Περίληψη |
Gene silencing, also known as RNA interference (RNAi), is a conserved
mechanism of regulation of gene expression mediated by small RNAs (sRNA), (Fire et
al., 1998). Silencing of transgenes and endogenous genes following introduction of
inverted repeats, antisense constructs and artificial miRNAs has been reported in diatom
species, including the model species P. tricornutum (De Riso et al., 2009; Kaur and
Spillane, 2015). The presence of an endogenous RNAi pathway has been suggested after
comprehensive and combinatorial analyses of sRNAs, gene expression and DNA
methylation in P. tricornutum (Veluchamy et al., 2013; Rogato et al., 2014). This RNAi
pathway in P. tricornutum may play a role in the regulation of protein coding genes and
TEs expression with possible consequences for the acclamatory response to nutrient
limitation (Maumus et al., 2009). Homologues of the RNAi-key genes DICER (DCR),
ARGONAUTE (AGO) and RNA-Dependent RNA polymerase (RDR) have been
previously identified by in silico analysis (De Riso et al., 2009). However, the validation
of their gene models, the characterization of their functions and the possible
physiological role of RNAi in diatoms are still lacking.
In this study, extensive in silico analysis of genomic and transcriptomic information
available in P. tricornutum suggests the presence of a single PtDCR, PtAGO and PtRDR
coding gene. Mining and phylogenetic analysis of DCR, AGO and RDR homologues in
diatoms from all publically available to date sequence datasets suggest an unanticipated
diversification of the RNAi pathway in these organisms. PtDCR/AGO/RDR cDNA were
cloned and splicing isoforms of PtDCR and PtAGO were identified. Subcellular
localization of PtDCR-/AGO-/RDR-YFP was investigated by confocal microscopy.
Functional characterization of PtDCR and PtAGO was first attempted by heterologous
expression in the yeast Saccharomyces cerevisiae and the plant Nicotiana bethamiana
hosts. In a second step, CRISPR/Cas9-mediated mutagenesis approach, recently
developed in P. tricornutum, was successfully harnessed to generate PtDCR-KO and
PtAGO-KO (KnockOut) lines. Growth phenotype of PtDCR-KO lines were investigated
under optimal and nitrate depleted culture conditions and during recovery from UVmediated stress. In parallel, mRNA and small RNAs whole transcriptome analyses were
carried out. Culture experiments suggest that PtDCR may play a role in the response to nitrate starvation. Transcriptomic analysis revealed that both sRNA and mRNA
transcriptomes were affected in PtDCR-KO line. At the global scale, sRNA size
distribution was found to shift towards larger fragment size in PtDCR-KO line. In
addition, the abundance of sRNA mapped to TEs was found dramatically reduced in
PtDCR-KO mutant and a concomitant increase in mRNA abundance of some TEs was
observed. Interestingly, PtDCR-KO sRNA transcriptome also presented changes in
tRNA-derived sRNA populations, suggesting a possible role of DCR in their processing
in diatoms. TE mobilization has been proposed to play a pivotal role in diatom species
diversification and capacity for adaptation to various environments. Taken together, our
results indicate that the single DCR encoding gene present in P. tricornutum plays a
major role in the production of TE-derived sRNAs and possibly TE mobilization, with
important consequences for diatom acclamatory response and evolution.
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