Small Non-coding RNA and Gene Expression

While we've been taught: don't shoot the messenger, our cells haven’t 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 MechanismsControl 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 double-stranded RNAs were injected into a worm’s 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 protein-coding genes are transcribed by RNA polymerase II, which generates pre-mRNAs 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, RNA-specific 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 RISK-mediated 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, single-stranded 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 RNA-induced silencing complex (RISC). The proteins in RISC unwind siRNA and remain bound to a single antisense strand, which then binds to mRNA in a sequence-specific 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 RNA-dependent 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 RNAEvolutionary 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 FunctionSmall, 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 works—including 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.