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Abstract
The remarkable discovery of small noncoding microRNAs (miRNAs) and their role in
posttranscriptional gene regulation have revealed another fine-tuning step in the expression of
genetic information. A large number of cellular pathways, which act in organismal development
and are important in health and disease, appear to be modulated by miRNAs. At the molecular
level, miRNAs restrain the production of proteins by affecting the stability of their target mRNA
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and/or by down-regulating their translation. This review attempts to offer a snapshot of aspects of
miRNA coding, processing, target recognition and function in animals. Our goal here is to provide
the readers with a thought-provoking and mechanistic introduction to the miRNA world rather
than with a detailed encyclopedia.
Keywords
MicroRNA; Noncoding RNA; Splicing; Intron; RNA interference; RISC; Knock down; Gene
silencing; Argonaute; Dicer; Drosha
Introduction
The discovery of miRNA is one of the most significant landmarks in modern molecular
biology. MicroRNAs (miRNAs) are small, regulatory, noncoding RNA molecules that
control the expression of their target mRNAs predominantly by binding to the 3'
untranslated region (UTR). A single UTR may have binding sites for many miRNAs or
multiple sites for a single miRNA, suggesting a complex post-transcriptional control of gene
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expression exerted by these regulatory RNAs. The founding members of the miRNA family
were identified in the Ambrose and Ruvkun laboratories and originally termed "small
temporal RNAs" (1, 2). The genome-wide identification and expression of miRNA in
mammals, fly, and worm has catapulted the field of small RNAs to the forefront of modern
biology (3–5). Furthermore, differential miRNA expression in diverse cell and tissue types
makes these molecules ideal biomarkers of disease detection and progression as well as
targets for therapeutic intervention. Considerable amount of data generated in a short period
show that miRNAs mainly exert their function post-transcriptionally by modulating mRNA
stability and/or protein translation.
Correspondence: Dr. Sailen Barik, Center for Gene Regulation in Health and Disease, and Department of Biological, Geological and
Environmental Sciences, Cleveland State University, 2121 Euclid Avenue, Cleveland, Ohio 44115, USA. Tel. 216-523-7326.
s.barik@csuohio.edu.
Conflicts of interest
The authors declare no conflict of interest.
Shukla et al. Page 2
gene expression connected with complex gene regulatory networks. miRNA are conserved
from amoeba to humans. Thus far, use of large-scale small RNA cloning strategies have led
to the discovery of miRNAs in a wide variety of organisms including brown alga, nematode,
mollusk, tunicates (Ciona intestinalis), sea lamprey, vertebrates, insects, amoeba, monocots,
dicots, large DNA viruses (Epstein-Barr and herpes viruses), and recently in T. gondii, a
unicellular protozoan parasite of the Apicomplexan family (6–10). More than two-thirds of
all human miRNAs are encoded in the intervening regions (introns) of protein-coding genes
as well as in long noncoding transcripts (Fig. 1). miRNAs can also be encoded in exons or
introns, depending upon the alternative splicing status of the pre-mRNA (11). In addition,
miRNAs can be of intergenic origin, i.e. found in the chromosomal regions between two
genes (Fig. 1) (12, 13).
As mentioned above, a large number of miRNAs are encoded in the introns of host genes,
suggesting that the RNA polymerase II-mediated transcription of these miRNAs and the
host genes is coupled (Fig. 1) (14–15). There are also examples of miRNA genes with RNA
polymerase III promoter. C19MC, the largest known miRNA cluster containing 46 tandemly
repeated miRNAs, appears to be transcribed by RNA polymerase III (16). These miRNAs
are presumably formed by repetitive sequences and interspersed by positive- and negative-
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strand Alu repeats (17, 18). Intergenic miRNA clusters are likely to be associated with
complex transcriptional loci; however, their transcriptional activation and processing remain
undefined. Interestingly, another report suggested that C19MC cluster is derived from an
intronic non-protein-coding RNA Polymerase II transcript (19). Many miRNAs are coded
from single standalone genes however, and clusters containing multiple miRNAs (e.g.,
miR-106b-25, miR 17–92; miR 302–367) appear to be common (Fig. 2). The question
remains unanswered as to why a significant number of miRNAs are expressed from clusters,
although there is little evidence supporting a common pathway or regulatory function of
miRNAs originating from the same cluster. It has been suggested that the expression of the
intronic miRNAs may be coupled with that of the host mRNA and the two may be
functionally related (20–25). In studies of other genes, expression of the intronic miRNAs
differed from their host gene expression, especially in the pathological states (26). A
significant intraclustral differential expression pattern of miRNA has also been observed
(27). Overall, these observations point to diverse mechanisms and the existence of additional
steps in the posttranscriptional processing of miRNA involving proteins in the maturation
process. Many of the RNA-binding proteins, including Lin 28, hnRNP L, KSRP, and
hnRNP A1, bind to conserved loop sequences in the miRNA, and therefore, may form part
of the gene regulatory network that controls the level of miRNA expression in response to
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Interestingly, the tumor suppressor protein p53 modulates p68- and Drosha-mediated
miRNA processing. Both Drosha and p68 associate with a subset of miRNAs and their
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processing is enhanced in the presence of the p53 transcription factor (41). This explains
why TDNA damage leads to posttranscriptional induction of several miRNAs as p53 is
induced in DNA damage.
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The p68 protein mentioned above is a DEAD-box RNA helicase and found to be associated
with the Drosha complex in prostate cancer cells (42). It is in fact highly expressed in
prostate cancer cells and is also associated with human androgen receptor (AR) coactivator
complexes in the nucleus. These findings give rise to the notion that AR may be involved in
the post-transcriptional modification of a set of miRNAs that are likely associated with
castration-resistant prostate cancer. However, this hypothesis is yet to be tested
experimentally.
homolog is known as Pasha (43, 44). The two RNase domains of Drosha help cleave the 5'
and 3' ends of the pri-miRNA, which determines the length of pre-miRNA (44). The
resultant pre-miRNA is exported to the cytoplasm by a complex of Exportin-5 and Ran-GTP
(45). The final maturation of miRNA occurs with the help of Dicer, another RNase III
nuclease that processes the pre-miRNA into a 22 bp double-stranded RNA. The processing
is often coupled with the formation of the ribonucleoprotein complex known as RISC
(miRNA-Induced Silencing Complex) (Figs. 1, 3, 4). The RISC minimally consists of one
strand of the miRNA (called "guide strand") in addition to Dicer, TRBP, PACT and
Argonaute (Ago) proteins. The complex engages with the target to execute silencing while
the other strand of the miRNA (called "passenger strand") is generally, but not always,
destroyed. It is unclear how the asymmetric strand selection and passenger strand
destruction occurs (extensively reviewed in 46–48 and references therein).
Interestingly, there exists a subset of intronic miRNA, known as "mirtron" (49–52), whose
processing appears to be independent of Drosha (Fig. 1). Mirtrons were initially identified in
Drosophila and C. elegans (49, 50), but more recently identified in human, macaque,
chimpanzee and mouse as well (52). Unlike the pri-miRNAs, the mirtrons lack the lower
hairpin and the flanking single-stranded regions that recruit the microprocessor complex.
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Instead, they contain 5' and 3' splice sites that are presumably recognized by small nuclear
RNAs (snRNA). After the debranching of the lariate intron, the spliced introns fold back
into a stem-loop structure that obviates the need for the Drosha-mediated microprocessor
function. It will not be surprising if refined methods of high-resolution deep-sequencing
reveal many more mirtrons in mammalian genomes, including human.
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contrast, the miRNA constitute only a tiny fraction of information content within the intron
that could be several hundred kilobases long and hence, additional internal intronic
promoter-mediated transcription, independent of the host gene, cannot be ruled out. Finally,
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these in vitro studies are unable to answer if mutations of splice sites of miRNA hosting pre-
mRNA introns may abolish the splicing, hence creating a stable transcript that might "over-
produce” the resident miRNAs, resulting in increased translational repression of its target
mRNA. Only in vivo experiments will be able to shed light on the so-called “fine-tuned”
gene expression by intronic miRNAs in response to splicing defects of host pre-mRNAs.
tRNase Z, which cleaves 3' to the tRNA to liberate pre-miRNA hairpins that are then
processed by Dicer to yield the mature viral miRNAs (58).
prediction, the ultimate proof of a miRNA-MRE pair must await experimental verification.
As the coding sequence of an actively translated mRNA is covered by ribosomes, one would
expect that the MREs would be located in the untranslated regions. Indeed, a comprehensive
study (60) showed that >45,000 miRNA target seed sites are particularly conserved in the 3'
UTR of target mRNAs, suggesting that the predominant regulatory functions of miRNAs are
exerted from the 3' UTR. An interesting study (61) found that after cell proliferation is
activated, mRNAs tend to terminate upstream to normal polyadenylation sites. Not
unexpectedly, the resultant shorter 3' UTRs lost some MRE sequences, in agreement with a
generally negative role of miRNA acting through the 3' UTR and a need for elevated gene
expression in proliferating cells. However, subsequent studies revealed that functional
MREs may also occur in the 5' UTR (62–64) as well as in the coding sequence (65). In
either case, it is possible that the efficiency of the MRE to engage the miRNA will be
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modulated by the kinetics of ribosomal movement across the site and/or the local secondary
structure of the mRNA, which has not been investigated thoroughly.
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In contrast, recent studies provide overwhelming evidence that the miRNA-bound mRNA is
also subject to degradation, mainly through deadenylation (Figs. 3, 4) (1, 2, 83, 84). These
studies suggested that in addition to translation repression, miRNA-mediated mRNA
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degradation exerts an enhanced and robust block in the production of the target gene’s
protein product. Many studies also suggested that miRNA-supported mRNA deadenylation,
degradation and translation repression may occur independently of each other (85, 86).
Interestingly, miRNA has also been implicated in the removal of the 5'-cap structure (86,
88). In two recent genome-wide studies, both target protein and mRNA levels of miRNAs
were studied (89, 90). By overexpression and knockdown of various miRNAs, these studies
together demonstrated the downregulation of both target protein and mRNA in HeLa cells
and mouse neutrophils. A more recent study showed that mammalian miRNA
predominantly act to decrease target mRNA levels (91). This study used a novel method of
ribosome profiling which essentially determines the positions of ribosomes at specific codon
nucleotide resolution coupled with deep-sequencing (92). The results suggested that the
miRNA-mediated destabilization of target mRNAs is likely the predominant reason for
reduced protein production in mammalian cells.
Multiplicity is a common feature in miRNA-mediated regulation such that: (a) one 3' UTR
often contains multiple MREs, and (b) the same miRNA affects dozens, if not hundreds, of
targets. Multiple MREs on the same 3' UTR generally promote stronger silencing when
situated at an optimal distance from each other (93, 94); although the mechanism for this is
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unknown, it has been speculated that multiple RISC complexes may stabilize each other
through cooperative contacts (93).
Intriguingly, recent evidence showed that miRNAs may target the promoter regions as well
(48) and have the potential to activate gene expression (95). Finally, the gene silencing
properties of miRNAs appear to be cell cycle-dependent such that they may globally up-
regulate translation in times of cell growth (96). The molecular mechanisms of these
apparently novel functions of miRNA remain to be elucidated.
In summing up, miRNAs appear to use multiple mechanisms for silencing of gene
expression. Nevertheless, the majority of studies overwhelmingly support two consensus
models: (i) translation repression at the initiation or elongation stages (Fig. 3), and (ii)
deadenylation and subsequent degradation of the message (Figs. 3, 4). It may be envisioned
that these mechanisms function together and are also regulated in a gene- or cell type-
specific manner.
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Conclusion
Naturally occurring miRNAs have emerged as important regulators of cell differentiation,
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proliferation and survival. Reciprocally, aberrant and altered miRNA expression has been
linked to many pathological conditions including cardiovascular disorders, diabetes and
cancer. The biogenesis of miRNAs may be regulated not only by transcriptional regulation
of their promoters that generate the precursor but also by proteins representing important
cellular pathways that participate in the processing of the precursor to generate the mature
miRNAs. Although remarkable progress has been made on miRNA biogenesis and mode of
action, it is still unclear how miRNAs find and bind to their cognate mRNAs and how other
cis-acting elements within the close proximity of an MRE may affect miRNA function. It is
also unclear how miRNAs in some context actually stimulate gene expression. A
comprehensive pathway-focused analysis of miRNA expression and function should aid in
our understanding of the cellular and molecular processes that are modified during the
course of pathogenesis.
Acknowledgments
Authors like to thank Dr. Ajay Singh for offering the opportunity to contribute to this special issue of Molecular
and Cellular Pharmacology, and to Dr. Kavleen Sikan for critical reading of the manuscript. Research in the
authors' laboratory was supported by grants from Department of Defense prostate and breast cancer research
programs as well as from National Science Foundation (to GCS), and by National Institute of Health (AI059267 to
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SB).
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Figure 3. Two modes of action of miRNAs. miRNAs can establish (A) imperfect or (B) perfect
complementarity within the 3’ UTR of its target mRNAs
Depending upon the complementarity, miRNA can then modulate the translation and mRNA
destabilization (mostly in animals) or degrade the target message (common in plants). Also
see Fig. 4 for detailed mechanisms.
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