Small Non-coding RNA and Gene Expression

While we've been taught: don't shoot the messenger, our cells havent gotten the
message. See how small bits of non-coding RNA target mRNA for destruction and
regulate gene expression.

All cells in a single organism carry the exact same genome, so how do we end up
with so many varieties of tissues and organs? Scientists know
that transcription of many genes in eukaryotic cells is repressed, or "silenced,"
but in some cases, genes are transcribed into mRNA that never gets translated.
Various post-transcriptional mechanisms are in place to add another level of
control over the already complex systems that regulate eukaryotic gene
expression. These mechanisms are the result of small, noncoding pieces of RNA
called siRNA (small inhibitory RNA), or interference RNA, and miRNA (microRNA),
or antisense RNA.

siRNA and miRNA Inhibit Translation by Parallel Mechanisms

Control of gene expression by these small, noncoding RNA molecules was first
observed in 1993, when a team of scientists discovered a small, double-stranded
RNA (dsRNA) in nematode (Caenorhabditis elegans) larvae that complemented
the sense strand of a larger mRNA and bound to its 3' untranslated region, thus
inhibiting translation (Lee et al., 1993). Since then, a number of different
mechanisms for translational control by small RNAs have been discovered. In
particular, mRNA is either targeted for cleavage by an siRNA-protein complex, or
translation is prevented by miRNA. Either way, the mRNA is eventually destroyed
by the cell.

RNA interference was popularized by work in C. elegans. When long doublestranded RNAs were injected into a worms gonad, a standard way of
introducing trans-genes into worms, they blocked the expression of
endogenous genes in a sequence-specific manner. In eukaryotes, most proteincoding genes are transcribed by RNA polymerase II, which generates premRNAs that are then processed to form mature mRNAs. These mRNAs are then
transported from the nucleus to the cytoplasm where they are translated. RNAi
can regulate endogenous gene expression. RNAi can be set in motion by
genomically-encoded short regulatory RNAs known as microRNAs. In algae,
worms, and flies, RNAi can be activated by endogenous transposition. In plants
and cultured insect cells, RNAi also has a role in antiviral defense in which viral
double-stranded RNAs are targeted for destruction by the RNAi machinery.

When long double-stranded RNAs enter a cell, they are recognized and cleaved
by Dicer, which is a member of the RNAse III family of double-stranded, RNAspecific endonucleases. Cleavage by Dicer creates short double-stranded RNAs
that are characterized by two nucleotide-long 3 overhangs. These are called
small interfering or siRNAs. siRNAs can form a ribonucleoprotein complex called
RISC, or RNAi silencing complex. This complex includes Slicer, an Argonaute
protein with an RNAse H-like domain called PIWI. RISC first mediates the
unwinding of the siRNA duplex. A single-stranded siRNA that is coupled to RISC
then binds to a target mRNA in a sequence-specific manner. The binding
mediates target mRNA cleavage by Slicer. The site of the cleavage falls in the
middle of the region of siRNA complementarity. The cleaved mRNA can be
recognized by the cell as being aberrant and then destroyed. This prevents
translation from occurring, silencing the expression of the gene from which the
mRNA was transcribed. In plants, the aberrant RNA that results from the RISKmediated cleavage can also serve as a template for RNA-dependent RNA
polymerase, or RDRP. This process relies on unprimed RNA synthesis, in which
the aberrant RNA is used as a template. The resulting double-stranded RNA is a
substrate for Dicer activity, which generates more siRNAs. In some organisms
with endogenous RNAi mechanisms, for example fungi, plants, worms, and
mammals, RNAi also involves another amplification step. In this step, singlestranded siRNAs not associated with RISC bind to their target mRNAs in a
sequence-specific way and serve as a primer for RDRP to polymerize the
antisense RNA strand. Such specificity is intrinsically sensitive to natural
sequence variation. The double-stranded RNA molecule that is created serves
as a substrate for Dicer, which cuts it into siRNAs. In turn, these can either
unwind and prime RNA-dependent RNA polymerization or, together with RISC,
mediate the cleavage of target mRNAs. This amplification, coupled with RNAi
spreading between cells, is thought to underline germline transmission of RNAi
in worms. RNAi spreading has also been described in plants but not in
mammals.
siRNAs begin as small, double-stranded RNA molecules (about 20 base pairs in
length), generated by the cleavage of dsRNA by an enzyme called Dicer, a
member of the RNase III family. siRNAs have two nucleotide overhangs at each 3'
end. miRNAs, on the other hand, originate as small hairpin-shaped precursor
molecules that are cut to size by a Dicer enzyme.
siRNA and miRNA inhibit translation by two different mechanisms while working
in association with a protein, forming a ribonucleoprotein complex called RNAinduced silencing complex (RISC). The proteins in RISC unwind siRNA and remain
bound to a single antisense strand, which then binds to mRNA in a sequencespecific manner, at which time a protein component of RISC called Slicer cuts the
mRNA in the middle of the binding region. The cut mRNA is recognized by the cell
as being abnormal and is subsequently destroyed. In the case of miRNA, a
microRNA-induced silencing complex (miRISC) associates with the mature miRNA,
and the complex binds to mRNA and physically blocks translation. Many miRNAs
form imperfectlycomplementary stem-loop structures on the target sense strand
of mRNA, as opposed to siRNAs, which require near-perfect matches.
In general, only one miRNA is produced from one precursor. In contrast, siRNA is
proposed to moderate its own amplification in plants and certain animal species,
such as C. elegans (Sijen et al., 2001). Proposed models suggest that either the
double-stranded mRNA-siRNA hybrid or the sense strand of siRNA (which is
released by RISC) undergo elongation or transcription, respectively, by RNAdependent RNA polymerase (RdRP) to generate a new double-stranded piece of

RNA, which acts as a new substrate for Dicer and can ultimately lead to the
formation of a new RISC (Figure 1).

Figure 1: A model for the mechanism of RNAi.

Silencing triggers in the form of double stranded RNA may be presented in the
cell as synthetic RNAs or replicating viruses, or may be transcribed from nuclear
genes. These are recognized and processed into small interfering RNAs by Dicer.
The duplex siRNAs are passed to RISC (RNA-induced silencing complex), and the
complex becomes activated by unwinding of the duplex. Activated RISC
complexes can regulate gene expression at many levels. Almost certainly, such
complexes act by promoting RNA degradation and translational inhibition.
However, similar complexes probably also target chromatin remodeling.
Amplification of the silencing signal in plants may be accomplished by siRNAs
priming RNA-directed RNA polymerase (RdRP)-dependent synthesis of new
dsRNA. This could be accomplished by RISC-mediated delivery of an RdRP or by
incorporation of the siRNA into a distinct, RdRP-containing complex.
Evolutionary Research Involving Small, Noncoding RNA
Evolutionary research and studies of gene expression - specifically, how
evolutionary changes in gene-regulatory networks affect phenotypic changes in
an organism - have given scientists an idea of the role of miRNA in
cell differentiation. A number of techniques for combining computational and
experimental work in order to study the rates of evolutionary changes, and link
them, have been developed (Chen & Rajewsky, 2007). Although, as of yet, there
is little evidence of the extent of miRNAs' involvement in cell differentiation, the
current theory is that they function to reinforce more powerful factors that control
developmental processes, particularly because many transcription factors are
highly conserved between distant species while miRNA is not found in some
species, such as budding yeast. Evolutionary studies also indicate that humans
alone might have over 1,000 species-specific (or primate-specific) miRNAs, each

of which can bind to hundreds of different mRNA strands. However, in animals,

miRNA-mediated control of gene expression is often relatively weak compared to
repression by transcription factors. To what extent, then, do we depend on
miRNAs to control gene expression that we need to have so many? Some theories
are that, over time, new miRNAs were acquired in sync with the development of
new tissue types and organs.
Additional roles that miRNA and siRNA have in gene expression involve control of
the inheritance of epigenetic modifications during cell division(Kloc et al., 2008),
and in activation of translation, in certain circumstances, depending on the cell
cycle stage (Buchan & Parker, 2007).

siRNA and Antiviral Defense

Evidence has also shown that siRNA is involved in antiviral defense in certain
plant and animal species. Plants are able to fight viral infection by using the viral
ssRNA to generate dsRNA, some of which is then chopped into small pieces of
siRNA, which can interfere with translation of other mRNA and inhibit viral
replication. siRNA spreads throughout the plant, but the survival of the plant and
the virus is an ongoing battle, as both are constantly evolving to "outwit" the
other (Sadava et al., 2006).
siRNA is also involved in antiviral defense in C. elegans (Wilkins et al., 2005).
Both rde-1 and rde-4 are genes known to be required for RNA interference in the
nematode. In studies, cells derived from rde-1 and rde-4 null mutants of C.
elegans were infected with a specific virus called vesicular stomatitis virus (VSV).
The virus could be tracked because it had been genetically engineered to
express green fluorescent protein (GFP), a frequently used biological
protein marker. Lower levels of fluorescence were observed in mutant cells with
an enhanced RNAi response (Figure 2).
Using Noncoding RNA to Investigate Gene Function
Small, noncoding RNAs have proven to be valuable tools for studying the roles of
specific proteins in the cell. When certain sequences are used to target specific
genes, thus shutting off expression of the protein product, the effects of the
deficiencies on the body can be observed. This approach is being used to study
the effects of abnormal RNAi expression on fetal development. Medical
researchers are also studying ways to control expression of different proteins
linked to various diseases by injecting manufactured dsRNA or antisense siRNA
strands into cells (Whalley, 2006). The mRNA targets of miRNA can be
determined by using bioinformatics methods and bioassays for monitoring the
amounts of target mRNA. In plants, miRNA binding sites are usually found in the
coding regions, while in animals, they are often in the 3' untranslated region of
mRNA (Chen & Rajewsky, 2007).
However, manipulating these different forms of RNA to effectively reduce gene
expression is not always so easy. Investigators have suggested that there are at
least eight different steps to the algorithm for designing the most effective RNAi
molecules to use in order to reduce expression (Reynolds et al., 2004).
Interestingly, many of the elements that need to be considered for optimization
are a direct reflection of what we know about how RNAi worksincluding
recognition and degradation of the target mRNA and interaction between the
siRNA and RISC.
Information provided by studies such as these may lead to the development of
drugs to treat the inappropriate expression of certain genes, or perhaps to
development of RNA-injection therapies for commercially important plants and for

human and animal diseases. Already, efforts are underway to use small,
noncoding RNAs for treatment of a wide array of diseases including cancer, heart
disease, and various infectious diseases (Boyd, 2008). For example, a number of
studies have indicated that small RNAs can act as tumor suppressors in the
treatment of cancer. However, there is also evidence that some miRNAs can act
as oncogenes (Boyd, 2008). It is clear that there is still a lot to learn about the
hundreds of small RNAs in our bodies and what roles they play in gene
expression.