Summary:
The present PhD Thesis has focused on the study of the small RNA pathways in
the silkworm Bombyx mori, highlighting the factors that affect the efficiency
of the RNAi mechanism. The factors examined here, mostly, involved the
expression of key-genes belonging to the RNA machinery and the presence of a
viral infection (persistent or pathogenic), while the mechanisms of the innate
immune response and the dsRNA uptake ability from the extracellular medium were
also investigated. Analysis of the results yielded important findings and
conclusions regarding the impact of the aforementioned factors on the
efficiency of silkworm’s RNAi mechanism.
Data resulting from the functional analysis of RNAi mechanism response in Bm5
cells support a possible overlap of well-characterized pathways in other
insects (for example Drosophila), such as miRNA, siRNA and piRNA, under certain
conditions. More specifically, four basic genes that participate in the
dsRNA-mediated silencing process in the silkworm are highlighted here: BmAgo1
of the miRNA pathway, BmDcr2/BmAgo2 of the siRNA pathway and BmAgo3 of the
piRNA pathway. The implication of BmDcr2 and BmAgo2 was expected due to the
literature available in Drosophila. However, the indicated role of BmAgo3 in
gene-silencing caused by dsRNA, and not ssRNA, molecules motivated the
necessity of a systematic analysis regarding the role of the two genes in piRNA
pathway of B. mori.
Based on the acquired data, BmAgo3 seems to be implicated into a dynamic
network together with other genes (BmAgo2, BmeIF-4E1 and BmHSP90), which are
important for the effective function of RNAi in the silkworm, thus providing
more evidence on the possible synergistic action of siRNA and piRNA pathways.
Nevertheless, while BmAgo3 clearly plays a substantial role in RNAi, its
expression at normal levels is not necessary, at least for the survival of
silkworm cells, where it has been permanently suppressed by 2/3 through the
expression of a RNA hairpin. In sharp contrast, according to our results, BmAub
is not considered as important for gene-silencing through RNAi, but it could be
possibly crucial for the survival of silkworm cells, as it was impossible to
obtain a transformed cell line where this gene could be permanently
downregulated.
While being aware of the susceptibility of Bm5 cells to dsRNA-mediated gene
silencing by transfection, it was additionally attempted to investigate the
dsRNA uptake ability from the extracellular medium through a likely novel and
unknown dsRNA transfer channel, with similar mode of action to CeSID-1 protein
in the nematode C. elegans. Nevertheless, preliminary experiments of cell
soaking in a dsRNA-containing medium did not show significant impact on the
expression of each target gene, while expression of BmR2D2 and BmTranslin2
factors did not seem to improve the capacity of the cells to take up dsRNA, and
therefore the efficiency of gene-silencing following soaking.
Next, the effect of expression levels of certain RNAi factors of B. mori on
viral infections in Lepidopteran cells was examined. It was demonstrated that
under persistent infection with the RNA virus BmMLV, the permanent suppression
of the piRNA gene BmAgo3 favors the increase of viral levels. Conversely, in
case of an acute infection with the recombinant DNA virus AcMNPV-EYFP,
expression of BmR2D2 and BmTranslin2 leads to a reduction of viral growth
inside the cells, and to a concomitant increase of viral particles
concentration in the extracellular medium. Moreover, it is interesting that the
hairpin RNA-mediated suppression of Ago3 and Aub genes favors the growth of
AcMNPV-EYFP virus. Hence, it is concluded that certain siRNA and piRNA factors
control crucial functions that are related to the progress of a viral infection
in Lepidopteran cells.
To further elucidate the involvement of RNAi mechanism in viral infections, the
capability of the RNA virus BmCPV to auto-regulate its own genes’ levels by
expressing precursor microRNAs from its genome was investigated. As it was
shown, this particular virus expresses a pre-miRNA that targets the genome
segment S10 coding for polyhedrin, which is able to regulate the levels of this
specific protein (Zografidis et al., 2015). Here, it was examined whether the
expression of the recombinant protein VP1 is being controlled by the expression
of pre-miRNA S1.2. However, the results did not lead to clear conclusions, as
the protein expression levels were either unaffected, or increased due to the
presence of pre-miRNA S1.2.
Next, the discovery of persistent infection by BmCPV virus in the silkworm
colony of Daizo strain maintained in our lab was accordingly used, so as to
study its effect on a pathogenic infection with the same virus. The existence
of a persistent infection preceding the pathogenic one does not seem to
significantly affect the antiviral response against BmCPV, as it is concluded
by the comparison with already published reports in the literature. This study
revealed that the transcriptome response of the silkworm midgut against the
pathogenic infection with BmCPV is a complex process, which embraces a variety
of different mechanisms, with the RNAi mechanism being critically represented
among them (BmDcr2, BmAgo2 and BmeIF-4E1) and the innate immunity one to a
lesser extent as well. Moreover, regarding the RNAi mechanism, the present
study has shown that BmDcr2 and BmAgo2 genes under normal conditions present
elevated expression levels in the midgut, in comparison to tissues which do not
form a normal infection route for pathogens in the rest of animal’s body, such
as epidermis. Apart from the aforementioned mechanisms, the response against
BmCPV includes genes controlling cellular processes such as: immune response,
proteolysis, apoptosis, stress, detoxification, hormonal signaling, ecdysis and
DNA replication. Finally, the detection of 20 nucleotides (nt) viral small RNAs
(vsRNAs), through next generation sequencing (NGS), constitutes a strong proof
of the RNAi mechanism activation, as a defense mechanism against the viral
infection by BmCPV, irrespectively the type of infection (persistent or
pathogenic).
Our findings reveal possible overlaps of the main RNAi pathways in the silkworm
in vitro, during the gene-silencing process mediated by dsRNA (miRNA, siRNA and
piRNA), or hairpin RNA (siRNA and piRNA). Interestingly, except for their
implication in the dsRNA-mediated silencing, the genes BmDcr2 and BmAgo2 of the
siRNA pathway have proved to operate as key factors in silkworm’ s midgut, both
under normal and viral infection (BmCPV) conditions. Additionally, BmAgo3 of
the piRNA pathway is an important component affecting the efficiency of the
RNAi mechanism in the silkworm, while it can also, together with BmR2D2 and
BmTranslin2, and likely BmAub as well, play a crucial role in the antiviral
response (BmMLV and AcMNPV) in Lepidoptera. Furthermore, the presence of a
persistent infection does not seem to negatively influence the efficiency of
the RNAi mechanism (BmMLV) and the antiviral defense (BmCPV) of the silkworm.
Finally, the classical pathways of the endogenous innate immunity of B. mori do
not respond strongly during antiviral defense (BmCPV) and therefore they do not
seem to significantly interact or crosstalk with the RNAi machinery, so as to
affect its proficiency. However, the present research highlights new genes that
presumably play strategic roles in the antiviral response of the silkworm in
combination with the RNAi mechanism.
