RNA silencing


RNA silencing or RNA interference refers to a family of gene silencing effects by which gene expression is negatively regulated by non-coding RNAs such as microRNAs. RNA silencing may also be defined as sequence-specific regulation of gene expression triggered by double-stranded RNA. RNA silencing mechanisms are conserved among most eukaryotes. The most common and well-studied example is RNA interference, in which endogenously expressed microRNA or exogenously derived small interfering RNA induces the degradation of complementary messenger RNA. Other classes of small RNA have been identified, including piwi-interacting RNA and its subspecies repeat associated small interfering RNA.

Background

RNA silencing describes several mechanistically related pathways which are involved in controlling and regulating gene expression. RNA silencing pathways are associated with the regulatory activity of small non-coding RNAs that function as factors involved in inactivating homologous sequences, promoting endonuclease activity, translational arrest, and/or chromatic or DNA modification. In the context in which the phenomenon was first studied, small RNA was found to play an important role in defending plants against viruses. For example, these studies demonstrated that enzymes detect double-stranded RNA not normally found in cells and digest it into small pieces that are not able to cause disease.
While some functions of RNA silencing and its machinery are understood, many are not. For example, RNA silencing has been shown to be important in the regulation of development and in the control of transposition events. RNA silencing has been shown to play a role in antiviral protection in plants as well as insects. Also in yeast, RNA silencing has been shown to maintain heterochromatin structure. However, the varied and nuanced role of RNA silencing in the regulation of gene expression remains an ongoing scientific inquiry. A range of diverse functions have been proposed for a growing number of characterized small RNA sequences—e.g., regulation of developmental, neuronal cell fate, cell death, proliferation, fat storage, haematopoietic cell fate, insulin secretion.
RNA silencing functions by repressing translation or by cleaving messenger RNA, depending on the amount of complementarity of base-pairing. RNA has been largely investigated within its role as an intermediary in the translation of genes into proteins. More active regulatory functions, however, only began to be addressed by researchers beginning in the late-1990s. The landmark study providing an understanding of the first identified mechanism was published in 1998 by Fire et al., demonstrating that double-stranded RNA could act as a trigger for gene silencing. Since then, various other classes of RNA silencing have been identified and characterized. Presently, the therapeutic potential of these discoveries is being explored, for example, in the context of targeted gene therapy.
While RNA silencing is an evolving class of mechanisms, a common theme is the fundamental relationship between small RNAs and gene expression. It has also been observed that the major RNA silencing pathways currently identified have mechanisms of action which may involve both post-transcriptional gene silencing as well as chromatin-dependent gene silencing pathways. CDGS involves the assembly of small RNA complexes on nascent transcripts and is regarded as encompassing mechanisms of action which implicate transcriptional gene silencing and co-transcriptional gene silencing events. This is significant at least because the evidence suggests that small RNAs play a role in the modulation of chromatin structure and TGS.
Despite early focus in the literature on RNA interference as a core mechanism which occurs at the level of messenger RNA translation, others have since been identified in the broader family of conserved RNA silencing pathways acting at the DNA and chromatin level. RNA silencing refers to the silencing activity of a range of small RNAs and is generally regarded as a broader category than RNAi. While the terms have sometimes been used interchangeably in the literature, RNAi is generally regarded as a branch of RNA silencing. To the extent it is useful to craft a distinction between these related concepts, RNA silencing may be thought of as referring to the broader scheme of small RNA related controls involved in gene expression and the protection of the genome against mobile repetitive DNA sequences, retroelements, and transposons to the extent that these can induce mutations. The molecular mechanisms for RNA silencing were initially studied in plants but have since broadened to cover a variety of subjects, from fungi to mammals, providing strong evidence that these pathways are highly conserved.
At least three primary classes of small RNA have currently been identified, namely: small interfering RNA, microRNA, and piwi-interacting RNA.

small interfering RNA (siRNA)

siRNAs act in the nucleus and the cytoplasm and are involved in RNAi as well as CDGS. siRNAs come from long dsRNA precursors derived from a variety of single-stranded RNA precursors, such as sense and antisense RNAs. siRNAs also come from hairpin RNAs derived from transcription of inverted repeat regions. siRNAs may also arise enzymatically from non-coding RNA precursors. The volume of literature on siRNA within the framework of RNAi is extensive. One of the potent applications of siRNAs is the ability to distinguish the target versus non-target sequence with a single-nucleotide difference. This approach has been considered as therapeutically crucial for the silencing dominant gain-of-function disorders, where mutant allele causing disease is differed from wt-allele by a single nucleotide. This type of siRNAs with capability to distinguish a single-nt difference are termed as allele-specific siRNAs.

microRNA (miRNA)

The majority of miRNAs act in the cytoplasm and mediate mRNA degradation or translational arrest. However, some plant miRNAs have been shown to act directly to promote DNA methylation. miRNAs come from hairpin precursors generated by the RNaseIII enzymes Drosha and Dicer. Both miRNA and siRNA form either the RNA-induced silencing complex or the nuclear form of RISC known as RNA-induced transcriptional silencing complex. The volume of literature on miRNA within the framework of RNAi is extensive.

Three prime untranslated regions and microRNAs

s of messenger RNAs often contain regulatory sequences that post-transcriptionally cause RNA interference. Such 3'-UTRs often contain both binding sites for microRNAs as well as for regulatory proteins. By binding to specific sites within the 3'-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript. The 3'-UTR also may have silencer regions that bind repressor proteins that inhibit the expression of a mRNA.
The 3'-UTR often contains microRNA response elements. MREs are sequences to which miRNAs bind. These are prevalent motifs within 3'-UTRs. Among all regulatory motifs within the 3'-UTRs, MREs make up about half of the motifs.
As of 2014, the miRBase web site, an archive of miRNA sequences and annotations, listed 28,645 entries in 233 biologic species. Of these, 1,881 miRNAs were in annotated human miRNA loci. miRNAs were predicted to have an average of about four hundred target mRNAs. Freidman et al. estimate that >45,000 miRNA target sites within human mRNA 3'UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs.
Direct experiments show that a single miRNA can reduce the stability of hundreds of unique mRNAs. Other experiments show that a single miRNA may repress the production of hundreds of proteins, but that this repression often is relatively mild.
The effects of miRNA dysregulation of gene expression seem to be important in cancer. For instance, in gastrointestinal cancers, nine miRNAs have been identified as epigenetically altered and effective in down regulating DNA repair enzymes.
The effects of miRNA dysregulation of gene expression also seem to be important in neuropsychiatric disorders, such as schizophrenia, bipolar disorder, major depression, Parkinson's disease, Alzheimer's disease and autism spectrum disorders.

piwi-interacting RNA (piRNA)

piRNAs represent the largest class of small non-coding RNA molecules expressed in animal cells, deriving from a large variety of sources, including repetitive DNA and transposons. However, the biogenesis of piRNAs is also the least well understood. piRNAs appear to act both at the post-transcriptional and chromatin levels. They are distinct from miRNA due to at least an increase in terms of size and complexity. Repeat associated small interfering RNA are considered to be a subspecies of piRNA.

Mechanism

The most basic mechanistic flow for RNA Silencing is as follows:
1: RNA with inverted repeats hairpin/panhandle constructs --> 2: dsRNA --> 3: miRNAs/siRNAs --> 4: RISC --> 5: Destruction of target mRNA
  1. It has been discovered that the best precursor to good RNA silencing is to have single stranded antisense RNA with inverted repeats which, in turn, build small hairpin RNA and panhandle constructs. The hairpin or panhandle constructs exist so that the RNA can remain independent and not anneal with other RNA strands.
  2. These small hairpin RNAs and/or panhandles then get transported from the nucleus to the cytosol through the nuclear export receptor called exportin-5, and then get transformed into a dsRNA, a double stranded RNA, which, like DNA, is a double stranded series of nucleotides. If the mechanism didn't use dsRNAs, but only single strands, there would be a higher chance for it to hybridize to other "good" mRNAs. As a double strand, it can be kept on call for when it is needed.
  3. The dsRNA then gets cut up by a Dicer into small strands of miRNAs or siRNAs A Dicer is an endoribonuclease RNase, which is a complex of a protein mixed with strand of RNA.
  4. Lastly, the double stranded miRNAs/siRNAs separate into single strands; the antisense RNA strand of the two will combine with another endoribonuclease enzyme complex called RISC, which includes the catalytic component Argonaute, and will guide the RISC to break up the "perfectly complementary" target mRNA or viral genomic RNA so that it can be destroyed.
  5. It means that based on a short sequence specific area, a corresponding mRNA will be cut. To make sure, it will be cut in many other places as well. It has also been shown that the repeated-associated short interference RNAs have a role in guiding chromatin modification.