Principal Hallmark characteristics of small and long non-coding RNAs
“Mutations have been crucial for geneticists, as day and night for astronomers. Whithout the successions of days and night we would not know about stars. Whithout mutations we would know very little about inheritance and the existence of genes.”
Gustavo Hoecker Salas
(December 5, 1915- March 19, 2008 )
National Prize of Science of Chile in 1989
The idea of variation in nature is very old, in Heraclitus of Ephesus (504-500 BC) we find the first ideas of changes when he stated: “we never bathed in the same river”. However in the field of biology, the Greeks considered that the species were immutables. This concept changes with the first scientific ideas of organic evolutions and heredity. Lamarck proposed the first evolutionary theory where the organisms evolved from simple forms. Also he proposed an hereditary model in which the environmental influences are very important as an agents of evolutionary change and proposed the Theory of acquired characters. With the Mendelism advent, Lamarcks’s Theory was left behind and all the mutations in the living organisms were attributed to Mendelian “factors”. However in recent years with the development of epigenesis, genomic imprinting and the horizontal transferences of the genes, Lamarck’s ideas have resurfaced.
The concept of mutation was coined by Hugo De Vries in 1901, whom worked with plants species of the genus Oenothera where he discovered some phenotypic hereditary characteristics that he coined as “mutations” and “mutants” to those individuals that have these phenotypic alterations. In opinion of De Vries, these mutations give origin to a new species that he named “elementary species” , . Thus, this gave birth to the saltacionist Theory of Evolution that he described in his book entitled “ Mutations”. The harmony between Mutation Theory and Mendel model of heredity, the simplicity of the experimental method and the vast accumulation of supporting data, explain the big impact in the biological world . Also, De Vries ventured with a hipothesis: “ With the knowledge of the principles of the mutations will be possible in the future to induce mutations artificially” . Wilhelm Johannsen argued that evolution consisted of discontinuous changes between “pure lines” and carried out their classic experiments in the beans
Mutations have been historically the cornerstone of biological disciplines: in basic science, to understand biodiversity and evolution of species, in medicine to explain phenotypic variation and diseases, in education to justify the individual differences found between the students within a classroom and also in agriculture and veterinary in the improvement of plants and animals useful to man. Thus, Mutations have allowed the explosive growth of genetics as an experimental science. In multicellular organisms the cell differentiation requires a series of genetic and epigenetic changes. The mutations (epimutations) can occurs also post transcriptionally in the different type of RNAs that constitute the epigenome. This article explores this theme, in the framework of the adaptation, phenotypic plasticity and evolution of eukaryotes.
2. Mutations at genome level
At the beginning of the genetics as an experimental discipline, mutations have been associated to the classic Mendelian genes and, with the advent of molecular genetics these genetic changes are produced in the coding area of the DNA. A gene occupied a definite place in the chromosome that was associated with a well determined phenotype,thus the gene was simultaneously a unit of mutation and function and were indivisible by recombination . Archivald Garrod in 1909 was interested in to explain the origins and inheritance of human diseases. Also he was the first proposing the concept that a gene is in direct relationship with the production of a specific protein and that establishes the genetic control of some inborn error of the metabolism. He showed that an alteration in an enzyme was linked to amino acid metabolism. In 1941 Beadle and Tatum postulated the hypothesis “one gene-one enzyme”. Thus, each gene control the production, function and specificity of a particular enzyme. Studies conducted in differents organisms proves that the capacity to synthetize the appropriate amino acid is caused by the modification or loss of a single enzyme. This concept was changed by Vernon Ingram who postulated the hypotesis “one gene-one polypeptide” in base to the sickle cell anemia disease in humans. Also Ingram postulated that this disease is caused by a single gene mutation which is letal in homozygous with severe sickle cell anemia, and is semiletal in heterozygous that show an attenuated sickle cell anemia. Normal homocigotes individuals are normal for the form of their blood cells and their hemoglobin in an electrophoretic analysis migrated differently in comparation to those heterozygotous individuals. The fingerprint show that the differences between normal and diseased individuals was only a single amino acids substitution in one of the beta chain of polypeptide. The glutamic acid in normal individuals is replaced by valina in individuals with sickle cell anemia. The difference between valina and glutamic acids is only one base in the codon. Moreove, the amino acid changes in one chain is independent of changes in the other chain, suggesting that the gene determining the alpha and beta chain are located in different loci. Thus one gene codes for one polypeptide and several polypeptides may be necessary for a functional enzyme of the organism. In 1961, Seysmour Benzer studing the fine structure of genes by using mutants in the phage T4 of E.coli, use for first time the concept of cistron. Inside of a gene, there are differents cistrons or “functional units”. Benzer demostrated the hypotesis of Ingram, the cistron corresponds to a sequence of nucleotides that code for a polypeptidic chain [9, 10]
The ideas about the genetic action and its mutability were complemented by Goldschmidt in1940  who defined the gene on the basis of its physiological action. With the first DNA sequencing by Frederick Sanger, it was clearly demostrated by C. Yanofsky that the gene is a nucleotide sequence that encodes for proteins. Thus, within the genes there are information for the amino acid sequence of the primary structure of protein.  Any mutation at nucleotides of a gene may cause an alteration in the primary structure of the protein. Depending on the phenotypic effect causing these mutations can be lethal, semiletal, deletereos or innocuous (silent mutation). Many researchers were interested in inducing mutations with differents agents in plants and animals such as Hermann Muller in
An important step in the process of regulation of gene expression were the Jacob and Monod experiments in
3. Epimutations at epigenome level
The concept of epigenome is a recent concept in genetics that arises with epigenesis concept. The epigenome involved the chemical changes at DNA level such as methylation and also histones acetylation, chromatin remodeling and phenotypic changes that originate by ncRNAs . The epigenesis is a old concept that was coined in 1942 by Conrrad H. Waddington to explain as an adults can be formed from a cygote by cell differentiation and gene regulation. In a multicellular organism each cell has an epigenotype that is determined by which genes are functioning in that particular cell. The differentiation of multicellular organisms is controlled by epigenetic markers and are transmitted through cell division. However, have been demonstrated that epigenetic changes in germ cell line could be hereditable transgenerationally. Epigenesis is a heritable changes in the expression of genes that not involve a change in the nucleotide structure of DNA but only changes in the chromatin. These changes alter the capacity of genes to respond to external signals . Epigenetic changes allows heritable or transgenerational modifications in the expression of genes without the need of mutations at DNA level and not necessarily following the Mendelian model of heredity. In classical model of Mendelian heredity a gene’s effects were assumed to be independent of its parental origin, but is know that some genes have differents effects depending if gene was inherited via a sperm or an egg. This process is know as genomic imprinting. At present there is a lot of evidence that genomic imprinting inclusive may influence human behavior. Is know that children who inherit a chromosomal deletion of 15q11-q13 from their father have behavior different of children who inherit a similar deletion from their mother [18, 19, 20]. Also, experimental animal models in mouse shows that in utero or early life environmental exposures produce effects that can be inherited transgenerationally and are accompanied by epigenetic alterations . These changes in the epigenome have been named as “epimutations”. In humans there are just a few reports that have been used to suggest inheritance of epimutations and the search of these epigenetic inheritance is under way . Some evidences have been described in colorectal cancer [ 22, 23, 24, 25].
Epigenesis and epimutation concepts also extend to ncRNAs that have different functions and in human genome constitute about of 60% of the total transcriptional output [26, 27, 28, 29, 30]. The ncRNAs are short single-stranded between 18 to 30nt length such as micro RNA(miRNAs) Small interfering RNA (siRNAs), small nuclear RNA (snRNAs), Small nucleolar RNAs(snoRNAs), piwi- interacting RNAs (piRNAs) and long nc RNA (lncRNA) 200-2800 nt length. All these ncRNAs are hairpin that are paired in some places similar to tRNAs. The homologies detected between the ncRNAs with endogenous viruses, tramposons and introns revealed that ncRNAs probably originates from RNA viruses . In the eukaryote genome, the ncRNAs are located in the non coding areas of mRNAs, endogenous viruses, tramposons and also transcribed from non coding DNA areas. The ncRNAs not transcribed for proteins and are characterized for a great variety of processes that included genomic imprinting, as enhancers of transcriptional regulation, mRNA processing and modification, sex determination by dosage compensation, protein degradation, oncogenic, tumor-suppresive, neural and synaptic plasticity of learning and memory and cognitive capacity by regulating dendrite morphogenesis during early development and also viral and tramposons defense [28,29,30,32,33,34]. Most of the mRNA stability elements are considered to be located in the 5′- and 3′- untranslated regions (UTRs) of genes where are located ncRNAs [35, 36] In the following paragraphs are detailed the features and the functions of each ncRNAs in eukaryotes. Also describes the effects of the mutations in the origin of disease, and also in the adaptation and evolution of the species. In Table 1 are shows the principal hallmark characteristics of these smalls and long ncRNAs.
|Name||Length in nucleotides (nt)||Principal functions||References|
|siRNAs||21-23 nt||mRNA cleavage|||
|miRNAs||21-23 nt||Regulate developmental timing||[50-52]|
|piRNAs||29-30 nt||Tramposons silencing in gametes|||
|snRNAs||90-216 nt||Efficiency of splicing, maintaining telomeres||[69-70]|
|snoRNAs||< 70 nt||Guide methylation of rRNAs,tRNAs and other snRNAs||[69,72]|
|lnc RNAs||200-2800 nt||X chromosome inactivation, human brain development||[76,77,94]_|
4. The mutations at non-coding RNAs level
4.1. Short interfering RNAs
The eukaryotic genome encode an ample amount of short interfering RNAs, in different cells and tissues principally miRNAs, siRNAs and piRNAs that have less than 200 nt length and are highly conserved. These short ncRNAs are engaged in specific gene regulation and modulate the development of several eukaryote organisms including mammals and are involved in gene silencing in higher eukaryotes [27,37]. They act by binding to complementary sites on targets mRNAs to induce cleavage or repression of transcription in
a specific manner. Thus these ncRNAs could participate in the degradation of some specific sequence of mRNA. Also, a mutation in proteins required for miRNAS function or biogenesis can affect animal development [ 37, 38, 39,40 ]. Generally the target genes and the mechanism of target suppression are unknown, the reason for this is that miRNAs have a very short sequence of nucleotides, and also the interaction of base pairs with target mRNAs may be affected by a protein complex . Unlike miRNAs of animals, miRNA target of plants are more easily identified because of near-perfect complementarity to their target sequences and act as siRNAs and destroy its target mRNA . In plants, the miRNAs target sites are generally found into the protein–coding segment of the target mRNAs but in animals are found in untranslated region 3’UTR [40, 41]. MiRNAs and siRNAs are processed from a double-stranded RNA precursors about 70 nt by a specific ribonuclease, DICER that excises long RNA into short duplexes of 21-23 nucleotides called siRNAs and miRNAs. Only one type of DICER is found
5. Mutations in Piwi interacting non-coding RNAs
PiRNAs are other class of small ncRNAs molecules that have 29-30 nt lenght and form the piRNA-induced silencing complex (piRISC) protein in the germ line of many animal species. Piwi proteins bind to piRNAs, which map to transposons. PiRNAs are important regulators of gametogenesis and have been proposed to play roles in transposon silencing .
PiRNAs are produced by the primary processing of single-stranded transcripts of heterochromatic master loci  The piRISC complex protects the integrity of the genome from invasion of transposable elements and other genetic elements as viruses and silencing them. They express only in gonads, specially during the spermatogenesis regulating the meiosis.[ 63,64] but also has been described during de ovogenesis . As a result of the loss of piRNAs silencing, in
Piwi proteins and piRNAs have conserved functions in transposon silencing in the embryonic male germ line. Piwi proteins are proposed to be piRNAs-guided endonucleases that initiate secondary piRNA biogenesis.The biogenesis and piRNA amplification is fundamental for the silencing of LINE1 transposons. Experimental data in mice in base to mutations in Mili and Miwi 2 alleles revealed that the defective piRNAs results in spermatogenic failure and sterility. .The relevance of the non-coding genome in human disease has mainly been studied in the context of the widespread disruption of miRNAs expression and function that is seen in human cancer. At present we are only beginning to understand the nature and extent of the piRNAs, snoRNAs, transcribed ultraconserved regions (T-UCRs) and large intergenic non-coding RNAs (lincRNAs) are emerging as key elements of cellular homeostasis . Genomic imprinting causes parental origin–specific monoallelic gene expression through differential DNA methylation established in the parental germ line. However, the mechanisms underlying how specific sequences are selectively methylated are not fully understood. Has been found that the components of the piRNAs pathway are required for de novo methylation of the differentially methylated region (DMR) of the imprinted mouse Rasgrf1 locus, but not other paternally imprinted loci. A retrotransposon sequence within a ncRNAs spanning the DMR was targeted by piRNAs generated from a different locus. A direct repeat in the DMR, which is required for the methylation and imprinting of Rasgrf1, served as a promoter for this RNA. Has been proposed a model in which piRNAs and a target RNA direct the sequence-specific methylation of Rasgrf1.
6. Mutations in small nuclear ncRNAs
SnRNAs are short molecules of RNA that are located within the nucleus of cells and participate in a variety of processes such as RNA splicing, regulation of transcription factors (7SK RNA) or RNA polimerase II (B2 RNA) and maintaining the telomeres . RNA-RNA interactions between snRNAs or between snRNAs and the pre-mRNAs play critical roles in the accuracy and efficiency of the splicing. The snRNAs also are combined with the protein factors, they make an RNA-protein complex called small nucleoriboprotein (snRNP).The presence of dynamic RNA-RNA interactions within a ribonucleoprotein (RNP) complex like the spliceosome suggests that the snRNAs themselves may need to adopt more than one RNA conformation in order to execute their functions during splicing. Not all of these interactions are established simultaneously, nor do they persist once established. Rather, interactions are formed, modified, disrupted, and replaced during spliceosome assembly and splicing. . The complex structure of spliceosome and the varied interactions between their protein subunits make than any mutations in the nucleotide structure of the snRNAs cause alterations in some of its interactions and functions. Thus, it has been demostrate that in yeast alternative RNA folding can cause cold sensitive function of RNA and that in the case of U2 snRNA, for which the potential to form the alternative structure is conserved, disrupting the alternative folding relieves the cold sensitive defect. This finding suggests that alternative RNA folding may provide a general explanation for the common occurrence of cold-sensitive mutations in RNA and RNA binding proteins . In the yeast
7. Mutations in macro or long non-coding RNAs
Macro or long coding RNAs are conserved and unlike the short RNA, always act in Cis position in the chromosomes and can be up to several hundred thousand nucleotides long, about 200-2800 nt. In the eukaryotic genome and, specially in mammals there are thousands of lncRNAs that are expressed in different cell lines and tissue and exhibit tissue-specific expression patterns. At moment there are a small amount of lncRNA in which are know in its function and stability, althought has been assumed that they are generally unstable. Reciently an genome-wide analysis in the mouse neuroblastoma cells, using a custom ncRNAs array has been determined that lncRNA show a similar range of half-lives to proteins-coding transcripts, suggesting that lncRNAs are not unstable and also that the stability of lncRNAs is a regulated process and depend of where are located in the genome these lncRNAs. Thus, the intergenic RNAs show more stability that those originated from introns of mRNA . Also it is know that in mammals these lncRNAs have different regulatory functions, principally X chromosome inactivation by heterochromatinization (Xist gene) and coats the inactive X chromosome from which it is transcribed. This represents part of the mechanism by which transcriptional silencing is achieved . The lncRNAs roX in flies plays a role in dosage compensation in sex determination similar to XIST gene in mammals . Also the lncRNAs are involves in the regulation of transcriptional and post transcriptional pathway programming, regulation of mRNA splicing, epigenetic gene activation in the regulation of Hox genes that regulate development and also in genomic imprinting and as enhacers of gene expression and in the length of telomere in the chromosomes [79,80,81,82,83,84,85,86,87,88,89,90].
In addition, several lncRNAs have been shown to be mis regulated in various diseases including cancer and neurological disorders [83,91]. One such alterations in an lncRNA, is Malat1 RNA (metastasis-associated lung adenocarcinoma transcript ). Malat1 also is highly abundant in neurons and It is enriched only when RNA polymerase II-dependent transcription is active. Knock-down studies revealed that Malat1 modulates the recruitment of SR family pre-mRNA-splicing factors to the transcription site of a transgene array. Malat1 controls the expression of genes involved not only in nuclear processes, but also in the function of the synapse. In cultured hippocampal neurons, knock-down of Malat1 decreases synaptic density, whereas its over-expression results in a cell-autonomous increase in synaptic density. These results suggest that Malat1 regulates synapse formation by modulating the expression of genes involved in synapse formation. . lncRNAs are present not only in animals but also in plants where they are involved in gene silencing and in the phenotypic plasticity . In mouse a lncRNAs that has been coined as Rubie (RNA upstream of BMP4 expressed in inner ear) originate malformation in the vestibular apparatus. The Mutation is expressed in developing semicircular canals. However, was discovered that the SWR/J allele of Rubie is disrupted by an intronic endogenous retrovirus that causes anormal splicing and premature polyadenylation of the transcript. Rubie lies in the conserved gene desert upstream of Bmp4, within a region previously shown to be important for inner ear expression of Bmp4 . Also in vertebrates and specifically in humans has been described mutations in transposables elements that are related to neurodegerative diseases. The mutation was located in a degenerated long interspersed elements (LINES). This mutation expressed in the brain and causes lethal infantil encephalopathy suggesting that these repetitive elements are important in human brain development .
8. The RNA editing
The epimutations at ncRNAs are very important for the adaptation of organism and could be also heritable. Traditionally has been considered that mutations are nucleotide changes that occur at the DNA level and also that are the only new source of genetic variation. However, an special epigenetic regulatory mechanism was discovered from the mitochondria of protozoa
9. The post-transcriptional nc RNAs epimutations and their role in the norm of reaction and phenotypic plasticity
Until recently it was thought that in eukaryotes the mutations important for the organism were located into the areas of DNA that code for proteins. Under this framework, protein were the only molecules that regulate the action of genes and, a mutation into the a structural gene could cause a change in the primary structure of proteins. A single amino acid change could cause a serious disease. With the advances in molecular genetics and the discovery of ncRNAs, now we know that In the ncRNAs also occurs epimutations that can also cause phenotypic changes and diseases. These epimutations are more difficult to interpret at a molecular level because they do not affect the protein sequence. Generally the epimutation in ncRNAs alter the RNA structural ensamble between ncRNAs and mRNAs and, alter the message of genetic information in the cells [101,102]. Similar to proteins, the epimutations produced in the ncRNAs into cells that belonging to differents organs and tissues within the body in eukaryotes can cause a great variety of illness.
The non-coding region of DNA previously thought was garbage, we now know it is not. An exception to this rule is the contribution of by the transposable elements described in maize by Barbara McClintock in 1947, dubbed as controlling elements. The merit of her discovery was the realization that the genome is not static and there are genes that are unstable in terms of location in the genome and could promote its own transposition. Now we know that these transposable elements are found in unicellular and multicellular organisms and have a viral origin . Also the discovery of transposable elements and horizontal transferences of genes had led to the understanding that the genome is a “fluid mosaic of genetic information” from different origins‚ where the horizontal transfer mediated by virus, tramposons and viruses play an important role in the genic flow between the organisms, not necessarily related genetically . Reciently, in prokaryotes and eukaryotes there are many evidences in that another class of molecular interaction occurs in the regulation of gene action and cellular processes, principally manifested by small ncRNAs that base pairs with mRNAs and regulate the gene expression postranscriptional [101,103]. NcRNAs are a very good tool for the inactivation of specific messages, for example some classes of these ncRNAs such as siRNAs and miRNAs have been found in the regulation of of development and cell death. The nc RNAs act also in prokaryotes, in the replication and maintenance of extrachromosomal elements they have an epistatic effect to any transcriptional signals for their specific mRNAs.Thus, a single ncRNA can regulate multiple genes and have profound effects on cell physiology.
The mutations not only occur in the structural genes but also in those areas that code for ncRNAs, in the mRNA messenger ( RNA editing) and also in the introns and in both ends of mRNA, specifically in the 3’UTR and 5’UTR regions where as well are located ncRNAs. Thus mRNA is not only an intermediary between DNA and protein, as is expressed in the classic Crick’s Central Dogme of Molecular Biology, but also correspond to a relevant producer of miRNAs and siRNAs. In addition the transcription of all eukaryotic genome generates a large amount of differents ncRNAs which together with proteins regulating the expression of genes. The experimental evidences show that ncRNAs do not occur randomly in all cells but there are an enrichment of a particular ncRNAs depending of their function and cell where they act. There is now evidences that the environmental and developmental influences have effects on the phenotype. The epigenetic changes at DNA and RNA level such as DNA methylation, acetylation of histones, epimutation and RNA editing have an importance in the Darwinian fitness and could be adaptative . Also many of these changes are inherited in a different way that the classic Mendelian model of heredity. One of the assumptions of population genetics is that genes are vertically transmitted to the progeny according to the laws of Mendelian inheritance. In this context, and based on Weissmann’s barriers between somatic and germinal cells, only genetic changes that take place within gametes are inherited by the next generation. However at present there are evidences about a non-Mendelian model of heredity which has a close proximity to a neo-Lamackian inheritance model.
This model is based on epigenetic changes induced by the environment, in the epimutations at ncRNAs level, in the mRNA editing and also in horizontal gene transfers. Thus epimutations could be heritable. In this type of heredity there must be no barriers that prevent the changes in somatic cells could be integrated into the genomic information that resides in the nucleus of germ cells. The transposable elements, viruses and ncRNAs can be vectors incorporating somatic mutations within the genome and epigenome of the germ cells. Thus could be evade the Weissman’s barriers between somatic and germ cells through retrovirus . Also a mutation in piRNAs which block the action of a virus or transposable element of somatic origin could facilitate the negative impact of mobile elements in germ cells and this change may be inherited.
In humans has been postulated that cardiovascular and metabolic function and that elements of the heritable or familial component of susceptibility to cardiovascular disease, obesity and other non-communicable diseases (NCD) can be transmitted across generations by non-genomic means. Placenta’s inaccurate nutritional cues,increases the risk of NCD. Endocrine or nutritional interventions during early postnatal life can reverse epigenetic and phenotypic changes induced, for example, by unbalanced maternal diet during pregnancy. Elucidation of epigenetic processes may permit perinatal identification of individuals most at risk of later NCD and enable early intervention strategies to reduce such risk .
Unlike prokaryontes,the eukaryote genome expresses numerous types of ncRNAs that play a fundamental role in the regulation and gene expression. Those small molecules have the possibility of interact with differents kinds of proteins generating a homeostatic system that can respond quickly to environmental changes. Both class of molecules, protein and ncRNAs, are the manifestation of a great amount of information accumulated within the genetic and epigenetic programs. The epigenetic plasticity protects individuals from environmental changes and explain the classic concepts of reaction norm and phenotypic plasticity that previously had been poorly explained on its genetic basis. But now we know that if there is an epigenetic control for these phenotypic changes. Also, these ncRNAs contribute to the processing of information in at least two form: a) Saving a lot of information on their small molecules with a minimal of energy cost.b) Rapid acquisition of information from environmental with a rapid response and adaptation. Further ncRNAs appear to facilitates the acceleration of the evolution of an organism’s information contained and functional computanional system. This new picture provides a new dimensions about information processing in the brain  and in other cells belonging to other tissues where the ncRNAs can mitigate the negative effects of the environment, increasing adaptability and acceleration in the organic evolution.
Financed by the project code B-12-1, Direction of Extension of the Metropolitan University of Educational Sciences, Santiago, Chile