Cholinesterase-Targeting microRNAs Identified in silico Affect Specific Biological Processes

MicroRNAs (miRs) have emerged as important gene silencers affecting many target mRNAs. Here, we report the identification of 244 miRs that target the 3′-untranslated regions of different cholinesterase transcripts: 116 for butyrylcholinesterase (BChE), 47 for the synaptic acetylcholinesterase (AChE-S) splice variant, and 81 for the normally rare splice variant AChE-R. Of these, 11 and 6 miRs target both AChE-S and AChE-R, and AChE-R and BChE transcripts, respectively. BChE and AChE-S showed no overlapping miRs, attesting to their distinct modes of miR regulation. Generally, miRs can suppress a number of targets; thereby controlling an entire battery of functions. To evaluate the importance of the cholinesterase-targeted miRs in other specific biological processes we searched for their other experimentally validated target transcripts and analyzed the gene ontology enriched biological processes these transcripts are involved in. Interestingly, a number of the resulting categories are also related to cholinesterases. They include, for BChE, response to glucocorticoid stimulus, and for AChE, response to wounding and two child terms of neuron development: regulation of axonogenesis and regulation of dendrite morphogenesis. Importantly, all of the AChE-targeting miRs found to be related to these selected processes were directed against the normally rare AChE-R splice variant, with three of them, including the neurogenesis regulator miR-132, also directed against AChE-S. Our findings point at the AChE-R splice variant as particularly susceptible to miR regulation, highlight those biological functions of cholinesterases that are likely to be subject to miR post-transcriptional control, demonstrate the selectivity of miRs in regulating specific biological processes, and open new venues for targeted interference with these specific processes.


INTRODUCTION
MicroRNAs (miRs) are small RNA molecules which target many mRNA transcripts, leading to their post-transcriptional silencing (Bartel, 2009). Many mRNAs can be silenced by multiple miRs and miRs often target more than one mRNA participating in a particular biological function (Bartel, 2009). Together, this suggests that the miR networks affecting specific mRNA transcripts may provide useful information on the biological roles in which these transcripts are involved. Cholinesterases are involved in many biological functions (Massoulie, 2002). However, miR-132 is the only miR so far that has been experimentally validated as targeting AChE, with consequences on inflammatory responses (Shaked et al., 2009). To delineate additional miRs which might regulate cholinesterase functions, we explored the 3 -untranslated regions (3 -UTR) of human cholinesterase transcripts (acetyl-and butyrylcholinesterase, AChE, BChE; Soreq and Seidman, 2001).
Given that several of the proteins involved in a specific function are often repressed by the same miR (Girardot et al., 2010), changes in a particular miR might down-regulate the entire process. Hence, we surmised that those functions that are shared by cholinesterases and the other targets of the cholinesterase-complementary miRs would be more susceptible for being affected by miR control than other processes. That concept is schematically presented as a workflow in Figure 1.

MATERIALS AND METHODS
MicroRNA candidates were identified on each of the 3 -UTR sequences of AChE and BChE, which are 235, 1030, and 478 nucleotides long for BChE, the major "synaptic" AChE-S variant and the stress-inducible AChE-R variant, respectively (Figure 2A). We used the PicTar 1 , miRanda 2 , miRbase 3 , and microCosm 4 algorithms to identify these transcript-specific miRs. All predictions ensured a threshold P-value < 0.05, and analysis specifications allowed both evolutionarily conserved and non-conserved miRs, which enabled us to include primate-targeting miRs as well.
Validation of miR-target interactions generally involved a 3 UTR luciferase assay. In some cases, it was complemented by protein blots, real-time RT-qPCR, microarrays, transgenic technology, β-galactosidase, or GFP-tagged targets. See, for example the Shaked et al. (2009) report for several of the latter technologies used to explore the miR-132 target AChE, and  for the "classical" 3 -UTR and transgenic approaches, in exploring p250GAP which is also a miR-132 target.
To search for gene ontology (GO) categories which are also relevant for the other mRNA targets of cholinesterase-related miRs, we used the DAVID functional annotation clustering tool 5 . For each of the miRs identified as targeting one of the cholinesterases we searched for other experimentally validated targets; and we then used the lists of the other validated targets as gene lists for the DAVID search. Each list was normalized to the entire human genome, which served as a background.

RESULTS
We identified 116,81,and 47 miRs (24,8, and 20 miRs/100 nucleotides) that are complementary to the 3 -UTR domains of the BChE, AChE-R, and AChE-S transcripts, respectively. Of these, 6 miRs target both BChE and AChE-R whereas 11 miRs are common to both AChE-R and AChE-S, but BChE and AChE-S do not share any miR ( Figure 2B). Positions of the identified miRs are presented in Figure 2C, with miR-132 targeting a similar seed domain localized at the very 3 -end of the 3 -UTR in both the AChE-S and AChE-R transcripts. Of the cholinesterase-targeting miRs, seven had multiple binding sites to the target AChE-S, nine to AChE-R, and seven to the BChE transcript, suggesting that they have a higher prospect for being functional (John et al., 2004). Compatible with the different conceptual principles on which each of the algorithms employed is based, only 8.6, 17, and 13.7% (7/81), (8/47), (16/116) of the miRs identified as targeting AChE-R,AChE-S, and BChE, respectively, were predicted by more than one of the algorithms. For AChE-R, these are hsa-miR-  These cholinesterase-targeting miRs and their other validated non-cholinesterase targets are listed in Tables 1-3 with the corresponding functions attributed to these other targets. The relevant citations appear in Tables A1-A4 in Appendix. Of note, numerous cholinesterase-targeting miRs have no experimentally validated targets at this time, yet others have more than one validated target and associate with more than one biological function. Examples include miR-124 which targets both the AChE-S and IQGAP1- (Furuta et al., 2010), a GTPase activating protein which promotes neurite outgrowth ( Table 1). Additionally miR-152 and miR-148a, which target AChE-R, also target the calmodulin regulating kinase CaMKIIα  Table 2). Lastly, the BChE-targeting cluster of miRs-222 and -221 also target the neuronal early immediate protein c-fos (Ichimura et al., 2010;    hsa-miR-146b-3p IRAK1 (IL1 receptor-associated kinase 1) EGFR (epidermal growth factor receptor) MMP16 (degrades extracellular matrix)  We focused our survey on those functions of those miRs for which experimental validation is available. Table 4 presents these miRs which are shared for AChE-R and AChE-S or AChE-R and BChE and some of their additional targets, highlighting the multitude of miR targets with predicted regulatory functions (e.g., the chromatin modulator zinc finger proteins ZEB1 and ZEB2 targeted by miR-200b, miR-200c, and miR-429 that are also directed to both AChE-R and AChE-S; Gregory et al., 2008). Likewise, the AChE-S-targeted miR-132 (Shaked et al., 2009;Soreq and Wolf, 2011) also targets the GTPase regulator p250GAP Frontiers in Molecular Neuroscience www.frontiersin.org involved in neurite extension (Vo et al., 2005;Hansen et al., 2010; Table 4).
The process-regulation hypothesis of miR function predicts the existence of biological functions in which both cholinesterases, and those other targets which share miRs with cholinesterases, would be involved. To challenge this hypothesis, we first identified the GO categories in which AChE and BChE are involved, and found 24 and 11 biological processes for these two proteins, respectively. Twenty-three, 13, and 18 enriched biological processes emerged as shared processes for the other validated targets of AChE-R, AChE-S, and BChE-targeting miRs, respectively (P-value threshold < 0.05).