Small Non-coding RNA and 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.

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.

RNAi technique animation tutorial from Nature Reviews Genetics.

© 2009 Nature Publishing Group All rights reserved.

Transcript

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 imperfectly complementary 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.

© 2002 Nature Publishing Group Hannon, G. J. RNA interference. Nature 418, 249 (2002). All rights reserved.

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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).

Figure 2

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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).

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 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.