Epigenetics is the study of meiotically and mitotically heritable changes in gene expression which are not coded for in the DNA. Exactly how these epigenetic modifications are directed to the particular gene and the local chromatin has remained enigmatic. Three distinct mechanisms appear to be intricately related and implicated in initiating and/or sustaining epigenetic modifications; DNA methylation, RNA-associated silencing, and histone modifications.

In human cells RNA can specifically direct epigenetic modifications to targeted loci (the promoter regions) and modulate silencing. This regulatory effect is through RNA-associated silencing, can be transcriptional in nature, and is operable through an RNA interference based mechanism (RNAi) that is specifically mediated by the antisense strand of small-interfering RNAs (siRNAs). These recent observations represent a paradigm shift in which a hidden layer of complexity is involved in gene regualtion and is operative via the action RNA essentially epigenetically regulating DNA.

from Kevin V. Morris (2008). RNA Mediated Transcriptional Gene Silencing. In: Morris, K.V. (Ed.) RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press, Norfolk, UK.

Further reading:

1. Epigenetics
2. RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity
3. Molecular Biology Books

Labels: epigenetics, expression, gene regulation, regulation, RNA, RNAi, siRNA

Designed molecules that recognize specific sequences within chromosomal DNA could provide useful probes for natural cellular processes, tools for laboratory experimentation, and lead compounds for therapeutic development. It was discovered that duplex DNA could be recognized by conjugates consisting of DNA oligonucleotides and cationic proteins or peptides. Similarly efficient recognition by neutral peptide nucleic acids (PNAs) was observed. It was found that duplex RNAs could also mediate efficient recognition of duplex DNA. RNAs can target transcription start sites and either inhibit or activate gene expression. This indicates that promoter-targeted RNAs can be powerful tools for regulating gene expression.

from Corey, DR (2008) RNA-Mediated Recognition of Chromosomal DNA. In: Morris , K.V. (Ed.) RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press, Norfolk, UK.

Labels: expression, gene regulation, PNA, promoter, regulation, RNA, therapeutics

A pyknon is a new type of putative regulatory motif that named from the greek adjective for dense. By definition, pyknons are variable length sequences with a statistically significant number of intact copies in the intergenic and intronic regions of the genome and additional copies in the untranslated or amino acid coding regions of known transcripts. Even though the original presentation discussed pyknons in the context of the human genome, pyknons likely represent a more general architectural component of eukaryotic genomes. The exact role of pyknons is currently unclear but the findings so far support a regulatory responsibility. The possibility has been raised that pyknons hint at a previously unseen layer of cell process regulation.

from Rigoutsos, I (2008) Pyknons as putative novel and organism-specific regulatory motifs In: Morris , K.V. (Ed.) RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press, Norfolk, UK.

Labels: gene regulation, pyknon, regulation, RNA

MicroRNAs are short, ~22 nucleotide regulatory RNAs, first discovered in Caenhorhabditis elegans. Hundreds of microRNAs have been identified in plants and animals. Based on the current number of predicted microRNAs, one to three percent of genomic DNA is believed to encode these small, regulatory RNAs.

MicroRNAs inhibit protein synthesis by binding to their target mRNAs and regulating gene expression in a post-transcriptional manner. The exact mechanism by which target gene expression is down-regulated is unclear; however, experimental evidence has led to several different theories to explain microRNA-mediated mRNA repression. These possible mechanisms include target degradation, localization to P-bodies, inhibition of translation initiation or elongation, mRNA deadenylation, and mRNA destabilization.

from Chin and Slack in RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity

Further reading: RNA and the Regulation of Gene Expression

Labels: gene regulation, microRNA, mRNA, regulation, RNA

Short RNAs play multiple roles in affecting gene expression at many levels as illustrated by work in Drosophila. RNA interference uses double stranded RNA which is cleaved by Dicer to produce small interfering RNAs (siRNAs) as guides to cleave homologous mRNAs. This process occurs in the cytoplasm and is used in the endogenous process of viral resistance. In addition, many of the same gene products are also involved in transcriptional gene silencing processes. This was first documented for cosuppression of white-Alcohol dehydrogenase transgenes, which is associated with the Polycomb repressive complex of chromatin proteins. Genetic studies of RNA silencing genes also implicate a role in heterochromatin silencing. Some gene products involved in RNAi are also involved in the formation of repeat associated small RNAs (rasiRNAs), whose formation appears to be Dicer independent and critical for repressing transposon expression particularly in the germline. Roles for small RNAs are also implicated in chromatin insulator activity, the integrity of the nucleolus and long-range associations of homeotic genes.

from Kavi et al in RNA and the Regulation of Gene Expression

Labels: Drosophila, regulation, RNA, RNAi, siRNA

Heterochromatin is a prevalent chromatin state among eukaryotes that has critical functions in chromosome segregation, control of genomic stability and epigenetic regulation of gene expression. In the fission yeast Schizosaccharomyces pombe, two RNAi complexes, the RNAi-induced transcriptional gene silencing (RITS) complex and the RNA-directed RNA polymerase complex (RDRC), are part of a RNAi machinery involved in the initiation, propagation and maintenance of heterochromatin assembly. These two complexes localize in a siRNA-dependent manner on chromosomes, at the site of heterochromatin assembly. RNA polymerase II (RNApII) has a central role in RNAi-dependent heterochromatin assembly. RNApII synthesizes a nascent transcript that serves as an RNA platform to recruit RITS, RDRC and possibly other complexes required for heterochromatin assembly. Both RNAi and an exosome-dependent RNA degradation process contribute to heterochromatic gene silencing. Recently reported findings challenge the widely accepted view that heterochromatic gene silencing is caused strictly by chromatin compaction. As RNAi-dependent chromatin modifications have been observed throughout the eukaryotic kingdom the mechanisms described here may be utilized in a large range of eukaryotes.

from Vavasseur et al in RNA and the Regulation of Gene Expression

Further reading: Epigenetics

Labels: epigenetics, regulation, RNA, RNAi, siRNA, yeast

Regulation of gene expression is a complex, multi-layered process that is crucial to correctly drive and maintain cell identity during development and adult life. RNA interference (RNAi) and the Polycomb system are two well-conserved gene silencing pathways. RNAi participates in post transcriptional as well as transcriptional gene silencing of natural genes as well as transposons and viruses. Polycomb group (PcG) proteins are well-known for their role in silencing HOX genes through modulation of chromatin structure. Both mechanisms are involved in specific epigenetic processes like cosuppression in Drosophila melanogaster and the formation of C. elegans mes and SOP-2 complexes. There are molecular links between RNAi components and Polycomb-mediated silencing in human cells and in Drosophila. RNA polymerase II and Argonaute 1 interact to bring about chromatin modifications on endogenous Polycomb target gene promoters in human cells, while Drosophila RNAi components modulate the nuclear organization of PcG target DNA elements, thereby affecting the strength of PcG-mediated silencing. Several microRNAs and non coding RNAs have been found in human and fly HOX gene loci. They may regulate HOX gene expression both post-trascriptionally and co-transcriptionally.

from Portoso and Cavalli in RNA and the Regulation of Gene Expression (Chapter 3)

Labels: Drosophila, epigenetics, polycomb, regulation, RNA, RNAi

Epigenetics is the study of meiotically and mitotically heritable changes in gene expression which are not coded for in the DNA. Three distinct mechanisms appear to be intricately related and implicated in initiating and/or sustaining epigenetic modifications; DNA methylation, RNA-associated silencing, and histone modifications. While chromatin remodeling and DNA methylation have been studied for several years now far less is know about how these epigenetic marks are directed to each particular gene. Recently, however the role of RNA in epigenetic gene regulation has begun to become apparent.

from Kevin V. Morris in "Chapter 2 Epigenetic Regulation of Gene Expression" from RNA and the Regulation of Gene Expression

Further reading:

1. RNA and the Regulation of Gene Expression
2. Epigenetics

Labels: epigenetics, expression, regulation, RNA

NASBA is an isothermal nucleic acid amplification method which is particularly suited to detection and quantification of genomic, ribosomal or messenger RNA. The product of NASBA is single-stranded RNA of opposite sense to the original target. The initial NASBA methods relied on liquid or gel-based probe-hybridisation for post-amplification detection of products. More recently, real-time procedures incorporating amplification and detection in a single step have been applied to a wide range of RNA and some DNA targets. Real-time NASBA has become a sensitive and specific method for detection, quantification and differentiation of RNA and DNA targets. Molecular beacons have been used in real-time NASBA in commercially-available kits in published assays. The increase in availability of fluorimeters suitable for real-time NASBA ensures that this methodology will become a realistic alternative to real-time reverse transcriptase PCR.

NASBA technology is an alternative method to standard proceduresfor the amplification and detection of a range of nucleic acid targets. The majority of applications have been developed for detection and analysis of RNA targets including viral genomes, viroids, ribosomal RNA (rRNA) and messenger RNA (mRNA). Advantages of NASBA over methods such as reverse transcriptase PCR include fast amplification kinetics and selective amplification of RNA in a background of DNA. The amplification is isothermal and thus there is no requirement for thermocycling during the procedure. Single-stranded RNA amplicons are produced by NASBA which can be used directly in subsequent rounds of amplification or probed for detection without the need for denaturation or strand separation.

Real-time NASBA assays are rapid, specific and sensitive with RNA amplification and a target-specific fluorescent signal achieved simultaneously in one tube with measurements obtained through a fluorimeter. Qualitative, quantitative, monoplex and multiplex formats of real-time NASBA have now been described. The methodology seems to be a suitable alternative to other amplification procedures without the need for expensive thermocyclers. Since the original reports of real-time NASBA in 1998 the number of applications, available kits and expansion to include DNA targets is apparent and likely to continue over the next few years.

from Fox et al in "Chapter 12: Real-Time NASBA" from Real-Time PCR: Current Technology and Applications

Further reading: Real-Time PCR: Current Technology and Applications

Labels: DNA, NASBA, real-time PCR, RNA, RT-PCR

Hammerhead ribozymes are the smallest known naturally occurring ribozymes which are capable of catalyzing the endonucleolytic trans-esterification of RNA. A recent re-examination of the catalytic properties of naturally-derived hammerhead ribozymes has resulted in a better understanding of the catalytic efficiency of this enzyme in vitro and in vivo. The minimal trans-cleaving hammerhead ribozyme has been a ubiquitous tool in both genomics and therapeutics research over the last twenty years and these new insights into hammerhead ribozyme biochemistry may offer hope for the generation of improved trans-cleaving ribozymes which function effectively in vivo. Next-generation hammerhead ribozymes may play an important role as therapeutic agents, as enzymes which tailor defined RNA sequences, as biosensors, and for applications in functional genomics and gene discovery.

from Hean and Weinberg in Chapter 1 from RNA and the Regulation of Gene Expression

Further reading: RNA and the Regulation of Gene Expression

Labels: biosensor, genomics, hammerhead ribozyme, regulation, ribozyme, ribozymes, RNA, therapeutics