The central nervous system (CNS) is a remarkably complex organ system, requiring an equally complex network of molecular pathways controlling the multitude of diverse cellular activities. Gene expression is a critical node at which regulatory control of molecular networks is implemented. As such, elucidating the various mechanisms employed in the physiological regulation of gene expression in the CNS is important both for establishing a reference for comparison to the diseased state and for expanding the set of validated drug targets available for disease intervention. MicroRNAs (miRNAs) are an abundant class of small RNA that mediates potent inhibitory effects on global gene expression. Recent advances have been made in methods employed to study the contribution of these miRNAs to gene expression.37 Here we present a methodological workflow from the perspective of an investigator studying the physiological regulation of a gene of interest. We discuss methods for identifying putative miRNA target sites in a transcript of interest, strategies for validating predicted target sites, assays for detecting miRNA expression, and approaches for disrupting endogenous miRNA function. We consider both advantages and limitations, highlighting certain caveats that inform the suitability of a given method for a specific application. Through careful implementation of the appropriate methodologies discussed herein, we are hopeful that important discoveries related to miRNA participation in CNS physiology and dysfunction are on the horizon.
Our lab has studied the mechanisms of cell-type-specific regulation of APP, APOE, and BACE1 genes, focusing primarily on promoters and 5'-flanking regions38–43. The present novel findings of miRNA's role on protein expression, focusing on the 3’-UTR of APP and BACE1, add significantly to our previous studies on gene regulation. Herein, we posit that although miRNA binds a specific seed sequence in target mRNA 3'- UTR, the variation in UTR length might prevent a miRNA's binding and, thus, affects its activity in a particular cell type. In this study, we demonstrated that miR-298 significantly reduced APP and BACE1 levels in human astrocytes at both protein and mRNA levels. However, miR-298 does not alter APP or BACE1 levels in other human cell lines. Surprisingly, the effects of one miRNA could be so diversified and even contrasting in different cells. The cell-type-specific regulation by miRNAs has not been well studied. Few cases have been reported, but the exact mechanism of the phenomenon remains unclear.44
We embarked on this study to answer a fundamental question. It is known that miRNA regulates protein levels in a cell-type specific manner but how miRNA functions differently in various cell types remains elusive. We postulated that although miRNA binds a specific seed sequence in the 3'-UTR of a target mRNA, the variation in UTR length could prevent a miRNA's binding and thus affect its activity in a particular cell type. We reasoned that the natural 3'- UTR could be a full length (e.g., 1.12kb for APP-3'UTR and 4.36kb for BACE1-3'UTR) in a particular cell type or truncated (undefined) in another. The main objective of our work was to address this issue as far as miRNA's function is concerned.
We deliberated other methods to determine the exact sequences of target mRNA 3’-UTR in order to account for differential miRNA activity. For example, the RACE technique is generally used to obtain the full-length sequence of an RNA transcript found within a cell. RACE produces a cDNA copy of the RNA sequence of interest, generated through reverse transcription, followed by PCR amplification of the cDNA copies. It involves using one common primer that takes advantage of mRNA transcript poly(A) tail and another customized primer. In essence, the PCR amplification is one-sided PCR with single-sided specificity. Further, such a sequence is not sufficient to understand the activity of miRNA and hence its function in regulating cellular protein.
Instead of RACE, we utilized a miRnape to determine a natural "UTR stop" in a cell type. In essence, we selected several miRNAs that are located at different locations within APP 3'-UTR. We checked the function of each miRNA on UTR activity using the full-length UTR and by dual reporter assay. Then we checked the function of each miRNA on native protein levels. Transfection results of known miRNA on native protein expression would determine whether UTR is fully active (positive results) or truncated (negative results) in a particular cell type. We then matched the results by doing transfection experiment with a known full-length UTR. Using a range of miRNAs with binding sites spread across the same target mRNA transcript 3’-UTR, we could map the functional miRNA’s site within 3’-UTRmRNA. Our miRnape technique is useful and may prove to be complementary to RACE in understanding cellular function of a miRNA.
To explain our work, we also propose several potential hypotheses and additional explanations.
Scenario 1: Endogenous miR-298 levels vary in various cells. It is possible that in some cells miR-298 levels are sufficiently high that its targets are already saturated. Additional exogenous miR-298 mimics transfected into cells could not further reduce endogenous APP and BACE1 levels. However, this hypothesis is not favored in our case based on the two pieces of evidence. First, endogenous miR-298 levels vary a little compared to exogenous miR-298 transfected. An about 5-fold elevation in neurons is not likely to saturate its target. Second, even if endogenous miR-298 had already saturated to its targets, exogenous miR-298 inhibitors should disengage that interaction and significantly increase APP and BACE1 levels, which is not the case.
Scenario 2: Alternative polyadenylation sites or SNPs are present within mRNA 3’-UTR targeted by miR-298. Our results suggest that endogenous APP protein was reduced only in U373 but not HMC3 (Suppl. Figure 2A-C) or neurons, and that a full-length APP 3’-UTR activity reporter activity was reduced in neurons when co-transfected with miR-298 mimics. These results indicate that endogenous APP 3’-UTR is not identical in all cell types. We consider two possibilities, either alternative polyadenylation site makes the APP 3’-UTR shorter in some cells or APP 3’-UTR has some SNPs in miR-298 binding region that reduces miR-298 binding affinity. However, the SNPs reported in the NCBI SNP database are located within or close to miR-101, miR-298, and miR-339 seed sequence binding sites on APP and BACE1 mRNA 3’-UTR, are of very low frequency (< 0.03%). The presence of such a low frequency of related SNPs would unlikely explain the miRNA effect differences observed by these rare SNPs, some of which are listed in Table 1.
Table 1
SNPs located within or close to miRNA seed sequence binding sites*
miRNA | Target | SNP | Frequency |
miR-101 | APP 3’-UTR | rs1568997318 | 0.000007 |
miR-101 | APP 3’-UTR | rs1439565783 | 0.000057 |
miR-101 | APP 3’-UTR | rs1260407227 | 0.000007 |
miR-101 | APP 3’-UTR | rs2036975815 | 0.000007 |
miR-298 | APP 3’-UTR | rs1479517600 | 0.000043 |
miR-298 | APP 3’-UTR | rs191651536 | 0.0002 |
miR-298 | APP 3’-UTR | rs2036942563 | 0.000004 |
miR-298 | APP 3’-UTR | rs2036942344 | 0.000004 |
miR-298 | APP 3’-UTR | rs1216676820 | 0.000007 |
miR-339 | BACE1 3’-UTR | rs570503330 | 0.000091 |
miR-339 | BACE1 3’-UTR | rs2034340128 | 0.000004 |
miR-339 | BACE1 3’-UTR | rs2034340051 | 0.00006 |
miR-339 | BACE1 3’-UTR | rs1159500293 | 0.000004 |
miR-339 | BACE1 3’-UTR | rs1053615732 | 0.000008 |
miR-339 | BACE1 3’-UTR | rs755195807 | 0.00015 |
miR-298 | BACE1 3’-UTR | rs910101318 | 0.000004 |
miR-298 | BACE1 3’-UTR | rs1037052324 | 0.000004 |
miR-298 | BACE1 3’-UTR | rs922820472 | 0.000019 |
* SNP information was obtained from dbSNP of National Center for Biotechnology Information. |
https://www.ncbi.nlm.nih.gov/snp/rs1568997318?vertical_tab=true |
Scenario 3: Some cis-acting elements bind to APP and BACE1 mRNA 3’-UTR and may prevent miR-298 from binding. Indeed, miRNA machinery in different cells work differently, even miR-298 and its target mRNA 3’-UTR are identical, its results would differ in different cells. In the case of exogenous APP 3’-UTR transfection experiments, since APP 3'-UTR are over-expressed transiently into the cells compared to endogenous APP mRNA, the abundance of exogenous APP 3’-UTR may make endogenous cis-acting elements, which bind native APP mRNA, unavailable. In other words, exogenous APP 3’-UTR might not be as tightly controlled as native APP mRNA.
To move the project further, sequence of native APP and BACE1 mRNA3’-UTRs would be important to check whether polyadenylation sites exist. Likewise, sequence of genomic DNA containing APP and BACE1 genes would confirm whether SNPs near miR-298 binding site might interfere with the binding. Further, transfection of miR-298 mimics in primary or induced pluripotent stem cell (ipsc) derived astrocytes and microglia as well as neurons derived from same patient would be an important model. The role of miR-298 in these cells would have critical biological significance.
In addition to Aβ generation, APP also plays critical roles in multiple biological and pathological processes, including synaptic pruning, inflammation, iron regulation and mild traumatic brain injury (mTBI).45–47 Indeed, insufficient pruning could be a potential cause of autism spectrum disorder.48,49
Likewise, BACE1 serves more than an APP cleaving enzyme and a pathogenic role in AD. BACE1, an aspartic protease, has many other native substrates in the brain, such as neuregulin50, seizure protein 651 and sodium gated voltage channel β2 (Navβ2)52, which are important for neuron function and biology. Other BACE1 substrates are involved in cell signaling and immunity including Golgi localized membrane-bound α−2,6–sialyltransferase (ST6Gal I)53, interleukin-1 type II receptor (IL1R2),54 P–selectin glycoprotein ligand–1 (PSGL-1)55 and low density lipoprotein receptor–related protein (LRP).56 By reducing BACE1 levels in astrocytes instead of neurons, we could possibly inhibit pathological Aβ production while saving the physiological functions BACE1 has in neurons. Same line of reason, APP is also not just amyloid producing. APP is involved in multiple signaling pathways by interacting with other proteins.
We have also considered the translational implication of our work based on miRnape. We suggest that truncated but natural, 3’-UTR found provides an avenue to regulate native protein levels by a particular miRNA in a cell type-specific manner. In short, while a traditional chemical or drug would have access to any cells, miRNA's (e.g., miR-298) biological effects can be tailored to a specific cell type (e.g., astrocytic line) over another undesired cell type (e.g., differentiated NB) with a 3'UTR truncated enough to lack a miRNA binding site. Likewise, other miRNAs and their target 3’UTRs can be tested by the miRnape method. Future work is also warranted to study different scenarios and potential outcomes, as described above, to achieve optimal miRNA activity in regulating native protein expression.