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microRNA strand selection: Unwinding the rules

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Abstract microRNAs (miRNAs) play a central role in the regulation of gene expression by targeting specific mRNAs for degradation or translational repression. Each miRNA is post‐transcriptionally processed into a duplex comprising two strands. One of the two miRNA strands is selectively loaded into an Argonaute protein to form the miRNA‐Induced Silencing Complex (miRISC) in a process referred to as miRNA strand selection. The other strand is ejected from the complex and is subject to degradation. The target gene specificity of miRISC is determined by sequence complementarity between the Argonaute‐loaded miRNA strand and target mRNA. Each strand of the miRNA duplex has the capacity to be loaded into miRISC and possesses a unique seed sequence. Therefore, miRNA strand selection plays a defining role in dictating the specificity of miRISC toward its targets and provides a mechanism to alter gene expression in a switch‐like fashion. Aberrant strand selection can lead to altered gene regulation by miRISC and is observed in several human diseases including cancer. Previous and emerging data shape the rules governing miRNA strand selection and shed light on how these rules can be circumvented in various physiological and pathological contexts. This article is categorized under: RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs
miRISC loading and miRNA strand selection. (a) Schematic representation of Argonaute domains and their relative contributions to miRNA duplex binding and strand selection. The 5′ end of the miRNA guide strand is anchored into a 5′ nucleotide binding pocket (5′NBP) located within the MID domain of Argonaute, which prefers to bind miRNA strands with Uracil at their 5′ ends. The interface of the MID and PIWI domains form a tract that senses the relative TS of each duplex end. The 3′ end of the miRNA guide strand associates with the PAZ domain of Argonaute (modeled after Gerbert and MacRae, 2018). (b) Consequences of miRNA strand selection. (top) The miR strand targets specific mRNAs based on sequence complementarity of the miRNA seed (nucleotides 2–7) to the 3′UTR of target mRNAs. (bottom) As miR* strands contain different seed sequences that their corresponding miR strands, they are expected to target a different set of mRNAs if they are loaded into miRISC. (c) Features associated with guide (miR) and passenger (miR*) strands. In general, the 5′ ends of miRNA guide strands tend to start with Uracil and be situated on the end of the miRNA duplex with lower thermodynamic stability, whereas passenger strands are located on the end of the miRNA duplex with higher thermodynamic stability and tend to start with less favorable nucleotides such as Cytosine (Modeled after Meijer, Smith, & Bushell, 2014)
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Overview of miRNA biogenesis. (1) Genomically‐encoded miRNAs are transcribed within the nucleus and form a stem‐loop structure that is (2) processed by the Microprocessor complex comprising Drosha and DGCR8/Pasha. (3) The processed miRNA is then transferred to the cytoplasm through Exportin‐5 (XPO5). (4) The stem‐loop precursor miRNA is processed again by Dicer to remove the loop, and the miRNA duplex is loaded into an Argonaute protein. (5) The guide strand of the miRNA duplex is selected for loading into Argonaute to form the miRNA‐induced silencing complex (miRISC), while (5′) the other passenger strand is ejected from the Argonaute and degraded. (6) The miRISC targets mRNAs for silencing based on the seed sequence of the loaded miRNA guide strand
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Summary of miRNA strand selection and regulatory mechanisms. Three major points of regulation for miRNA strand selection (1) altered miRNA processing by the Microprocessor complex (not shown) or Dicer, (2) remodeling of the miRNA duplex by nontemplated RNA modifications such as uridylation or A‐to‐I editing, and (3) changes in the strand preference of Argonaute. Once presented with a miRNA duplex, Argonaute makes a binary choice to load one miRNA strand and discard the other strand from miRISC. Alternate Argonaute programming leads to shifts in the target profile of miRISC based on the seed sequence of the loaded strand and is sometimes associated with human diseases
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Examples of dysregulated miRNA strand selection in human disease. (a) Under wild‐type conditions, the miRNA guide strand is selected and loaded into miRISC in a preferential fashion. As the passenger strand is degraded, mRNAs matching the seed of the miR* strand are maintained in an active state whereas seed‐matched targets of guide‐loaded miRISC are repressed. (b) In BRCA1‐BAP1 deficient breast cancer cells, which cannot perform homologous recombination, miR‐223 arm switching leads to down‐regulation of miR‐223* targets and expression of miR‐233 mRNA targets (Srinivasan et al., 2019). (c) In squamous cell carcinoma (SCC), miR‐21* is upregulated and both miR‐21 and miR‐21* are loaded into miRISC and independently repress mRNAs that contribute to SCC pathogenesis (Ge et al., 2016). (d) A point mutation in miR‐133 (U‐C) associated with human atrial fibrillation alters the relative thermodynamic stability of the miR‐133 duplex ends and leads to selection of miR‐133* (Ohanian, Humphreys, Anderson, Preiss, & Fatkin, 2013). Presumably, altered mRNA target specificity by miR‐133* contributes to atrial fibrillation associated with this variation
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Potential mechanisms influencing miRNA 5p/3p arm ratios. The two mechanisms that may affect the relative abundance of miRNA arms are strand selection and miRNA turnover. We propose that strand selection is likely the dominant mechanism leading to altered arm ratios, as miRNA turnover would be expected to influence miRNA arm ratios after a dominant miRNA strand is selected. Mechanisms influencing the production of isomiRs (highlighted in bold) have been experimentally demonstrated to influence 5p/3p ratios of miRNAs and can lead to miRNA arm switching, whereas other mechanisms remain largely hypothetical
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Regulation of miRNA strand selection. (a) Sequential processing of miRNAs by the Microprocessor complex (top) and Dicer (bottom) generates a miRNA duplex. (b) Altered miRNA processing leads to the production of templated isomiRs and may have changes in 5′ nucleotide identity or relative duplex‐end stabilities. C. elegans miR‐51 isomiRs are shown as an example. (c) Contribution of uridylation to strand selection of miR‐324 (Kim et al., 2020). In wild‐type tissues expressing low levels of terminal 3′ uridylases (TUTases), nonuridylated miR‐324‐5p is selected for miRISC loading. In Glioblastoma, high expression of TUTases leads to uridylation of miR‐324‐3p, altered Dicer specificity and reversed strand selection toward miR‐324‐5p. (d) Contribution of TRBP to miRNA strand selection. TRBP is proposed to maintain the fidelity of Dicer processing (Lee & Doudna, 2012). In the absence of TRBP, Dicer generates isomiRs, which may lead to altered duplex characteristics that influence strand selection
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Not all miRNAs follow the rules. Shown are the percentages of miRNAs that do not follow the 5′ nucleotide selection rule, thermodynamic duplex‐end stability rule, or either rule in (a) Caenorhabditis elegans (n = 189 miRNAs), (b) Drosophila melanogaster (n = 219), and (c) humans (n = 832). miRNA sequences were obtained from miRBase (Release 22.1) and analyzed for duplex formation using RNAduplex (ViennaRNA package v2.4.14) with default parameters. The RNA secondary structure represented in dot‐matrix format was first examined for unpaired, overhanging bases at each duplex end. To determine duplex‐end stabilities, four terminal base pairs at each duplex end were loaded into RNAduplex for terminal minimum free energy (MFE) calculation. ΔΔG for duplex ends was calculated as follows: ΔΔG = Δguide − Δpassenger, where Δguide was the four base‐pair MFE value for the 5′ end of the guide miRNA and Δpassenger was the four base‐pair MFE for the 5′ end of the miRNA*. miRNAs were not considered to follow the 5′ nucleotide selection rule if the 5′ nucleotide of the passenger strand was identical to or more favorable than the 5′ nucleotide of the guide strand. The preferred 5′ nucleotide was defined as U > A > C > G (Frank et al., 2010). miRNAs with equal or higher thermodynamic stability on the guide end of the duplex compared to the passenger end of the duplex were not considered to follow the thermodynamic stability rule. (d) miRNA reads observed in small RNAseq experiments (“Strand abundance”) are thought to originate from the same duplex. (e) Hypothetically, for any given miRNA, miRNA reads observed in small RNAseq experiments may originate from distinct duplexes generated through alternative processing
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Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs
Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
RNA Processing > Processing of Small RNAs

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