REVIEW

published: 03 June 2020

doi: 10.3389/fcell.2020.00409

MicroRNAs: From Mechanism to

Organism
Philipp J. Dexheimer and Luisa Cochella*
Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria

MicroRNAs (miRNAs) are short, regulatory RNAs that act as post-transcriptional

repressors of gene expression in diverse biological contexts. The emergence of small
RNA-mediated gene silencing preceded the onset of multicellularity and was followed
by a drastic expansion of the miRNA repertoire in conjunction with the evolution of
complexity in the plant and animal kingdoms. Along this process, miRNAs became
an essential feature of animal development, as no higher metazoan lineage tolerated
loss of miRNAs or their associated protein machinery. In fact, ablation of the miRNA
biogenesis machinery or the effector silencing factors results in severe embryogenesis
defects in every animal studied. In this review, we summarize recent mechanistic insight
into miRNA biogenesis and function, while emphasizing features that have enabled
multicellular organisms to harness the potential of this broad class of repressors. We
first discuss how different mechanisms of regulation of miRNA biogenesis are used, not
Edited by: only to generate spatio-temporal specificity of miRNA production within an animal, but
Alessandro Rosa, also to achieve the necessary levels and dynamics of expression. We then explore how
Sapienza University of Rome, Italy
evolution of the mechanism for small RNA-mediated repression resulted in a diversity of
Reviewed by:
Stuart A. Newman,
silencing complexes that cause different molecular effects on their targets. Multicellular
New York Medical College, organisms have taken advantage of this variability in the outcome of miRNA-mediated
United States
repression, with differential use in particular cell types or even distinct subcellular
Silvana Papagerakis,
University of Saskatchewan, Canada compartments. Finally, we present an overview of how the animal miRNA repertoire
*Correspondence: has evolved and diversified, emphasizing the emergence of miRNA families and the
Luisa Cochella biological implications of miRNA sequence diversification. Overall, focusing on selected
[email protected]
animal models and through the lens of evolution, we highlight canonical mechanisms
Specialty section: in miRNA biology and their variations, providing updated insight that will ultimately
This article was submitted to help us understand the contribution of miRNAs to the development and physiology of
Stem Cell Research,
a section of the journal multicellular organisms.
Frontiers in Cell and Developmental
Keywords: miRNA, evolution, development, Argonaute, Drosha, Dicer, biogenesis, silencing
Biology
Received: 24 February 2020
Accepted: 04 May 2020
Published: 03 June 2020
INTRODUCTION
Citation:
The Emergence of Small RNA-Guided Effector Systems
Dexheimer PJ and Cochella L
(2020) MicroRNAs: From Mechanism
Regulation of gene expression by small RNAs emerged as an ancient feature of cellular biology
to Organism. and is found in all three domains of life (bacteria, archaea and eukarya). Having evolved
Front. Cell Dev. Biol. 8:409. primarily as a means of defense against foreign nucleic acids (Shabalina and Koonin, 2008;
doi: 10.3389/fcell.2020.00409 Obbard et al., 2009) the principle of small RNAs specifically guiding effector proteins to selected

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Dexheimer and Cochella MicroRNAs: From Mechanism to Organism

nucleic acids via antisense-complementarity is a recurrent theme

in biology. At their core, these RNA-based interference (RNAi)
systems consist of two components, a nucleic acid allowing for
sequence-specific target recognition, and an effector protein that
mediates downstream effects with varying outcomes.
In eukaryotes, the effector proteins that mediate the silencing
of target nucleic acids are part of the Argonaute protein
family. After their origin in prokaryotes (Makarova et al., 2009;
Olovnikov et al., 2013; Swarts et al., 2014a,b; Willkomm et al.,
2015), Argonautes diversified into a versatile class of effector
proteins, forming the core of various multiprotein regulatory
systems or RNA-Induced Silencing Complexes (RISC). They
all share common structural elements and the ability to bind
short, single stranded RNAs in a conformation that enables base
pairing with target RNAs (Steiner et al., 2007; Farazi et al.,
2008; Takeda et al., 2008; Czech and Hannon, 2010). The other
critical protein component of eukaryotic RNA-induced silencing
pathways are nucleases that process precursor RNAs into small
RNAs that can be loaded onto Argonaut proteins. A major
player in multiple RNAi pathways is Dicer, an RNAse III type
endonuclease that cleaves double-stranded RNA molecules to
generate targeting-competent small RNAs that guide the effector
machinery. Although no prokaryotic homolog of Dicer has been
found to date, the origins of individual domains can be traced
back to prokaryotes (Shabalina and Koonin, 2008). Together,
RNase III type endonucleases and Argonaute proteins lie at
the heart of diverse small RNA based pathways involved in
multifaceted aspects of molecular biology. FIGURE 1 | The evolutionary origins of miRNAs in eukaryotes. Two key
players in small RNA-mediated silencing, Argonaute proteins and RNase III
The different modules that constitute the eukaryotic RNAi like enzymes, originated in prokaryotes. The miRNA pathway in animals
systems likely originated in archaea, bacteria, and bacteriophages emerged with the birth of the Microprocessor, composed of Drosha and
(Koonin, 2017). This core structure has diversified, specialized, Pasha, in unicellular holozoans (Bråte et al., 2018). Diverse lineages that
and acquired new functions in the course of eukaryotic evolution. branched from the last eukaryotic common ancestor (LECA) also evolved
A key innovation in diverse lineages was the ability to not miRNA-like pathways. However, it is still under debate whether these evolved
independently in four additional clades: Slime molds, Green algae, Brown
only load and target foreign or parasitic RNAs, but also to Algae, and Land plants (Kruse et al., 2016; Valli et al., 2016; Cock et al., 2017;
feed endogenously produced RNAs into the existing RNAi Bråte et al., 2018); or if the pathway was already present in the last common
pathways to achieve gene regulation. This formed the basis for the ancestor (Moran et al., 2017).
emergence and expansion of the endogenous class of small RNAs
called microRNAs (miRNAs), whose regulatory potential has
been harnessed in animals, plants, and other eukaryotic lineages
for the establishment of elaborate gene regulatory networks that levels these accumulate to act effectively; and what the molecular
control development and physiology (Figure 1). consequences of their repression are.
A comprehensive understanding of the contributions of
What Is the Purpose of This Review? miRNAs thus requires bridging biochemical and mechanistic
The complexity of multicellular organisms relies on the insights with the organismal level. When and where does miRNA-
segregation of functions across many distinct cell types, which mediated regulation take place? How is the production of a
requires intricate gene regulatory mechanisms. As versatile specific miRNA controlled in space and time to achieve that? And
repressors of gene expression, miRNAs are thought to facilitate how do miRNAs integrate into cellular gene regulatory networks
the generation of different cell types with highly specialized that control development and physiology? Recent technological
physiology (Alberti and Cochella, 2017). It comes as no advances have enabled more quantitative assessment of the
surprise that the evolution of multicellular organisms has been kinetics of miRNA production and turnover, and context-
accompanied by an increase in complexity of the miRNA pathway dependent differences in composition of miRISC during distinct
and the miRNA repertoires (Hertel et al., 2006; Heimberg et al., developmental stages. This allows a more nuanced view of when,
2008; Wheeler et al., 2009; Berezikov, 2011; Guerra-Assunção where and how miRNAs are utilized within animal development
and Enright, 2012; Hertel and Stadler, 2015). The use of this and post-developmental processes. Moreover, the sequencing of
increased diversity of repressors has been exploited by the an increasing number of genomes has upgraded our ability to
addition of multiple mechanisms that control which miRNAs place miRNAs and the associated protein machinery into an
are produced at specific times and in specific cell types; at what evolutionary context. These layers of understanding will lead

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Dexheimer and Cochella MicroRNAs: From Mechanism to Organism

us to broader concepts of what miRNAs contribute to the cytoplasm is further cleaved by Dicer to give rise to a short RNA
phenotypic complexity observed in present-day organisms. duplex with characteristic 2-nt 30 overhangs. From this duplex,
We do not attempt to provide a comprehensive survey of one strand is preferentially loaded into an Argonaute protein
all the miRNAs that have been studied and their functions in to generate a functional miRNA-induced silencing complex
different animals, for which we suggest the following starting (miRISC) (Figure 2).
points (Chen et al., 2014; Alberti and Cochella, 2017; Bartel, Drosha and Pasha/DGCR8 originated in unicellular holozoans
2018). Instead, we emphasize general concepts from diverse after fungi branched from the pre-metazoan lineage (Bråte et al.,
mechanistic studies to understand how multicellular organisms 2018), placing the appearance of miRNAs before the onset
have exploited the potential of this broad class of repressors. We of multicellularity. However, plants do not encode a homolog
first focus on how miRNA biogenesis is controlled and discuss of Drosha or Pasha but process primary miRNA transcripts
how the different modes of regulation can affect miRNA function via the Dicer-homolog DCL1 (Dicer like 1), which generates
within an animal. Next, we discuss how different composition mature miRNAs according to a similar principle involving two
of the RISC complex and different cellular contexts can result sequential endonucleolytic cleavage events (Voinnet, 2009). This
in distinct outcomes of miRNA-mediated repression. Finally, we led to the initial hypothesis that miRNAs evolved independently
present an updated overview of how the miRNA repertoire has in animals and land plants. However, the possibility that
evolved and what this teaches us so far about how miRNAs have the last common ancestor of these lineages might have
acquired functionality during evolution. already employed a common miRNA-like pathway for post-
transcriptional regulation is not to be dismissed (Moran et al.,
2017). Nevertheless, after their introduction, the number of
Evolutionary Origins of the miRNA miRNAs continuously expanded during the course of evolution,
Pathway providing multicellular organisms with a diversified toolset for
In 1993, the product of the Caenorhabditis elegans gene lin-4 regulation of gene expression.
was surprisingly found to give rise to a small non-coding RNA,
processed from a longer hairpin precursor, which functions by miRNAs Are Essential for Development
repressing protein production from the lin-14 mRNA via an Regulation by miRNAs evolved into a key feature of multicellular
antisense RNA–RNA interaction (Lee et al., 1993; Wightman organisms. As general repressors of gene expression, miRNAs
et al., 1993). First thought of as a nematode peculiarity, it have been incorporated into gene regulatory networks that
became clear in the early 2000s that such endogenous small control diverse developmental and post-developmental
RNAs like lin-4, in particular let-7, play a fundamental role processes. No higher metazoan lineage has tolerated the
across eukaryotic lineages (Pasquinelli et al., 2000; Reinhart loss of essential miRNA pathway components. In fact, defects in
et al., 2000). The discovery of lin-4 and let-7, followed by the the miRNA biogenesis machinery or the effector silencing factors
identification of hundreds of endogenous small RNAs derived results in severe embryogenesis defects in every animal studied.
from hairpin precursors in animal genomes, laid foundation In C. elegans, removal of the miRNA-specific Argonautes
to the miRNA field (Lee and Ambros, 2001; Lau et al., alg-1 and alg-2 causes embryonic lethality with a predominant
2001; Lagos-Quintana et al., 2003; Aravin et al., 2003; Lim arrest in the morphogenetic process of elongation (Vasquez-Rifo
et al., 2003). Not only is the protein machinery associated et al., 2012). Disruption of ago1 in Drosophila causes embryonic
with miRNAs highly conserved, but so are many miRNAs – lethality with notable nervous system abnormalities (Kataoka
with >30 miRNAs shared across all bilaterian animals, and et al., 2001). Loss of Dicer in Zebrafish results in abnormal
hundreds of others conserved within specific clades. Moreover, gastrulation causing severe defects in brain development and
at least 37% of Drosophila and 60% of human protein- organogenesis. Notably, injection of a miR-430 duplex into
coding transcripts are subjected to selective pressure to retain embryos considerably rescued neuronal defects, indicating an
miRNA binding sites (Friedman et al., 2008; Agarwal et al., outstanding role of a single miRNA in early fish development
2018), underscoring the biological significance of miRNA- (Giraldez et al., 2005). Mouse embryos lacking Dicer1 or Ago2
mediated gene silencing. fail to undergo gastrulation, accompanied by a malformation of
miRNAs are 21–23 nt small RNAs that elicit post- germ layers (Bernstein et al., 2003; Alisch et al., 2007). Moreover,
transcriptional downregulation of protein output from their this phenotype is recapitulated in animals bearing a deletion of
target mRNAs. This is typically achieved through a combination Dgcr8 (Wang et al., 2007). In plants, disruption of DICER-LIKE1
of translational inhibition and promotion of mRNA decay (Jonas leads to embryonic arrest early in development, likely caused by
and Izaurralde, 2015; Bartel, 2018). The origin of miRNA-biology a failure to prevent precocious differentiation events (Nodine
lies in the evolution of a mechanism that allowed for processing and Bartel, 2010). Interpretation of these experiments should
of long endogenous transcripts into short RNA duplexes that consider possible miRNA-independent functions of disrupted
are further recognized and cleaved by Dicer. In animals this miRNA-pathway factors, as such roles have been demonstrated
is achieved by the Microprocessor complex, composed of for Drosha, Pasha and Dicer (Chong et al., 2010; Macias et al.,
the RNase III endonuclease Drosha and its co-factor Pasha 2012; Gromak et al., 2013; Luhur et al., 2014; Rybak-Wolf et al.,
(Partner of Drosha, or DGCR8 in vertebrates). Cleavage of 2014; Cirera-Salinas et al., 2017; Kim et al., 2017; Marinaro et al.,
a primary miRNA transcript by the Microprocessor in the 2017). However, given that removal of different components
nucleus releases a hairpin precursor, that upon export to the in the miRNA-pathway causes highly similar phenotypes, it

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Dexheimer and Cochella MicroRNAs: From Mechanism to Organism

FIGURE 2 | Schematic of the miRNA biogenesis pathway. The different steps leading to production of a mature miRNA are shown. Kinetic studies in Drosophila
revealed that biogenesis is fast for most miRNAs but loading into Argonaute represents the rate-limiting step (based on Reichholf et al., 2019).

is strongly suggested that miRNA-deficiency underlies the expressed with very high spatial and temporal specificity and may
observed defects. function predominantly in specialized cell types within complex
Whereas miRNAs are collectively essential in animals, multicellular organisms (Alberti and Cochella, 2017).
uncovering the functions of many individual miRNAs has been
challenging (Miska et al., 2007; Park et al., 2012; Chen et al.,
2014). This is in part due to redundancy (Ge et al., 2012), miRNA PRODUCTION AND ITS
most notably among miRNAs that fall into so-called families REGULATION
and share targeting specificity (Alvarez-Saavedra and Horvitz,
2010), but also redundancy with other repressive mechanisms Multicellular organisms have taken advantage of the potential
that contribute to the output of gene regulatory networks of miRNA-mediated regulation employing multiple mechanisms
(acting either at the RNA or protein levels). For example, to control the production of miRNAs in distinct cell-types,
in Drosophila, the near-complete loss of miRNA-mediated under varying conditions, or during different developmental
repression is tolerated to a large extent if general metabolism is stages. miRNA biogenesis begins with transcription of a primary
simultaneously slowed down (Cassidy et al., 2019). This suggests transcript by RNA polymerase II (Figure 2). Transcriptional
that under conditions of slow developmental tempo, the timely regulation will thus determine whether a miRNA will be
downregulation of targets or the establishment of the right produced in the first place, and in general miRNA abundance
protein levels, to which miRNAs contribute, might be achieved correlates well with the rates of primary-miRNA transcription
through compensatory mechanisms. In a number of cases, the (Reichholf et al., 2019). At the organismal level, there is
contribution of miRNAs has been revealed only in sensitized evidence that for most miRNAs, transcriptional control is the
genetic backgrounds or upon environmental perturbation, such main determinant of cell-type specificity (Alberti et al., 2018).
as exposure to extreme temperatures or pathogen stress. This However, the maturation of primary miRNAs, initially by the
suggests that the activity of some miRNAs is redundant with Microprocessor in the nucleus and later by Dicer in the cytoplasm
other factors that are influenced by environmental conditions (Figure 2), can also be regulated, contributing significantly to
(Hornstein and Shomron, 2006; Brenner et al., 2010; Ebert and the dynamics of accumulation and steady-state abundance of
Sharp, 2012; Cassidy et al., 2013, 2016, 2019; Siciliano et al., different miRNAs (Conrad et al., 2014). Moreover, it became
2013; Ren and Ambros, 2015; Ilbay and Ambros, 2019). It is also evident that regulation of miRNA decay plays an important role
likely that the function of many miRNAs has not been uncovered, in determining the functional levels of individual miRNAs (Zhou
even if they play non-redundant roles, because they are often et al., 2018; Reichholf et al., 2019).

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Dexheimer and Cochella MicroRNAs: From Mechanism to Organism

Transcription of each pri-miRNA is under the control of miRNA function, and further investigation of how these different
specific transcription factors and enhancers that outline their rates come about will provide critical insight. Nevertheless, this
expression patterns. For example, an elaborate transcriptional quantitative understanding already provides a framework for
mechanism determines the expression of the miRNA lsy-6 in determining the ability of miRNAs to reach the necessary cellular
a single neuron in C. elegans (Cochella and Hobert, 2012). In concentrations to execute their repressive functions.
contrast, post-transcriptional regulation of miRNA biogenesis Effective repression by miRISC requires a high concentration
is achieved at two different levels: (i) globally, by controlling of a miRNA relative to its target (Ameres and Zamore, 2013).
the shared core machinery that produces most miRNAs, e.g., To achieve this, synthesis of a number of miRNAs begins long
modulating levels or activity of the Microprocessor, and (ii) at before the onset of their repressive function. For example, the
the level of individual miRNAs, exploiting unique sequence and miRNA lsy-6 in C. elegans, which functions in a sensory neuron
structural features of diverse miRNA precursors which impact by repressing the transcription factor COG-1 (Johnston and
interaction with the biogenesis machinery. Such interactions are Hobert, 2003), is produced in the mother of the sensory neuron
often facilitated by various RNA binding proteins (RBPs). (Cochella and Hobert, 2012). Transcription of cog-1 begins in
Recent advances in the use of metabolic labeling of RNA the postmitotic neuron several hours later in a cell that has
have enabled kinetic studies of different steps during the life high concentration of lsy-6 (Cochella and Hobert, 2012). This
of a miRNA, and provided insight into how the rates of likely contributes to the ability of lsy-6 to act as a genetic
these steps vary for distinct miRNAs (Reichholf et al., 2019). switch by completely preventing COG-1 expression (Johnston
An in-depth analysis in Drosophila S2 cells revealed that the and Hobert, 2003; Cochella and Hobert, 2012). Another example
rates of mature miRNA biogenesis range from 17 to >200 of this is the essential C. elegans miRNA let-7, which is required
molecules/minute/cell. However, the loading into Argonaute is for transition of the last larval stage to adulthood, yet starts
significantly slower and in fact represents a kinetic bottleneck being transcribed in the first larval stage (Martinez et al., 2008;
in the production of functional miRISC. As a consequence, Van Wynsberghe et al., 2011). Taking into account the relative
a large fraction of the miRNA duplexes produced by Dicer dynamics of accumulation of a given miRNA and its target/s
(approximately 40%) is degraded before loading. Other reports offers helpful insight for understanding the contribution of that
found only <10% of cellular miRNAs are bound by Argonaute miRNA to specific cellular processes.
(Janas et al., 2012; Stalder et al., 2013). This seemingly wasteful These are the general forces that determine whether a miRNA
strategy ensures specific loading of Argonaute with miRNAs, is made and if so, to what level it accumulates relative to its
which compete against other abundant duplex RNAs originating targets. The core mechanisms that control each of these steps
from tRNAs, rRNAs, snRNAs, or snoRNAs (Reichholf et al., have been studied in great detail in different cell-based systems.
2019). Consistently, Argonaute binding is a better indicator of Multicellular organisms take advantage of various ways to adjust
the inhibitory potential of a miRNA than its overall concentration these mechanisms to generate the necessary spatio-temporal
(Flores et al., 2014). For this specificity mechanism to be effective, specificity and the dynamics of miRNA expression that support
Argonaute levels need to be limited. This is achieved in large development and homeostasis.
part through the relatively short half-life of empty Argonaute,
which is efficiently ubiquitinated and degraded (Diederichs and
Haber, 2007; Derrien et al., 2012; Janas et al., 2012; Martinez Drosha/DGCR8 and Dicer: The
and Gregory, 2013; Smibert et al., 2013; Kobayashi et al., 2019; Gatekeepers of miRNA Production
Reichholf et al., 2019; Bose et al., 2020). Processing of primary-miRNA transcripts (pri-miRNAs) initiates
Another important contributor to miRNA abundance is their co-transcriptionally (Lee et al., 2003; Lee Y. et al., 2004; Morlando
stability. While overall miRNAs are among the most stable et al., 2008; Ballarino et al., 2009). In Drosophila, Pasha/DGCR8
cellular RNAs, individual species can range in stability from can associate directly with RNA pol II via its phosphorylated
minutes to more than a day (Reichholf et al., 2019). Rates of decay C-terminal domain, linking transcription with the first step of
can be modulated by external stimuli to remodel the miRNome, miRNA maturation (Church et al., 2017). Interaction of DGCR8
as is the case for light-regulated retinal miRNAs (Krol et al., and Drosha with pri-miRNAs occurs via recognition of a hairpin
2010). In general, neuronal miRNAs in mammals appear to have secondary structure flanked by single-stranded RNA and leads to
a high turnover rate compared to other cell types, and their cleavage by Drosha to release a precursor hairpin (Lee et al., 2003;
abundance can be rapidly regulated in connection with neuronal Han et al., 2004; Sohn et al., 2007). The efficiency and the accuracy
activity (Krol et al., 2010). Together with the biogenesis rates, of this reaction are crucial determinants of miRNA abundance
the different decay rates determine the steady-state abundance of and targeting specificity, respectively. Because miRNAs bind their
miRNAs, such that two miRNAs can reach the same abundance targets primarily through nucleotides 2–8 relative to the 50 end
through contrasting strategies: a miRNA with slow biogenesis of the miRNA (the “seed” sequence), a change in cleavage site
and slow decay can accumulate to the same concentration as that affects the 50 end of a miRNA even by a single nucleotide
one with fast biogenesis and fast decay. The first case will result can drastically alter target specificity of that miRNA. The choice
in a stable concentration that is less likely to change upon of cleavage site by Drosha is crucial for setting the 50 end of
small perturbations, while the second is energetically more costly both miRNA arms: the 5p arm directly and the 3p indirectly by
but provides potential for dynamic regulation. Such different determining the register for the subsequent cleavage by Dicer
strategies for accumulation have important consequences for (Auyeung et al., 2013; Partin et al., 2017; Kwon et al., 2019).

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Dexheimer and Cochella MicroRNAs: From Mechanism to Organism

Interestingly, recent mapping of Drosha cleavage sites with single the Microprocessor. Indeed, not all wild-type pri-miRNAs are
nucleotide resolution in human cell lines (HEK293T and HeLa optimal targets for Drosha or DGCR8 binding, resulting in
cells), revealed that some miRNAs undergo alternative processing a broad range of processing efficiencies (Han et al., 2006;
resulting in different 50 and 30 ends (Kim et al., 2017). This has the Auyeung et al., 2013; Fang and Bartel, 2015; Kim et al., 2017),
potential to further diversify the miRNA-repertoire. and different degrees of sensitivity to the presence of co-
Once a pri-miRNA is processed to a precursor hairpin (pre- factors like for example DGCR8’s co-factor, the iron-containing
miRNA) in the nucleus, it will be exported and further cleaved porphyrin heme (Partin et al., 2017; Nguyen et al., 2018).
in the cytoplasm by Dicer (Bernstein et al., 2001; Grishok A number of sequence motifs that increase the specificity
et al., 2001; Ketting et al., 2001; Lee et al., 2002). Consistent and efficiency of processing by the Microprocessor have been
with its evolutionary origin before the appearance of miRNAs identified; for instance, apical elements in the hairpin, most
(Mukherjee et al., 2013), Dicer has a wider range of substrates prominently a UGU motif (Auyeung et al., 2013) and the
than the Microprocessor – it can cleave RNA duplexes of any mGHG-motif on the basal side, which seems to be a key
length, as long as they have a 2-nt 30 overhang (and in some cases determinant of cleavage site selection in many pri-miRNAs
even blunt-ended duplex RNA). The domain structure of Dicer (Kwon et al., 2019). Highlighting the importance of the pri-
serves as a molecular “ruler,” such that the two RNAse III active miRNA sequence and structure for its correct processing, a
sites are positioned at a defined length from the 30 overhang of the single nucleotide change in the apical loop of pri-mir-30c-1
pre-miRNA and determine the length of the mature small RNA found in some patients with breast and gastric cancer results
(MacRae et al., 2006, 2007). Dicer cooperates with other RNA in increased processing and thus higher miR-30c-1 abundance.
binding proteins, PACT or TRBP in humans, and Loquacious in This was attributed to enhanced binding of SRSF3, a protein
Drosophila (Treiber et al., 2019). This interaction can affect not of the SR family that promotes Microprocessor cleavage
only the efficiency of the processing, but also the length of the (Fernandez et al., 2017).
mature miRNA produced (Lee et al., 2006; Chakravarthy et al., The diversity in pri- and pre-miRNA sequences means that
2010; Fukunaga et al., 2012; Lee and Doudna, 2012; Zhu et al., different steps might be rate-limiting for individual precursors,
2018). Therefore, together with Drosha, Dicer defines not only and this has enabled the evolution of additional regulatory
the abundance but also the ends of the mature miRNAs that will mechanisms. A number of individual miRNAs are subjected
be loaded into Argonaute. to unique modes of regulation via specific interactions with
Because of its broader activity, Dicer processes not only different RNA-binding proteins (RBPs) (Choudhury et al., 2013;
pre-miRNAs, but also different endogenous and foreign duplex Treiber et al., 2017; Kooshapur et al., 2018; Michlewski and
RNAs to produce other classes of short interfering RNAs Cáceres, 2018; Downie Ruiz Velasco et al., 2019). A recent mass
(siRNAs). Whereas Drosophila has two different Dicer proteins, spectrometry-based screen using various human cell line lysates
one specialized for miRNA (Dcr-1) and the other for siRNA identified numerous RBPs that impact processing of subsets
biogenesis (Dcr-2) (Lee Y. S. et al., 2004), many other animals of miRNA-precursors and ultimately the expression of target
have a single type of Dicer. The involvement of this enzyme in mRNAs (Treiber et al., 2017). The binding specificity of many
two different pathways creates a bottleneck that in some animals of these seems to be determined by the terminal loop, in which
leads to competition between the two different types of small the RNA is single-stranded and typically exposed for contact with
RNA precursors. In C. elegans for example, downregulation of the other proteins (Choudhury and Michlewski, 2012; Treiber et al.,
endo-siRNA pathway results in an increase of miRNA-biogenesis, 2017). However, interestingly a few RBPs also bind the stem of
whereas induction of exogenous RNAi competes with both endo specific miRNA hairpins (Treiber et al., 2017). A complementary
siRNA and miRNA production. This suggests that, at least in in silico approach using published eCLIP-data has also identified
some contexts, Dicer can be limiting for small RNA production a number of novel RBP:pre-miRNA interactions affecting
(Zhuang and Hunter, 2011). processing of specific miRNAs (Nussbacher and Yeo, 2018).
Processing by the Microprocessor and by Dicer is subjected Some RBPs have been shown to recruit terminal nucleotidyl
to diverse regulatory mechanisms. These can either affect Drosha transferases, most commonly uridyl transferases or TUTases
and DGCR8 or Dicer themselves to broadly impact the biogenesis (Hagan et al., 2009). Post-transcriptional modifications of
of multiple miRNAs, or specifically regulate the maturation of precursor or mature miRNAs can impact the kinetics of
individual miRNAs, typically through the action of RNA binding biogenesis or silencing. Uridylation of mature miRNAs by
proteins (RBPs) that recognize unique features of primary and TUTases tends to promote decay. However, uridylation of pre-
precursor miRNAs. miRNAs may either promote decay or have stabilizing effects
and affect further processing by Dicer, depending on the 30 end
structure of the precursor (Thornton et al., 2014; Bortolamiol-
Global and miRNA-Specific Regulation Becet et al., 2015; Kim et al., 2015; Reimão-Pinto et al., 2015).
Determine the Rates of miRNA Terminal miRNA modification by TUTases occur in Bilateria
Production as well as in more basal animal clades such as Cnidaria and
Pri-miRNAs share some broad structural features, but they Porifera. In fact, TUTases acting on small RNAs were already
can differ substantially in the length of their stems, the present in the last common ancestor of all animals, underscoring
sequences in their loops, and the sequences flanking the hairpin, the fundamental involvement of these mechanisms in miRNA
posing a challenge for efficient and specific processing by biology (Modepalli and Moran, 2017).

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Dexheimer and Cochella MicroRNAs: From Mechanism to Organism

The general activities of Dicer and the Microprocessor can which acts as a conserved post-transcriptional repressor of let-
also be regulated, with global consequences for miRNA output. 7-family miRNA biogenesis (Moss et al., 1997; Yang and Moss,
A common regulatory mechanism is through post-translational 2003; Heo et al., 2008; Newman et al., 2008; Viswanathan et al.,
modifications of the enzymes themselves, or their cofactors 2008). LIN28 recruits TUTases that ultimately prevent Dicer-
(Treiber et al., 2019). Regulation of Dicer activity for example recognition and promote decay via the exonuclease Dis3L2
is affected by phosphorylation of its binding partner TRBP, (Hagan et al., 2009; Chang et al., 2013). Release of LIN28-
resulting in increased stability of the Dicer-TRBP complex mediated repression of let-7 maturation provides a temporal
and enhanced pre-miRNA processing activity (Paroo et al., switch in different contexts during progression of animal
2009; Xu et al., 2015; Warner et al., 2016). Dicer itself can development (Thornton and Gregory, 2012).
be phosphorylated on two conserved residues, resulting in However, most of the RBPs that affect biogenesis and loading
its nuclear translocation and inhibition in worms, mice and are ubiquitously or very broadly expressed, suggesting that they
humans (Drake et al., 2014). This inhibitory mechanism has may rather contribute to achieving homeostatic levels of specific
been implicated in the reduction of mature miRNA levels miRNAs. This could be necessary to compensate for individual
in the C. elegans germline, contributing to precise gene miRNA features that make them better or worse substrates of
expression changes in the oocyte-to-embryo transition (Drake the biogenesis machinery. It is also possible that some of these
et al., 2014). In general, post-translational modifications may RBPs have different expression levels or modifications in different
serve to integrate diverse cellular signaling pathways with the tissues that might directly contribute to the spatio-temporal
production of miRNAs. specificity of miRNA production. Another possibility is that some
Moreover, levels of the processing machinery can vary across regulators are used to restrict the production of miRNAs to
different cell types or under different conditions, also impacting specific sub-cellular localizations. An extreme example of this
the global miRNA output. A survey of Drosha expression is the synapse-specific maturation of miRNAs by Dicer upon
levels across different mouse tissues revealed differences in the neuronal activation (Sambandan et al., 2017).
range of 4–10-fold, with the brain having the highest and the
liver the lowest level of Drosha mRNA and protein (Sperber
et al., 2014). Lower Drosha expression enhances the inherent SILENCING MECHANISM – VARIATIONS
differences in processing efficiency of different pri-miRNAs, ON A THEME
resulting in the deregulation of subsets of miRNAs with specific
properties (Sperber et al., 2014). The contribution of some of The ancestral mechanism of small RNA guided effector proteins
these mechanisms to the development and physiology of animals involves irreversible destruction of targeted nucleic acids by
remains to be tested. Nevertheless, the dysregulation of the cleavage (Park and Shin, 2014; Moran et al., 2017). This miRNA
miRNA biogenesis machinery has been associated with diverse mode of action, which is usually accompanied by near-perfect
forms of cancer, typically resulting in a global repression of target complementarity, is prevalent in plants (Voinnet, 2009)
miRNA maturation (Lin and Gregory, 2015). and also observed in basal metazoan lineages like Cnidaria
The final step for producing a functional miRISC is the loading (Moran et al., 2014; Mauri et al., 2017). Bilaterian animals on
of a miRNA into Argonaute. This seems to be the rate-limiting the other hand, predominantly employ a mechanism that relies
step in a number of contexts (Diederichs and Haber, 2007; Janas on partial base pairing between the “seed” region of a miRNA
et al., 2012; Reichholf et al., 2019; Bose et al., 2020), likely related (nucleotides 2–8) and sequences typically in the 30 UTR of
to the fact that loading does not simply reflect binding of an mRNAs. Recruitment of miRISC in bilaterians usually results in
RNA to Argonaute, but follows a number of steps that require the downregulation of protein output through a combination
assistance from other factors, including chaperones and energy of translation inhibition and target mRNA decay (Jonas and
from ATP (Kobayashi and Tomari, 2016). As with every other Izaurralde, 2015; Bartel, 2018; Figure 3).
step of miRNA biogenesis, miRNA loading can also be affected Appearance of the slicing-independent, seed-based
by specific features of the miRNA duplex, such as presence of a 50 mechanism laid the foundation for functional diversification
phosphate, identity of the 50 terminal nucleotide and stability of of miRNA biology in animals. Compared to a mode of target
the duplex. These features also determine which of the two duplex recognition that involves full sequence complementarity, a
strands is preferentially loaded on Argonaute and acts as a mature mechanism that requires only partial base-pairing has the
miRNA; the opposite strand, or miRNA∗ , is typically rapidly potential to greatly increase the target repertoire of any miRNA.
degraded. In addition, specific RNA binding proteins that impact This likely expanded the overall potential of miRNA-mediated
the loading on Argonaute either positively or negatively have repression in bilaterian animals, enhancing the connectivity
been described. For example, TDP43 disrupts loading of miR-1 of gene regulatory networks, and contributing to cell-type
and miR-206, two muscle-specific miRNAs (King et al., 2014), diversification and acquisition of morphological complexity
while hnRNPD0 supports loading of let-7b (Yoon et al., 2015). during evolution (Peterson et al., 2009; Moran et al., 2017).
The effects of all these regulatory mechanisms at the
organismal level can in principle have two different types of The Metazoan Seed-Based Mechanism
consequences. On the one hand, post-transcriptional regulation The best understood mechanism of miRNA-mediated repression
of miRNA biogenesis may be used to encode temporal or spatial in animals relies on a protein of the GW182 family, which
information in a developing animal. Such is the case of LIN28, together with Argonaute forms the core of what is considered

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Dexheimer and Cochella MicroRNAs: From Mechanism to Organism

FIGURE 3 | Canonical miRNA-silencing mechanism in animals. miRNAs elicit repression of target genes usually through a combination of translational repression
and promotion of mRNA decay. Argonaute is guided by a miRNA to a cognate target mRNA and tethers GW182, forming the core of the most common animal
miRISC. GW182 interacts with PABP and recruits the deadenylase complexes CCR4-NOT or Pan2-Pan3 (not shown), leading to deadenylation, decapping and
ultimately exonucleolytic decay. Inhibition of translation occurs mainly at the initiation step by interfering with assembly or activity of eIF4F, via eIF4E-T, and DDX6.

the canonical miRISC. Members of the GW182 protein family Djuranovic et al., 2012). The relative contribution of these two
(e.g., GW182 in Drosophila, AIN-1/2 in C. elegans and TNRC6 mechanisms to biological function remains a matter of debate
in humans) are rapidly evolving but share Gly-Trp repeats (Eichhorn et al., 2014), but as we discuss below multiple pieces
that bind to conserved pockets in Argonaute proteins (El- of evidence indicate that this may be determined by the cellular
Shami et al., 2007; Zhang et al., 2007; Eulalio et al., 2008; context and miRISC composition.
Pfaff et al., 2013). Through other domains, GW182 proteins Despite the conserved nature of the canonical miRNA
recruit RNA-processing factors, repressing translation as well targeting mechanism, there is an increasing number of examples
as enhancing mRNA turnover (Chekulaeva et al., 2011; Fabian highlighting context-dependent variability in the mode and
et al., 2011; Kuzuoğlu-Öztürk et al., 2012; Huntzinger et al., functional consequence of miRISC targeting (Figure 4). This
2013). The precise mechanism for translational repression variability can arise from two different mechanistic sources.
remains to be resolved; however, the emerging consensus is First, miRISC can have non-canonical composition in different
that it involves inhibition of cap-dependent translation initiation cell types or distinct developmental stages (e.g., differential
via interaction with eIF4F (Pillai et al., 2004; Mathonnet presence or abundance of GW182 and other proteins that act as
et al., 2007; Zdanowicz et al., 2009; Ricci et al., 2013; Fukaya facultative Argonaute interactors). In metazoans, such variations
et al., 2014). GW182 proteins also recruit the PAN2-PAN3 are observed most notably between soma and germline, and we
and CCR4-NOT deadenylase complexes, ultimately triggering expand on this in the next section. Second, as perfect target
mRNA decay (Behm-Ansmant et al., 2006; Guo et al., 2010; complementarity is not a prerequisite, there are different possible
Braun et al., 2013; Eichhorn et al., 2014; Jonas and Izaurralde, miRNA:mRNA interaction modes (Lal et al., 2009; Shin et al.,
2015; Kuzuoğlu Öztürk et al., 2016; Niaz and Hussain, 2018; 2010; Chi et al., 2012). Whereas the majority of miRNA binding
Figure 3). GW182 has been proposed to promote the formation sites are located in the 30 UTR and involve pairing to the
of phase-separated condensates containing miRISC and target seed region of a miRNA, these features can differ with varying
mRNAs, increasing the local concentration of deadenylases functional consequences; from efficiency of silencing depending
and other factors for efficient repression (Sheu-Gruttadauria on the relative location of miRNA-binding sites within the target
and MacRae, 2018). GW182 is also present in Nematostella, mRNAs (Grimson et al., 2007; Forman and Coller, 2010; Zhang K.
where it is able to interact with Argonaute and the CCR4- et al., 2018); to degradation of the miRNA itself, if target pairing
NOT complex, suggesting that this mechanism originated in the extends to the 30 end of the miRNA (Ameres et al., 2010; Cazalla
last common ancestor between Cnidaria and Bilateria (Mauri et al., 2010; De la Mata et al., 2015; Bitetti et al., 2018; Ghini et al.,
et al., 2017). Experimental uncoupling of translational inhibition 2018; Sheu-Gruttadauria et al., 2019).
and mRNA decay has proven challenging, as the two processes
are intimately linked (Subtelny et al., 2014; Radhakrishnan
and Green, 2016). Still, analyses of the dynamics of miRNA- Variations in miRISC Composition and
mediated silencing in zebrafish early embryos and in mammalian Functional Outcome
cells in culture, revealed that miRNAs first repress translation The mode of miRNA targeting and the effects that different RISC
initiation and then induce mRNA decay (Bazzini et al., 2012; complexes will exert on their targets vary significantly across

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Dexheimer and Cochella MicroRNAs: From Mechanism to Organism

different eukaryotic clades. Even within a single animal, the Animals like C. elegans have adopted variant miRNA activity
composition and functional consequences of miRISC can differ in the germline, yet other animals seem to have evolved ways
in specific biological settings. The first evident variation is in to dampen or abolish germline miRNA activity altogether.
the Argonaute component itself, e.g., C. elegans evolved more In Drosophila oocytes, Ago-1 is present at very low levels
than 25 Argonaute proteins (Youngman and Claycomb, 2014). and only increases upon maternal to zygotic transition (Luo
ALG-1 and ALG-2 are the main miRNA-related Argonautes. et al., 2016), coinciding with the production of a miRNA
They function redundantly to a large degree, and only loss cluster involved in maternal mRNA decay (Bushati et al.,
of both simultaneously causes penetrant embryonic lethality 2008). Zebrafish zygotes also have low miRNA levels, with
(Vasquez-Rifo et al., 2012). However, they do display some considerable accumulation starting around the blastula stage
functional differences which may stem from differences in (Chen et al., 2005). At that time, miRNAs are also involved
expression patterns and levels (ALG-2 appears to be dominantly in the maternal to zygotic transition. Most notably, miR-430
expressed throughout embryogenesis) as well as in miRNA- is expressed at the onset of zygotic genome activation and
binding preference (Vasquez-Rifo et al., 2012; Brown et al., 2017). promotes maternal mRNA clearance (Giraldez et al., 2006).
Interestingly, another Argonaute, ALG-5, has recently been The mouse germline also appears to lack essential miRNA
implicated in miRNA-mediated silencing in the worm. ALG-5 is functions, as depletion of DGCR8 results in normal oocytes
expressed in the germline, where it associates effectively with only that give rise to healthy offspring upon fertilization with
a subset of miRNAs and plays a role in gametogenesis (Brown wild-type sperm (Suh et al., 2010). In the absence of both
et al., 2017). Insects such as cockroaches, from a basal clade, also maternal and zygotic DGCR8, zygotes still undergo normal
have two partially redundant Argonautes for miRNA mediated pre-implantation development but then arrest prior to E6.5
silencing (Rubio et al., 2018). However, subsequent specialization (Suh et al., 2010). Clearly, miRNA-mediated regulation plays
took place in more derived clades like Drosophila, which encodes important roles in early animal development, yet the contribution
one Argonaute for siRNA and another one for miRNA-mediated and mechanisms of action in the developmental window
silencing (Förstemann et al., 2007; Iwasaki et al., 2009). around fertilization remain an active area of investigation
Most of the variation in outcome of miRISC binding to (McJunkin, 2017).
an mRNA stems from differential association of Argonaute miRNAs provide an outstanding way to confer specificity to
with other proteins. This seems to primarily affect the relative a variety of repressor complexes. Different effector mechanisms
contribution of translation inhibition vs. mRNA destabilization. may operate within one organism, or even within one cell,
In particular, the larger differences appear to occur between and may be dynamically regulated under different conditions.
the germline or early embryo versus somatic tissues. In C. It will be interesting to find out how different modes of
elegans, the prevalent form of miRISC in the germline has regulation are used in different cellular contexts within animal
been shown to lack the GW182 proteins AIN-1 and AIN-2 development and physiology.
and instead associates with P-granule constituents. The resulting
recruitment of target mRNAs to P-granules inhibits protein
production but does not promote mRNA decay (Dallaire et al., INNOVATION THROUGH EXPANSION OF
2018). This has been proposed to protect maternal mRNAs whose THE miRNA REPERTOIRE
translation products are not beneficial in the germline but are
required in the early embryo for robust development. Consistent In the course of animal evolution, the miRNA repertoire
with these observations, C. elegans embryos tolerate mutations expanded drastically in conjunction with complexity (Hertel
that severely impede association of Argonaute with AIN-1/2. et al., 2006; Heimberg et al., 2008; Wheeler et al., 2009; Berezikov,
Despite Argonautes being essential for embryonic development 2011; Guerra-Assunção and Enright, 2012; Hertel and Stadler,
of C. elegans (Vasquez-Rifo et al., 2012), AIN-1/2 play a secondary 2015; Figure 5). Often, miRNAs arose in bursts coinciding with
role at this stage (Jannot et al., 2016). However, the interaction major organismal innovations, for example the emergence of
between Argonaute and the GW182 orthologs is necessary for vertebrates, or cortical expansion in primates (Peterson et al.,
post-embryonic development (Jannot et al., 2016). 2009; Hertel and Stadler, 2015; Kosik and Nowakowski, 2018;
GW182-independent function of miRISC has also been Fromm et al., 2020). While the animal miRNA machinery
observed in other contexts. For instance, in Drosophila S2 cells, originated in unicellular holozoans (Bråte et al., 2018), the most
induction of mitogenic signaling via serum withdrawal results conserved animal miRNA, miR-100, first appeared in the last
in formation of an ER-associated, GW182-independent miRISC. common ancestor of cnidarians and bilateria (Grimson et al.,
This complex is a potent inhibitor of translation but has no 2008). Organisms diverging early in eukaryotic evolution tend
effect on mRNA deadenylation and decay (Wu et al., 2013). to have few, mostly non-conserved miRNA genes, indicating
In line with this, depletion of GW182 in Drosophila S2 cells a high evolutionary flux in basal clades (Grimson et al., 2008;
abolished miRNA-dependent deadenylation but had practically Cock et al., 2017). The availability of high-quality miRNA
no effect on translation repression (Fukaya and Tomari, 2012). annotations in diverse genomes has recently upgraded our
The diversity in miRNA mediated silencing mechanisms provides ability to place miRNAs into an evolutionary context (Berruezo
organisms with enhanced capacity for gene regulation, while et al., 2017; Cock et al., 2017; Wang et al., 2017; Ylla et al.,
posing additional challenges for a complete understanding of 2017; Fromm et al., 2019, 2020). This opens up exciting new
miRNA functions in vivo. avenues, enabling connections between miRNA age, expression

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Dexheimer and Cochella MicroRNAs: From Mechanism to Organism

FIGURE 4 | Alternative outcomes of miRNA-mediated targeting. In addition to the common miRISC effects on translation and stability of target mRNAs, other
functional outcomes of miRISC binding to an mRNA are possible. (A) Full sequence complementarity results in Ago-mediated target cleavage, a mechanism that
resembles the mode of action commonly employed in cnidaria and plants. (B) Target-mediated miRNA degradation is induced by interaction with targets through
extensive pairing, in particular extending to the miRNA 30 end. (C) Recruitment of Argonaute in the absence of GW182 results in inhibition of translation without
affecting mRNA stability (likely involving alternative co-factors).

and function during development, as well as the emergence of has not accumulated a single mutation between humans and
specific features at the organismal level. worms (Pasquinelli et al., 2000), or mir-9a, has remained identical
between Drosophila, mouse, and human (Li et al., 2006).
Diversification of the miRNA Repertoire miRNAs are much younger than protein coding genes (on
A widespread view in the field is that miRNAs tend to be rapidly average 169 Myr vs. 1195 Myr respectively), and many of them
gained and lost in the course of evolution (Nozawa et al., 2010; arose more recently, after the split of diverse phylogenetic groups.
Fromm et al., 2013; Meunier et al., 2013), although part of this For instance, an estimated 46% of human miRNAs are primate-
has been recently challenged (Tarver et al., 2018). Nevertheless, specific and 14% are human-specific (Patel and Capra, 2017). In
a substantial number of miRNAs have acquired important general, miRNA genes evolve de novo from hairpin-structures
functions and remain conserved. Because miRNA evolution is located within introns or intergenic regions (Lu et al., 2008;
intimately connected to evolution of target genes (Zhao et al., Nozawa et al., 2010), which in turn likely originate through one
2015; Nozawa et al., 2016), conservation of miRNAs is observed of three proposed models: (i) inverted gene duplication of a gene
primarily over the seed region, which determines targeting that will subsequently become target of the miRNA (Allen et al.,
specificity. However, features beyond the seed can critically 2004); (ii) transposon-insertion followed by derivatization (Li
impact miRNA function, underscored by the observation that et al., 2011; Qin et al., 2015); and (iii) spontaneous evolution
some mature miRNAs are conserved from the first to the last out of random sequences (De Felippes et al., 2008). However,
nucleotide across large evolutionary distances. For example, let-7 the majority of functionally important miRNAs arose from

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Dexheimer and Cochella MicroRNAs: From Mechanism to Organism

FIGURE 5 | Diversification of the metazoan miRNA repertoire. Shown are numbers of individual miRNAs/miRNA-families (in bold) annotated with high confidence for
various clades (https://mirgenedb.org, Fromm et al., 2020). Number of miRNA families present in the last common ancestors of branching clades are noted above
the split. Exemplary organisms depicted are Amphimedon queenslandica, Nematostella vectensis, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio,
Mus musculus, and Homo sapiens. Data for bilateria is derived from Fromm et al. (2020), for Amphimedon and Nematostella numbers were retrieved from the work
of Grimson et al. (2008); Moran et al. (2014), and Calcino et al. (2018).

duplications of existing miRNAs (Kim and Nam, 2006; Carthew (Giraldez et al., 2005), has evolved at least 57 family members
and Sontheimer, 2009; Berezikov, 2011). (according to mirgenedb2.0) located within a 16 kb region on
Diversification of the miRNA repertoire occurs both through chromosome 4. Mouse embryos lacking the miR-290-295 and
the addition of new miRNAs and the addition or change in miR-302-367 clusters (mammalian miR-430 homologs) arrest
targets. An improved understanding of the latter will require early in embryogenesis, with defects that are only partially
better knowledge of what are the biologically meaningful targets recapitulated if one or the other family is removed (Medeiros
of any given miRNA. Whereas for a few miRNAs we know of a et al., 2011; Parchem et al., 2015).
number of experimentally well-defined targets, for most miRNAs Underscoring the biological significance of miRNA
we rely on computational prediction algorithms. Because such duplication followed by organization into families, there
prediction tools yield numerous false positives, substantial seems to be an expansive trend among miRNAs with important
experimental validation of targets will be necessary for a deeper biological functions. Potential reasons include (i) increasing the
understanding of how miRNA-target interactions change over dose of mature miRNA to enhance efficient target silencing,
time (Fridrich et al., 2019). or (ii) evolutionary robustness and flexibility, e.g., mutations
in one family member are not immediately detrimental, and
miRNA Families individual copies can be further diversified in sequence or in
Duplication followed by sequence diversification of miRNAs expression pattern. Different family members share identical
can lead to target diversification, changes in expression pattern, seed sequences, and in some cases, paralogous miRNAs are
and pronounced increases in dose (Luo et al., 2018). In also conserved around nucleotides 13–17, which underscores
many cases, if the seed sequence is retained, miRNA gene the contribution of these positions to efficient targeting (Wee
duplication marks the birth of a (homo-seed) miRNA family. et al., 2012; Schirle et al., 2014; Sheu-Gruttadauria and MacRae,
Members of a family function largely redundantly on a shared 2017). However, in other cases family members differ in
set of target mRNAs. In many cases the full extent of sequences beyond the seed, which can affect targeting properties
the function of miRNA family becomes apparent only upon (Broughton et al., 2016; Brancati and Großhans, 2018; Zhang S.
removing all members (Miska et al., 2007; Alvarez-Saavedra et al., 2018) but also biogenesis or loading efficiency as discussed
and Horvitz, 2010; Parchem et al., 2015). Curiously, most above. Thus, while miRNA family members appear largely
animal miRNAs whose loss of function causes severe defects, redundant, individual members can acquire specific functions
occur in families with sometimes extreme copy numbers. In (Abbott et al., 2005; Alvarez-Saavedra and Horvitz, 2010;
C. elegans, two miRNA families are required for completion Brancati and Großhans, 2018; Zhang S. et al., 2018).
of embryonic development: the MIR-36 family (8 members A recent analysis found that out of 352 human miRNAs that
in C. elegans, 29 members in the closely related C. briggsae) are conserved among vertebrates, 207 (58.8%) are duplicates and
and MIR-100 family (miR-51-56 in the worm) (Alvarez- 125 (35.5%) are homo-seed family miRNAs (Luo et al., 2018).
Saavedra and Horvitz, 2010). In zebrafish, MIR-430, which miRNAs in families differ from singletons in their evolutionary
plays an outstanding role during embryonic development dynamics and functional roles, with family miRNAs tending to

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Dexheimer and Cochella MicroRNAs: From Mechanism to Organism

be: (i) older than singletons, (ii) more conserved at the sequence genetics, either from manipulations of the miRNA biogenesis
level than singletons, (iii) enriched for diverse expression in machinery or individual miRNAs. While it is clear that miRNAs
distinct tissues, (iv) broader in target range, and (v) implicated in are necessary for the correct development and function of
more diseases. These functional correlations are still significant, multiple cell types, in most cases we do not understand the
albeit less pronounced, when family vs. singleton miRNAs are functionally relevant relationships of miRNAs and their targets,
stratified by age, with older miRNAs tending to contribute more the consequences on gene regulatory networks, and how the
to disease, target more genes, and being expressed in significantly effect on specific cell types impacts the organism. Given the
more tissues (Patel and Capra, 2017). In addition, the efficiency broad implication of miRNAs in physiology and disease, a
of target repression correlates with degree of conservation as well deeper mechanistic understanding of the roles of miRNAs within
as evolutionary age for many miRNAs (Luo et al., 2018). complex organisms is highly desirable. We expect that new
The expansion of metazoan miRNAs was likely one of the technologies that enable this depth of analysis in animals will
factors that contributed to the evolution of complexity in present- make this possible.
day animals. In this context, de novo evolution but also miRNA
duplication followed by sequence diversification played an
important role laying the foundation for miRNA families. Among AUTHOR CONTRIBUTIONS
others, family membership and evolutionary age of miRNAs
coincides with functional trends, offering a useful context for PD and LC wrote the manuscript. PD designed, drew, and
elucidating the contribution of miRNAs to animal development painted the figures in ink and watercolor.
and homeostasis. Nonetheless, clade-specific singleton miRNAs
also provide a source of innovation. For example, miR-791
originated in a class of nematodes called Chromadorea, and FUNDING
at least in C. elegans it acquired an important function in
its CO2 -sensing neurons (Drexel et al., 2016). Similarly, lsy-6 The LC lab was supported by an FP7/2007-2013 grant from
originated in the last Caenorhabditis common ancestor and plays the European Research Council to LC (ERC-StG-337161) and
an essential role in sensory neuron diversification in C. elegans the Austrian Science Fund (SFB-F43-23 and P 32636-B). PD
(Johnston and Hobert, 2003). was supported by a grant from the Austrian Science Fund for
an RNA Biology Doctoral School (W-1207-B09). Basic research
at IMP was supported by Boehringer Ingelheim GmbH. This
CONCLUDING REMARKS manuscript is not influenced by the interests or views of
Boehringer Ingelheim GmbH.
Since the discovery of miRNAs, the field has made tremendous
progress toward understanding the molecular mechanisms of
biogenesis and action of this versatile class of repressors. The ACKNOWLEDGMENTS
vast majority of these mechanistic studies have been performed
in cell culture models, in which biochemical approaches are We thank members of the LC lab and Gijs Versteeg for
feasible. This has given us detailed snapshots of the possible critical feedback on the manuscript. We apologize to colleagues
roles of miRNAs at the molecular and cellular levels. At the whose work we did not include due to scope limitations and
organismal level however, most of our understanding comes from our own ignorance.

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Frontiers in Cell and Developmental Biology | www.frontiersin.org 18 June 2020 | Volume 8 | Article 409