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Pdsg1 and Pdsg2, novel proteins involved in developmental genome remodelling in Paramecium.

Arambasic M, Sandoval PY, Hoehener C, Singh A, Swart EC, Nowacki M - PLoS ONE (2014)

Bottom Line: We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny.Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development.Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University of Bern, Bern, Switzerland.

ABSTRACT
The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.

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Effect of PDSG1 depletion on the retention of injected IES (51A2591).Lanes 1 and 2, control non-silenced cells. Lanes 3 and 4, control silencing with empty vector (EV). Lanes 5 and 6, PDSG1 depleted cells. Lanes 7 and 8, DCL2 and DCL3 depleted cells. Lanes 2, 4, 6 and 8, IES 51A2591 injected cells. IES-mtA promoter (control mcIES).
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pone-0112899-g004: Effect of PDSG1 depletion on the retention of injected IES (51A2591).Lanes 1 and 2, control non-silenced cells. Lanes 3 and 4, control silencing with empty vector (EV). Lanes 5 and 6, PDSG1 depleted cells. Lanes 7 and 8, DCL2 and DCL3 depleted cells. Lanes 2, 4, 6 and 8, IES 51A2591 injected cells. IES-mtA promoter (control mcIES).

Mentions: In order to determine the role of Pdsg1 in the scanning process we monitored the retention of a specific IES (51A2591) during the depletion of Pdsg1. In wild type strain the IES-51A2591 is normally excised after development and its excision was not affected in PDSG1-KD strains (Fig. 1D, IES number 4). First, we challenged the system by injecting the IES-51A2591 into the maternal MAC, which will promote the retention of this specific sequence in the progeny MAC, as previously reported [8] (Fig. 4, lane 2). As a control, and to monitor the normal progression of development, both strains were subjected to feeding with E. coli producing RNA from empty vector construct (Fig. 4, lanes 3 and 4). Since empty vector control does not include target sequences for silencing, in the progeny of the wild type background IES-51A2591 was correctly excised (Fig. 4, lane 3). Conversely, under this condition in the injected strain the retention of IES-51A2591 was passed to the progeny as anticipated (Fig. 4, lane 4). Next, PDSG1 was silenced in wild type strain and compared to the IES-51A2591 injected strain (Fig. 4, lanes 5 and 6 respectively). As expected, the depletion of Pdsg1 in wild type background did not promote the retention of IES-51A2591 (Fig. 4, lane 5). However, the silencing of PDSG1 promoted complete excision of the IES-51A2591 in the injected strain (Fig. 4, lane 6). This suggests that the depletion of Pdsg1 has a negative effect on the scanning process, arguing that Pdsg1has a direct functional role in the scanning. Furthermore, elimination of the scnRNA production (Dcl2/3 silencing, Fig. 4, lanes 7 and 8) was used as a positive control of the disruption of the scanning process. In this case IES-51A2591 was retained in both strains due to the absence of scanning molecules (scnRNAs).


Pdsg1 and Pdsg2, novel proteins involved in developmental genome remodelling in Paramecium.

Arambasic M, Sandoval PY, Hoehener C, Singh A, Swart EC, Nowacki M - PLoS ONE (2014)

Effect of PDSG1 depletion on the retention of injected IES (51A2591).Lanes 1 and 2, control non-silenced cells. Lanes 3 and 4, control silencing with empty vector (EV). Lanes 5 and 6, PDSG1 depleted cells. Lanes 7 and 8, DCL2 and DCL3 depleted cells. Lanes 2, 4, 6 and 8, IES 51A2591 injected cells. IES-mtA promoter (control mcIES).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232520&req=5

pone-0112899-g004: Effect of PDSG1 depletion on the retention of injected IES (51A2591).Lanes 1 and 2, control non-silenced cells. Lanes 3 and 4, control silencing with empty vector (EV). Lanes 5 and 6, PDSG1 depleted cells. Lanes 7 and 8, DCL2 and DCL3 depleted cells. Lanes 2, 4, 6 and 8, IES 51A2591 injected cells. IES-mtA promoter (control mcIES).
Mentions: In order to determine the role of Pdsg1 in the scanning process we monitored the retention of a specific IES (51A2591) during the depletion of Pdsg1. In wild type strain the IES-51A2591 is normally excised after development and its excision was not affected in PDSG1-KD strains (Fig. 1D, IES number 4). First, we challenged the system by injecting the IES-51A2591 into the maternal MAC, which will promote the retention of this specific sequence in the progeny MAC, as previously reported [8] (Fig. 4, lane 2). As a control, and to monitor the normal progression of development, both strains were subjected to feeding with E. coli producing RNA from empty vector construct (Fig. 4, lanes 3 and 4). Since empty vector control does not include target sequences for silencing, in the progeny of the wild type background IES-51A2591 was correctly excised (Fig. 4, lane 3). Conversely, under this condition in the injected strain the retention of IES-51A2591 was passed to the progeny as anticipated (Fig. 4, lane 4). Next, PDSG1 was silenced in wild type strain and compared to the IES-51A2591 injected strain (Fig. 4, lanes 5 and 6 respectively). As expected, the depletion of Pdsg1 in wild type background did not promote the retention of IES-51A2591 (Fig. 4, lane 5). However, the silencing of PDSG1 promoted complete excision of the IES-51A2591 in the injected strain (Fig. 4, lane 6). This suggests that the depletion of Pdsg1 has a negative effect on the scanning process, arguing that Pdsg1has a direct functional role in the scanning. Furthermore, elimination of the scnRNA production (Dcl2/3 silencing, Fig. 4, lanes 7 and 8) was used as a positive control of the disruption of the scanning process. In this case IES-51A2591 was retained in both strains due to the absence of scanning molecules (scnRNAs).

Bottom Line: We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny.Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development.Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University of Bern, Bern, Switzerland.

ABSTRACT
The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.

Show MeSH
Related in: MedlinePlus