Subolesin, discovered and characterized in I. scapularis
as a tick protective antigen [7
], is an evolutionary conserved protein which is involved in modulation of tick blood digestion, reproduction and development [10
]. In other organisms, subolesin orthologs may be involved in the control of developmental processes [16
]. Although the function of subolesin is unknown, these results suggest a conserved function for subolesin. Because of the profound effect of subolesin knockdown in ticks and other organisms [10
], our hypothesis was that subolesin may have a role in gene expression, thus affecting multiple cellular processes. Therefore, the objective of this study was to provide evidence of the role of subolesin in gene expression. To test this hypothesis, three experiments were conducted. In the first series of experiments, subolesin-interacting proteins were identified and characterized in R. microplus
, suggesting the interaction of subolesin with regulatory proteins. Therefore, in the second series of experiments, the effect of subolesin knockdown was analyzed in I. scapularis
and showed the effect of subolesin on gene expression affecting different biological processes. Finally, post-translational modifications were predicted for tick subolesin. All together, the results of these experiments suggested a role for tick subolesin in gene expression.
To identify proteins that interact with subolesin in yeast two-hybrid experiments, we used a cDNA library obtained from tick eggs because subolesin is expressed in tick embryos and gene knockdown affects egg development [8
]. Two genes, GI and GII, were identified encoding for proteins that interact with tick subolesin. These proteins contained domains and post-translational modification sites found in proteins with regulatory functions. The transduction/transcription domain found in GI is present in phosphorylated proteins involved in transcriptional regulation and other cell functions related to gene expression [19
]. The EF1_alpha_II and EF1_alpha_III domains present in GII are found in proteins with different functions such as protein biosynthesis, DNA binding, transcriptional regulation, RNA processing, structural constituent of cytoskeleton as well as ATP and GTP binding that are also involved in gene expression (see for example proteins with accession numbers [Genbank:EAY95388
The results reported herein suggested that subolesin may interact with regulatory proteins. In other organisms, subolesin orthologs interact with proteins with gene expression regulatory activities. The human subolesin ortholog interacts with LNXp80 [Genbank:AK056823
], DIPA [Genbank:NM_006848
] and SPG21 [Genbank:NM_016630
] proteins. LNX is an E3 ubiquitin-protein ligase that mediates ubiquitination and subsequent proteasomal degradation of Numb, implicated in the control of cell fate decisions during development. DIPA interacts with the viral phosphoprotein hepatitis delta antigen (HDAG) and acts as a repressor of gene transcription [21
]. SPG21 binds to the hydrophobic C-terminal amino acids of CD4 which are involved in repression of T cell activation [22
]. In D. melanogaster
, the subolesin ortholog bhringi
; CG8580) may act to regulate Twist activity through recruitment of the chromatin remodeling Brahma complex [17
]. Therefore, subolesin may exert its effect on gene expression through the interaction with GI, GII and possibly other regulatory proteins. Interestingly, the GII knockdown phenotype was similar to that obtained with subolesin, suggesting that these proteins may functionally interact in ticks.
The results of the microarray analysis of gene expression profile in ticks after subolesin knockdown provided evidence for the role for subolesin in gene expression. Subolesin knockdown affected the expression of genes involved in multiple cellular pathways. The nonspecific effect of dsRNA injection on tick global gene expression was not addressed in these studies. However, the injection of an unrelated dsRNA did not affect the expression of selected genes differentially expressed after subolesin knockdown. Although we cannot rule out off-target effects of subolesin RNAi in ticks, evidence suggested that this was not a likely possibility to explain the effect of subolesin knockdown on tick gene expression pattern. Firstly, we did not find complementary sequences between subolesin and identified differentially expressed genes that could support off-target effects of subolesin RNAi. To search for complementary sequences between subolesin and identified differentially expressed genes, we used the approach proposed by Birmingham et al. [23
] who showed that although maximum complementarity by itself is an unsatisfactory predictor of off-target RNAi effects, a highly significant association exists between off-targeting and exact complementarity between the seed region (bases 2–8) of siRNA and their off-targeted gene 3' untranslated region (UTR). Secondly, the analysis of D. melanogaster
subolesin ortholog RNAi off-target effects demonstrated the presence of a single off-targeted gene [24
], suggesting that off-target effects of subolesin RNAi may also be minimal in ticks.
A common characteristic of many regulatory protein sequences is the presence of phosphorylation sites. Although we did not demonstrate phosphorylation of tick subolesin, there is evidence that the human ortholog protein undergoes phosphorylation at serine 21 (AS*PKRRR) [25
], a PKC phosphorylation site that is conserved in tick sequences. Therefore, as with other regulatory proteins, subolesin may be regulated by reversible phosphorylation by PKC.