Introduction
n
the opening session of the Annual Congress of Iranian Pathologists, there is usually
a brief report of the emerging technologies or scientific innovations on the
field of medicine and/or pathology. At the 9th Annual Congress held
in November 7 – 9, 2007 in Tehran, the subject was “New Advances
in RNAs.”1 The following is an overview of that report.
Traditionally, biologists believed that ribonucleic acid
(RNA) molecules, as mRNA, simply carried genetic information from DNA in the
nucleus to the places in the cell where proteins are made. The simple formula was
believed to be: DNA makes RNA, RNA produces protein, and proteins are major
cellular machinery that carry out all the crucial tasks, as structural substances,
enzymes, hormones, and so on. RNAs were also founded
in the protein factory themselves. Two other molecules of the RNAs family are
nonprotein coding and are called rRNA and tRNA. With this idea, the major efforts
of gene hunter researchers were to discover new structural genes whose products
encode proteins.2–4
When the human-genome project was completed,
biologists wondered to see that plants and animals, of any species, all seem to
have approximately the same amount of genes to produce proteins. Yet, the
complexities of all are very different. Flies are more complex than worms and
they are much less complex than mammals. In addition to the above, the recent
studies indicated that nearly 97% of the human genome is composed of noncoding
DNA, which varies from one species to another. Instead, many of them produce
important noncoding regulatory products. The most recent among this nonprotein-coding
RNA (npcRNA) class are genes that encode small or short- length RNA
molecules (approximately <40 nucleotide in length). An important group of
these are called microRNAs. These molecules unlike mRNAs, do not encode protein
but inhibit mRNA gene production.5–10
History
Short-length RNAs (small RNAs) include many different
classes of nonprotein-coding RNA molecules. Among these are microRNAs
(miRNAs) and small interfering RNAs (siRNAs), each with their own properties
and functions. These molecules function in many physiologic and pathologic
processes. The main function of this class of RNA is regulatory function in the
form of interfering with protein production of target mRNAs either via specific
cleavages of homologous mRNA or by specific inhibition of mRNA protein
synthesis.5
The research which led to discovery of these molecules
started in 1993.4,8 Investigators were working with a nematode
called Caenorhabditis elegans; they were looking how the developmental timing of this worm is
controlled. They found a small temporal RNA (stRNA) that was concerned with
this process, and labeled this as lin-4.11–14 They observed that
stRNAs do not encode protein but their actual function was not clear. Later on,
they found that the molecule was involved with down-regulation of
mRNA that specified the temporal progression during development. Wightman et al.
identified the first microRNA target lin-14 mRNA.8 Another stRNA
called let-7 was discovered in 2000 which has had the similar effects.14
Let-7 was expressed during the fourth stage of larval C. elegans
development. Still the importance of small RNAs was not known. Attentions grew
when they found that homologous of let-7 was present in other species such as
frogs, flies, mice, and human, and the sequences of all this let-7 RNA was
similar and in many cases identical. Lin-4 and let-7 were identified by their
mutant phenotype and until recently they were the only known small RNA.15 Flow
of investigations in various research laboratories in the world led to more
understanding and precise definition of lin-4, let-7, and short-length
RNAs. The finding in this regards showed that nonprotein-coding small
RNAs have mainly a gene-silencing property that is preserved in all livings,
and the genes have thus been called “silencing genes.” Shortly after
the discovery of the first short-length RNA gene, lin-4, in C. elegans, many such
regulatory RNA genes have been identified in worms, plants, flies, fish,
mammals, and human. Andrew Fire and Craig Mello, among other investigators,
have been awarded the 2006 Noble Laureate for Physiology and Medicine for their
discovery.16–17
The recent identification of small short-length
RNA, which is discovered in nearly all creatures, has revolutionized our understanding
of gene performance, activities, and regulation. Small RNA interference
(siRNAs) and related RNA silencing phenomenon with experimental introduction of
RNAi into cells to interfere with function of an endogenous gene have provided
great hope for understanding the physiology and pathology of cells.18–21
Definition
MicroRNAs are single-stranded small RNA molecules 22 –
23 nucleotides (nt) in length.4,22–24 These are noncoding RNAs that are
negatively regulating gene expression via sequence-specific base pairing with
target mRNAs. miRNAs are of RNA family, encoded by genes that are
transcribed from DNA (see below) but not translated into protein.
siRNAs are another small RNAs and mediate the
phenomenon of RNA interference called RNAi. It was first discovered in plants
and lower organisms as an indigenous evolutionary conserved biologic molecule.22–24
According to Lagos-Quintana et al. siRNAs are double-stranded but stRNA are
single-stranded.20 Small RNAs have diverse expression pattern during
development.
miRNAs and siRNAs are created in
the same pattern. Both stRNAs and siRNAs are generated by a process
requiring Dicer, a pronuclease III enzyme. Dicer is a multidomain protein with
tandem ribonuclease III (RNase III) domain. Dicer processes double-stranded RNA
into small fragments about 21 – 25 nucleotides (nt) in length. Dicer is
composed of three structurally rigid regions connected by flexible hinge.25
miRNAs and siRNAs mediate down-regulation of mRNA gene expression. But the way they
act in mRNAs are different. Since miRNAs have evolutionarily preserved
regulatory functions, they are featuring orthologs in other species.5
miRNA biogenesis and function specificity and potency rely on the nature of
protein-protein, protein-RNA, and RNA-RNA interactions found in different
complexes. Any of these will fulfill a specific function in a well-orchestrated
process.26
Production
of small RNAs
Two distinct pathways exist for the action of short-length
RNAs (stRNA and siRNAs), but their production is more or less similar. Here, we
briefly consider the production of miRNAs.4,27
MicroRNAs are produced through transcription of miRNA
genes in the nucleus known as microRNAs precursor genes (mir-gene). Rigoutsos
and his group of IBM Research Center Studies predicted that the number of
microRNA precursors in mammalian genomes likely ranges in the tens of thousands.25,
28, 29 The genes encoding microRNAs are much longer than the processed
mature miRNA molecules.
1-
Transcription
of miRNA genes (mir-gene) produces primary miRNA transcript (pri-miRNA).
Pri-miRNA has a cap and poly-A tail.
2-
Primary
miRNAs are processed in nucleus to short, 70-nt loop structure, the so-called “pre-miRNAs.”
This process is performed by a protein complex named microprocessor complex
consisting of the nuclease Drosha and stranded RNA- binding protein Pasha30
or as it is performed in mammals, this can be done through pronuclease III, a
Dicer. The microprocessor complex varies according to creature, for example,
the pathway in plants varies slightly due to their lack of Drosha homolog.
3-
With the help
of specific transporter proteins (i.e., exportin-5 complex) pre-miRNA is
exported to the cytoplasm.
4-
Pre-miRNAs
through an additional “cutting” by an enzyme Dicer generate mature miRNAs.
Dicer also initiates the formation of the RNA-induced silencing complex (RISC).30,31
Mature miRNAs are about 21–30 nt in length. At this stage, miRNAs are still
double stranded.
5-
With the
action of Dicer miRNAs unwind and make two single-stranded molecules.
6-
Single-stranded
molecules of this duplex are incorporated into a multiprotein complex known as RISC.
7-
Base pairing
between single-stranded miRNAs/RNAi and target mRNA direct RISC to either.
a-
repress target
mRNA translation,or
b-
mRNA
destruction (cleavage).
In either ways, the gene from which the target mRNA
was derived is silenced. A given miRNA can silence many target mRNA genes,
estimate range from one to hundred target genes, based on target predictions
using a variety of bioinformatics approaches.32,29 For example,
thousands of mammalian mRNAs are under selective pressure maintaining 7-nt
sites matching miRNAs. As a consequence a single miRNA, direct or indirect,
regulates many of these target sites.33
Silencing
performance
MicroRNAs are responsible for post-transcriptional
gene silencing as part of critical cellular pathways and intercellular
coordination, mainly during embryonic development.34–36
Silencing of gene expression by microRNAs is preserved
in all living creatures, from flowering plants37 to human. The basic
mechanism of silencing phenomenon is still not known. It is not understood to
what extent and through what process does the suppression of mRNA or reduction
of its activity occur. Gene silencing is a fundamental mechanism of gene
regulation, mastering creation development. In mammals, the majority of miRNAs
guide RISC to the 3’ untranslated regions (UTRs) of mRNA targets, with the
consequence that translation of the target mRNA is inhibited.38
miRNAs and
siRNAs involving in RNAi use the same RISC to direct silencing. For miRNAs
processing, its precursor double-stranded RNA (dsRNA), uses Dicer enzyme which
will produce single-stranded small RNA. This small RNA attaches to RISC and
directs to target mRNA. After that the mechanism diverges, miRNAs attach
imperfectly to mRNA and form a bulge that block mRNA to produce protein while
siRNA binds perfectly with the target mRNA and destroy mRNA.4
The cells are full of RNAs
The number and the diversity of RNAs are amazingly
numerous and many more miRNAs and siRNAs are being reported.
Tuschl group of Max Plank Institute identified 16
novel miRNAs which are only expressed in 0 –2-hr embryo of Drosophila
melanogaster.20 Each clusters and family of short-length small
RNAs have many subsets. New RNAs are crushing forth from research laboratories
rapidly. The subset of microRNAs have their own specific function. Examples
are:
·
siRNAs-like
scan RNA or scanRNAs (scnRNAs) are produced from nongenic, heterogeneous
micronuclear transcripts, take part in genome rearrangements, chromosome
segregation, and meiotic prophase.39,40
·
Small nuclear
RNAs (snRNAs) are constituent of spliceosome (a large,
dynamic ribonuclear protein complex, which contains five essential snRNAs from
U1 to U7), the mechanism involved to produce mRNA by removing introns region of
genes.41–43
·
Small
nucleolar RNAs (snoRNAs) have been identified in rice and other plants.
These are involved in rRNA processing. It modifies ribosomal RNAs by
orchestrating the cleavage of the long pre-rRNA into the functional subunits.44–46
·
Repeat-associated
siRNAs (rasiRNAs) silence the genetic repeat. It has been
suggested that rasiRNAs-based silencing is mechanistically different miRNAs and
RNAi pathway.47–49
·
Transacting
short interference RNAs (tasiRNA). They have been distinguished recently. There
are genes encoding tasiRNA cleavage plant developing proteins. TasiRNAs regulate
gene expression at the post-transcriptional level.49,50
·
Natural
antisense transcript-associated siRNAs (nat siRNAs). These were
discovered in plant and there are human homologous for it.49,50
·
piwi-intracting
RNAs (piRNAs) (which were introduced last summer), are
abundant in developing sex cells. piRNAs have been identified as key players in germline.
No male mammal, male fish or fly of either sex would be fertile without them.
Mammalian piRNAs are approximately 26 – 31 nt in lengh.51–53
·
X-inactive specific
transcript RNAs (xistRNAs) have important roles in X-chromosome
inactivation (XCI) and have the power to turn off an entire chromosome. In female,
the action is more severe because of two X-chromosomes.54–57
·
Pregnancy-induced
noncoding RNAs (pincRNAs). These newly-found miRNAs have been seen
in pregnancy.
Hundreds of papers during the last three to four
years have been published regarding various activities of these new molecules.
Small noncoding RNAs of the miRNAs class appear to be numerous and diverse.15
According to Isidro Rigoutsos of IBM, more than 37,000 different microRNAs
present compares with 21,000 or so protein-encoding genes.3 This is
not an exaggeration when we consider that 97% of human genome is composed of
noncoding DNA. Furthermore, the results of a project called ENCODE published in
Nature (searching the detail of only 1% of human genome) nearly accepted the
amount of genes dealing with protein coding may be less than that was estimated.
MicroRNAs
and embryonic stem cells (ESC)
Stem cells have the capability to divide throughout their life to
produce differentiated daughter cells while maintaining a population which are
undifferentiated and remain stem cell in its proper niche. Stem cells posses
the ability to produce any types of cells. They are divided as embryonic stem
cells (ESCs), adult tissue stem cells, and many
cancers also have small population of cells with the character of stem cells.
These cells have been called cancer stem cells and thought to be the cause of
cancer relapse.58 MicroRNAs are considered crucial and thought to be
the key to understanding how stem cells remain in a state from which they can produce any
types of cells. miRNAs have also been implicated in the development and
pathology of stem cells.58 Multipotential mesenchymal stromal cells (MSCs) ,which can
be isolated from adult tissue, have the capability for self-renew and differentiate
into different lineages. Both of them are under regulation of miRNAs.59
Dr. Haifan Lin, one of the chief investigators in cell
biology at Yale University, studied the mechanism of stem cell self-renewal in
fruits, flies, mice, and human cancer cells. He
discovered that a group of piRNAs may play an important role in stem cell
proliferation and germline development. Around 20 miRNAs are made only in human
ESC. The miRNA expressions vary in both differentiated and undifferentiated
stem cells.58–61
RNA
interference (RNAi)
Development of gene-specific double-
stranded RNAs (dsRNAs) is the base of these newly-recognized silencing RNAs.
These are a unique class with relative stability, that have the ability to
activate the interferon pathway and in particular production of RNA
interference (RNAi).62–63 RNAi, was discovered in 199864;
it is a preserved ancient silencing gene with diverse actions and
activities.
RNAi’s action is to turn down the production of any single
protein to very low levels and that distinguishes it from miRNAs which control
multiple proteins simultaneously. RNAi mechanistically knocks down
gene expression via different actions including mRNA degradation, inhibition of
its translation, or chromatin remodeling. It seems that these could be
powerful tools for generating deficient phenotypes without mutating genes. In
this way it becomes promising hope for future treatment of the diseases. This
will provide a roadmap launches RNAi from research bench to the bedside
medicine24,62–64 ,and drugs based on RNAi are moving fast toward
pharmaceutical market (see below).
Functional
features of miRNAs
In plants and animals, microRNAs are essential
post-transcriptional gene regulators of various processes such as proliferation,
differentiation, development, programmed cell death, and
interaction between virus and host cells.64 miRNAs regulate the
expression of downstream gene targets including transcriptional factors. miRNAs
regulate the activity of at least one third of human protein-encoding genes. But there is
very little cellular proteins that do not happen under direct microRNA watch,
since other microRNAs derivatives such as siRNAs do.
MicroRNAs are also regulated by extracellular
signaling pathways that are important for differentiation into specific tissue,
suggesting that they play a role in specifying tissue identity. Expression of a
known miRNA in a specific cell type plays a useful marker for identifying a
particular cell type.59
The
complexities of creature
As is known, the current concept is that DNAs store
data that will get translated into living organisms. In this manner, the
complexity of the development related to genes is presented in DNAs: more genes
give more complexities.
Now, it become clear that all living creatures have
about the same number of genes with little differences to produce proteins;
nevertheless, their complexities vary. C. elegans, a tiny worm that
lacks a proper brain or a Drosophila, a
fruit fly, have nearly the same number of genes that human has—only a little
bit shorter. Thus, it seems that the traditional gene propensities are not as
only important molecules as it was believed. Instead, there must be other
molecules involved in this connection that is the regulatory genes, which are
related to RNA operating system. The researchers discovered that it is not the function of
protein-encoding RNAs (mRNAs) per se, but it is the regulatory RNA
system which is grouped as short-length RNAs or microRNAs family. In regard of complexities,
it does not matter how many protein-coding genes you have; it is important how
they are regulated. By silencing target protein-coding RNAs, microRNAs explain
why some creatures are more complex than others. What exactly those microRNAs
molecules do, have been the subjects of tremendous investigations. Single-stranded
miRNAs regulate the levels and qualities of hundred different proteins. As has
been mentioned earlier, they are like “powerful strings controlling copious
protein puppets”.3 Some types of these regulatory miRNAs (or
siRNAs) edit other kinds of microRNAs. So far, approximately 500 known
mammalian miRNAs gene families have been identified; each of these gene
families may regulate hundreds of different protein-coding genes. According to
a guess estimate, there are approximately 1,000 genes out of 30,000 human genes
that encode small RNAs and up to 30% of human protein-coding genes may be
regulated by microRNAs.
MicroRNAs
and our understanding of diseases
An interesting consequence of recognition of small
RNAs is that researchers found a new source for understanding disease
processes.32 Accordingly, they have found aberrant or altered miRNAs
expressions involving a number of human diseases including malignant, viral,
infectious, metabolic, and respiratory and heart diseases.65–68 Many
miRNAs are deregulated in human tumors67; many tumor miRNAs are located
in the genomic regions linked to cancer.53,68 The role of miRNAs in
cancer includes oncogenes and tumor suppressor genes. Identifying proper
biomarkers makes them appealing targets for diagnosis, therapy, and
prediction of the outcome. miRNAs signature can be used to detect and classify
various neoplastic tumors and with certain profiles of miRNA expression one can
predict the outcome of a disease. It become a new promise for anticancer gene
therapy.38,52 The discovery of miRNAs encoded by a family of virus
oncogene, and the finding that numerous DNA viruses also encode miRNAs,
attracted scientists’ attention to the probability that viruses themselves
have their own miRNAs and miRNAs as critical mediator for viral action
including oncogenecity.69,70 In another view, researchers showed
that viral gene expression is down-regulated by host miRNA which could be a
promise for design and manufacturing new antiviral drugs.
RNAs
in the world of treatment
The new RNAs world is also a source for the treatment
of diseases. In this approach, the basic technology is RNAi technology that is
the sequence-specific gene silencing. RNAi is made of small interfering RNAs
(siRNAs) which are derived from longer dsRNA by a Dicer. RNAi can be applied as
man-made or be used as endogenous molecules. RNAi becomes widely applied as experimental
tools to analyze, both in vitro and in vivo, the function
of genes. RNAi has been produced synthetically in mammalian cells by
introduction of manufactured either double-stranded siRNAs or by plasmid and
viral vector systems that express short hairpin double-stranded RNAs (shRNA)
which are subsequently by cell machinery processed to siRNAs.24
Experimentally, in mammalian cells, RNAi has been used to clarify the
functional role of individual genes. This has been done in different diseases
particularly in cancer cells and brain degenerative diseases. As is known,
cancer involves mutant genes that cause uncontrolled cell proliferation. The
aim for researchers is to silence those genes with RNAi. Most of these trials
have been done on cell culture in the laboratory.71 The main
objectives are to turn from laboratory to bedside. Apart from direct
therapeutic action of RNAi, some RNAi therapies may be used to support
conventional chemotherapy. Whitehurst and his group at the University of Texas
used RNAi for this purpose. They converted tumoral cells that did not respond
to anticancer drug paclitaxel into cells that become sensitive to this drug71
and this could be promising for other drugs or multidrug- resistant
diseases.
RNAi drugs stop the production of disease-related
protein at source. In this regard, they are now being tested as new tools in
many pharmaceutical companies searching for new drugs, though it is currently
in its introductory phase. Many companies including Opko Corporation, Novartis,
and Glaxo Smith Kline, are planning to gain new markets in this field. Some of
them doing final round of their clinical trials for an RNAi-based drug.3 Novartis
already announced its successes on several diseases such as macular degeneration
of eye, hepatitis C, Huntington’s disease, respiratory infection,
and cancer.72
The most probable explanation for RNAi is that it acts
as a defense against viruses including HIV. RNAi is a vital component
interacting with the innate anti-inflammatory and antiviral immune response. In
human interferon beta (β-IFN) has been shown to be involved with the
expression of many cellular miRNAs and they have been sequenced in HCV genomic
RNA.73–74
RNAs, unlike DNAs, are single-stranded and double-stranded
RNA (dsRNA) is rare in nature. However, viruses often make it when they
reproduce. It appears that organisms with the ability to recognize and destroy
dsRNA molecules (such as virus) will destroy dsRNAs; RNAi has this
ability. Using siRNA in cell cultures, Bitko and his group in an experiment
induced prevention and inhibition of respiratory viruses (RSV and parainfluenza,
PIV) by nasally- administered siRNAs.68
Small
RNAs and evolution
Alvarez-Garcia and Miska from the Wellcome Trust
Cancer Research,32 reviewed the functions of microRNAs in animal
development and evolution. The action of this little molecule in the animal
developmental timing, patterning, embryogenesis, organogenesis, cell
differentiation, and
cell death has been well summarized. MicroRNA family, lin-4 and let-7 as
heterochromic genes, have potential roles during evolutionary changes. Many
investigators studied microRNAs’ roles in evolution.
It functions through the inhibition/inactivation of
effective mRNA translation of the target genes. The underlying mechanism and
microRNAs target is still unknown, but it relates to their importance in the
origin of complexities.36,37 Bartel of Massachusetts Institute of
Technology (MIT) prepared a list of microRNAs in different creatures from
plants to mammals in the hope to find which and when different families of
miRNAs in these creatures have emerged.3,7
Researchers discovered miRNA genes interspersed among
set of protein encoding genes called HOX clusters. These are revolutionarily
conserved molecules. Recent studies have identified new insight into how HOX proteins
cause morphologic diversity at the organismal and evolutionary levels. In this
occasion miRNAs function as intergenic regulatory transcripts. Cluster of HOX
genes, which number from four to 48 per genome—depending on animal—control the
patterning and morphogenesis of the main body axis.75–80 Recent
evidence shows that HOX genes are fragmented, become reduced, or
expanded in animals that correlate with their morphologic changes in evolution.80
miRNAs, involvement in regulating HOX genes of flies and mice is
supported by the fact that miR-196, an miRNA, represses HOXbB gene expression. These
miRNA regions are also conserved across species in the same way as other
mechanistic ways that regulate HOX gene expression.76–77 Yekta et al.
demonstrated that miR-196 encoded at three locations in mammalian HOX A, B, and
C clusters, has extensive cleavage effects to HOX8B, C, and D.79
MicroRNAs are important molecules in the evolution of
the human brain. Recent research showed the comparison between miRNA in human
and chimpanzee, and they found about 8% of miRNAs that are expressed in human
brain is exclusively related to humans. This finding suggests that evolution is
related to the changes in miRNA as much is related for protein-coding mRNA.81–83
Sometimes, RNA carries genetic information, by hitching a lift in the
germ-cells independently of DNA. Some investigators suggest that RNA could
itself provide an alternative evolutionary structure. The idea that RNA in
certain conditions is governed by environmental factors seems very important in
natural selection.
In the last decade, much laboratory works have
been done to investigate the regulatory and behavioral functions of noncoding
RNAs (ncRNAs) in various aspects of development (patterning, embryogenesis,
organogenesis, etc.) and their revolutionary roles on
evolution on variety of plants and animals. Many of these studies are dealing
with the role of microRNAs in different features of animal and plant
development. There are several recent studies in the field of microRNA
in development and evolution.4, 23,33,84
Conclusion
The discovery of the first short-length RNA gene
(lin-4, in C. elegans) in 1993, and the subsequent numerous scientific
reports indicating that many more “RNA of this kind which do not encode protein”
exist have announced a new world of RNA that subverts the traditional beliefs
about RNA molecule. These small short-length RNAs have a crucial
role as regulatory apparatus maintaining all cell activities. MicroRNAs are an
abundant class of endogenous nonprotein coding RNA molecules, have been found
in plants, animals, and
viruses, and preserved in these creatures; they play crucial roles in gene
silencing. This silencing includes translational repression, mRNA cleavage,
and mRNA decay initiated by miRNA-directed
deadenylation of the target mRNA. These are the main mechanisms of miRNA-guided
gene regulation at the post-transcriptional levels.
MicroRNAs have crucial roles in development,
developmental timing, cell differentiation, proliferation, programmed cell
death, defense against viruses, inflammation, and in pathogenesis of conditions such as cancer,
respiratory, mental, and cardiovascular diseases.
RNAi is becoming a new tool in the diagnosis and
treatment of diseases and new advances to prediction of the disease outcome.
Having this concept, the man-made RNAi manufacturing has got a powerful market
place in the interest of pharmaceutical companies.
Finally, now with the advances in new era of RNA
activities and recent discoveries, it is likely to have changes in the people’s
point of view about how cells regulate themselves, how life becomes more
complex, the features of creature developments, how a certain disease develops, how
the diseases can be cured or prevented, and even how the process of evolution
operates.
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