Cis-natural Antisense Transcript
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Natural antisense transcripts (NATs) are a group of
RNA Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...
s encoded within a cell that have transcript complementarity to other RNA transcripts. They have been identified in multiple
eukaryote The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...
s, including humans, mice, yeast and ''
Arabidopsis thaliana ''Arabidopsis thaliana'', the thale cress, mouse-ear cress or arabidopsis, is a small plant from the mustard family (Brassicaceae), native to Eurasia and Africa. Commonly found along the shoulders of roads and in disturbed land, it is generally ...
''. This class of RNAs includes both protein-coding and non-coding RNAs. Current evidence has suggested a variety of regulatory roles for NATs, such as
RNA interference RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by ...
(RNAi),
alternative splicing Alternative splicing, alternative RNA splicing, or differential splicing, is an alternative RNA splicing, splicing process during gene expression that allows a single gene to produce different splice variants. For example, some exons of a gene ma ...
,
genomic imprinting Genomic imprinting is an epigenetic phenomenon that causes genes to be expressed or not, depending on whether they are inherited from the female or male parent. Genes can also be partially imprinted. Partial imprinting occurs when alleles from b ...
, and X-chromosome inactivation. NATs are broadly grouped into two categories based on whether they act in cis or in trans. Trans-NATs are transcribed from a different location than their targets and usually have complementarity to multiple transcripts with some mismatches.
MicroRNA Micro ribonucleic acid (microRNA, miRNA, μRNA) are small, single-stranded, non-coding RNA molecules containing 21–23 nucleotides. Found in plants, animals, and even some viruses, miRNAs are involved in RNA silencing and post-transcr ...
s (miRNA) are an example of trans-NATs that can target multiple transcripts with a few mismatches. Cis-natural antisense transcripts (cis-NATs) on the other hand are transcribed from the same genomic locus as their target but from the opposite DNA strand and form perfect pairs.


Orientation

Cis-NATs have a variety of orientations and differing lengths of overlap between pairs. There have been five identified orientations for cis-NATs to date. The most common orientation is head-to-head, where the
5' end Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. In a single strand of DNA or RNA, the chemical convention of naming carbon atoms in the nucleotide pentose-sugar-r ...
s of both transcripts align together. This orientation would result in the greatest knockdown of gene expression if transcriptional collision is the reason for transcript inhibition. There are however some studies that have suggested that tail-to-tail orientations are the most common NAT pairs. Others such as tail to tail, overlapping, nearby head-to- head, and nearby tail-to-tail are less frequently encountered. Completely overlapping NATs involve the antisense gene being located completely over top of each other. Nearby head-to-head and tail-to-tail orientations are physically discrete from each other but are located very close to each other. Current evidence suggests that there is an overrepresentation of NAT pairs in genes that have catalytic activity. There may be something about these genes in particular that makes them more prone to this type of regulation.


Identification approach

Identification of NATs in whole genomes is possible due to the large collection of sequence data available from multiple organisms. ''
In silico In biology and other experimental sciences, an ''in silico'' experiment is one performed on a computer or via computer simulation software. The phrase is pseudo-Latin for 'in silicon' (correct ), referring to silicon in computer chips. It was c ...
'' methods for detecting NATs suffer from several shortcomings depending on the source of sequence information. Studies that use
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...
have sequences whose orientations are known, but the amount of mRNA sequence information available is small. Predicted gene models using algorithms trained to look for genes gives an increased coverage of the genome at the cost of confidence in the identified gene. Another resource is the extensive
expressed sequence tag In genetics, an expressed sequence tag (EST) is a short sub-sequence of a cDNA sequence. ESTs may be used to identify gene transcripts, and were instrumental in gene discovery and in gene-sequence determination. The identification of ESTs has pro ...
(EST) libraries but these small sequences must first be assigned an orientation before useful information can be extracted from them. Some studies have utilized special sequence information in the ESTs such as the poly(A) signal,
poly(A) tail Polyadenylation is the addition of a poly(A) tail to an RNA transcript, typically a messenger RNA (mRNA). The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In euka ...
, and splicing sites to both filter the ESTs and to give them the correct transcriptional orientation. Combinations of the different sequence sources attempts to maximize coverage as well as maintain integrity in the data. Pairs of NATs are identified when they form overlapping clusters. There is variability in the cut-off values used in different studies but generally ~20 nucleotides of sequence overlap is considered the minimum for transcripts to be considered and overlapping cluster. Also, transcripts must map to only one other mRNA molecule in order for it to be considered a NAT pair. Currently there are a variety of web and software resources that can be used to look for antisense pairs. The NATsdb or Natural Antisense Transcript database is a rich tool for searching for antisense pairs from multiple organisms.


Mechanisms

Molecular mechanisms behind the regulatory role of cis-NATs are not currently well understood. Three models have been proposed to explain the regulatory effects that cis-NATs have on gene expression. The first model attributes that base pairing between the cis-NAT and its complementary transcript result in a knockdown of mRNA expression. The assumption of this model is that there will be a precise alignment of at least 6 base pairs between the cis-NAT pair to make double stranded RNA. Epigenetic modifications like
DNA methylation DNA methylation is a biological process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoter (genetics), promoter, DNA methylati ...
and post-translational modification of core
histone In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei and in most Archaeal phyla. They act as spools around which DNA winds to create structural units called nucleosomes ...
s form the basis of the second model. Although it is not yet clearly understood, it is thought that the reverse transcript guides methylation complexes and/or histone-modifying complexes to the promoter regions of the sense transcript and cause an inhibition of expression from the gene. Currently it is not known what attributes of cis-NATs are crucial for the epigenetic model of regulation. The final proposed model that has gained favour due to recent experimental evidence is the transcriptional collision model. During the process of transcription of cis-NATs, the transcriptional complexes assemble in the promoter regions of the gene.
RNA polymerase In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that catalyzes the chemical reactions that synthesize RNA from a DNA template. Using the e ...
s will then begin transcribing the gene at the transcription initiation site laying down nucleotides in a 5' to 3' direction. In the areas of overlap between the cis-NATs the RNA polymerases will collide and stop at the crash site. Transcription is inhibited because RNA polymerases prematurely stop and their incomplete transcripts get degraded.


Importance

Regulation of many biological processes such as development, metabolism and many others requires a careful co-ordination between many different genes; this is usually referred to as a
gene regulatory network A gene (or genetic) regulatory network (GRN) is a collection of molecular regulators that interact with each other and with other substances in the cell to govern the gene expression levels of mRNA and proteins which, in turn, determine the fu ...
. A flurry of interest in gene regulatory networks has been sparked by the advent of sequenced genomes of multiple organisms. The next step is to use this information to figure out how genes work together and not just in isolation. During the processes of mammalian development, there is an inactivation of the extra X-chromosome in females. It has been shown that a NAT pair called Xist and Tsix are involved in the hypermethylation of the chromosome. As much as 20–30% of mammalian genes have been shown to be the targets of
miRNA Micro ribonucleic acid (microRNA, miRNA, μRNA) are small, single-stranded, non-coding RNA molecules containing 21–23 nucleotides. Found in plants, animals, and even some viruses, miRNAs are involved in RNA silencing and post-transcri ...
s, which highlights the importance of these molecules as regulators across a wide number of genes. Evolutionary reasons for utilizing RNA for regulation of genes may be that it is less costly and faster than synthesizing proteins not needed by the cell. This could have had a selective advantage for early eukaryotes with this type of transcriptional regulation.


Disease

Antisense transcription might contribute to disease through chromosomal changes that result in the production of aberrant antisense transcripts. A documented case of cis-NATs being involved in human disease comes from an inherited form of α-
thalassemia Thalassemias are a group of Genetic disorder, inherited blood disorders that manifest as the production of reduced hemoglobin. Symptoms depend on the type of thalassemia and can vary from none to severe, including death. Often there is mild to ...
where there is silencing of the
hemoglobin Hemoglobin (haemoglobin, Hb or Hgb) is a protein containing iron that facilitates the transportation of oxygen in red blood cells. Almost all vertebrates contain hemoglobin, with the sole exception of the fish family Channichthyidae. Hemoglobin ...
α-2 gene through the action of a cis-NAT. It is thought that in malignant cancer cells with activated
transposable element A transposable element (TE), also transposon, or jumping gene, is a type of mobile genetic element, a nucleic acid sequence in DNA that can change its position within a genome. The discovery of mobile genetic elements earned Barbara McClinto ...
s creates a large amount of transcriptional noise. It is likely that aberrant antisense RNA transcripts resulting from this transcriptional noise may cause stochastic methylation of
CpG island The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG sites occur with high frequency in genomic regions called CpG isl ...
s associated with
oncogene An oncogene is a gene that has the potential to cause cancer. In tumor cells, these genes are often mutated, or expressed at high levels.
s and
tumor suppressor gene A tumor suppressor gene (TSG), or anti-oncogene, is a gene that regulates a cell (biology), cell during cell division and replication. If the cell grows uncontrollably, it will result in cancer. When a tumor suppressor gene is mutated, it results ...
s. This inhibition would further progress the malignancy of the cells since they lose key regulator genes. By looking at upregulated antisense transcripts in tumor cells, researchers are able to look for more candidate tumor suppressor genes. Also, aberrant cis-NATs have been implicated in neurological diseases such as
Parkinson's disease Parkinson's disease (PD), or simply Parkinson's, is a neurodegenerative disease primarily of the central nervous system, affecting both motor system, motor and non-motor systems. Symptoms typically develop gradually and non-motor issues become ...
.


References

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