORFs was used, otherwise it would appear empty. Because GFP was fused C-terminally to the DIRS-1 ORFs, this result indicates that distribution of an RNA silencing signal in the 3′ direction exists in D. discoideum and depends on the occurrence of RrpC. We note that a weak signal for small GFP RNAs is also present in strains where the fusion protein is not silenced, but not in the untransformed AX2 wild-type strain (Fig. 4E).


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