Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia

Abstract

In many eukaryotes, RNA-dependent RNA polymerases (RdRPs) play key roles in the RNAi pathway. They have been implicated in the recognition and processing of aberrant transcripts triggering the process, and in amplification of the silencing response. We have tested the functions of RdRP genes from the ciliate Paramecium tetraurelia in experimentally induced and endogenous mechanisms of gene silencing. In this organism, RNAi can be triggered either by high-copy, truncated transgenes or by directly feeding cells with double-stranded RNA (dsRNA). Surprisingly, dsRNA-induced silencing depends on the putatively functional RDR1 and RDR2 genes, which are required for the accumulation of both primary siRNAs and a distinct class of small RNAs suggestive of secondary siRNAs. In contrast, a third gene with a highly divergent catalytic domain, RDR3, is required for siRNA accumulation when RNAi is triggered by truncated transgenes. Our data further implicate RDR3 in the accumulation of previously described endogenous siRNAs and in the regulation of the surface antigen gene family. While only one of these genes is normally expressed in any clonal cell line, the knockdown of RDR3 leads to co-expression of multiple antigens. These results provide evidence for a functional specialization of Paramecium RdRP genes in distinct RNAi pathways operating during vegetative growth.

The 5′ Spreading of Small RNAs in Dictyostelium discoideum Depends on the RNA-Dependent RNA Polymerase RrpC and on the Dicer-Related Nuclease DrnB

“…One explanation for this latter observation could be that RrpA and RrpB be responsible for the siRNAs that are seen in the antisense RNA expressing rrpC gene deletion strain (Fig. 5A), similar to the recently reported specialization of RdRPs in P. tetraurelia [16]. If that was the case, it might serve to explain the disappearance of antisense RNA derived siRNAs in the triple rrp deletion strain.…”

“…The siRNAs that trigger the amplification are referred to as primary siRNAs and they can lead to the generation of secondary siRNAs by RdRP activity, which thus amplifies the silencing signal, as summarized in [15]. Individual RdRPs have been shown to have distinct functions in a given organism, as exemplified by the three enzymes of the ciliate Paramecium tetraurelia [16].…”

RNAi Effector Diversity in Nematodes

“…Further examples of RdRP-catalyzed amplification mechanisms have recently been reported in Paramecium tetraurelia , where multiple RdRPs appear to exist [53], and in Drosophila , where a non-canonical RdRP has been identified [54]. The most well-conserved RdRP in our dataset is EGO-1 ( Enhancer of Glp-One [ glp-1 ]), which appears in seven species (Table 2).…”

Diversity of RNA interference pathways in regulation of endogenous and exogenous sequences expression in ciliates Tetrahymena and Paramecium

“…Введение в клетки T. thermophila методом электропорации трансгенной последовательности, с которой транскрибируются гомологичные тому или иному гену формирующие двунитевые шпильки молекулы РНК, вызывает накопление 23-24 н. siРНК и значительное снижение количества мРНК гомологичных эндогенных последовательностей [11]. Как и большинство siРНК, микроРНК и piРНК других эукариот, эти малые РНК почти всегда содержат U на 5'-конце, и для половины этих siРНК 3'-концевой нуклеотид, обыч но тоже U, видимо, добавляется внематрично [10] [12,13], или кормлением инфузорий бактериями Escherichia coli, продуцирующими дцРНК (так называемый feeding) [14,15]. В последнем случае нужную последовательность клонируют в вектор между направленными навстречу друг другу промоторами T7 и трансформируют им E. сoli, которые затем при индукции IPTG усиленно продуцируют дцРНК.…”