In biology, the complexity of systems seems to be a constant. We now well know that DNA contains all the information we need in the form of genes and that they are finely activated in different cells and conditions. We also know that the passage of information from genes reside in messenger RNA (mRNA), transcribed from DNA, which is the only coding molecule that is transported outside the nucleus to be translated into proteins. However, there is a multitude of DNA that does not associate with any genes and therefore any proteins, considered as “junk DNA”. Such a definition led sceptic researchers to investigate further and finally, a key role in biology for the non-coding RNAs was discovered.
There are two main classes of non-coding RNAs; microRNAs and endogenous small interfering RNAs (endo-siRNAs). Contrary to mRNA, both are translated from non-coding regions in the DNA (Figure1). However, some of them have been found inside coding regions too. The peculiar feature of non-coding RNAs is that they form a double-stranded molecule of RNA by partial matching of two complimentary sequences connected with a hairpin loop at one end (microRNAs), or complete matching with no hairpin (endo-siRNAs).
Several molecules and mechanisms are involved in the biogenesis of these small RNAs before translocation outside the nucleus. Once in the cytosol, an RNAse III enzyme called Dicer binds and cleaves both microRNAs and endo-siRNAs into their short (just 18-25 nucleotides), mature form that lacks a hairpin loop. The small RNAs are now biologically active in the cell. They associate with the riboprotein RISC forming a special ribonucleoprotein complex that goes searching for complementary mRNA sequences, associates with them (in the 3’ UTR region) and interferes with the generation of the protein it codes for. By repressing the formation of new proteins (microRNAs) or sending the mRNA to be degraded (endo-siRNAs) these small molecules can modulate the destiny of stem cells to a level that we are just beginning to understand.
The non-coding RNAs, via a post-transcriptional regulation, can do a lot for events that require dramatic and fast cell fate transition, like those occurring during early embryonic development. When a zygote is formed by fusion of the two gametes, the genome is kept transcriptionally silent, no information from the genes are passed to coding mRNAs. Therefore, it’s not surprising that regulation of parental mRNA by non-coding RNAs will drive the very first phases of development
When researchers mutate both the maternal and zygotic Dicer in mice (the genes required for the production of microRNAs and endo-siRNAs), no major developmental phenotypes have been seen until the blastocyst stage. This is due to the low levels of microRNAs present in the early phases of pre-implantation development. Over time, levels of microRNAs increase and become more important in the blastocyst. Conversely, levels of endo-siRNAs decrease in the same short developmental window. Although it seems that microRNAs are not important for pre-implantation development, endo-siRNAs could be the effectors of a reprogramming that gradually ‘switches on’ the genome and the microRNAs. Although the loss of microRNA and endo-siRNA is not well understood, the hypothesis is that different “silencing machinery” operates for these non-coding RNAs, allowing endo-siRNA to work in conditions where microRNAs cannot, and vice-versa later on in development.
Why noncoding-RNAs are so important for development?
In cells derived from a mammalian blastocyst, where microRNAs are abundant, the suppression of Dicer is enough to cause a developmental defect. Embryonic stem cells (ESc) are derived from the inner cell mass of the blastocyst and are defined by two key features: the potential to differentiate into all the embryonic tissues, pluripotency, and the ability to regenerate their own population, self –renewal. However, in vivo, both pluripotency and self-renewal have to be silenced to allow embryonic development to occur by transformation of the pluripotent pool of cells into the specialised multipotent and finally, unipotent cells of the body. A lack of Dicer stops ESc following their developmental destiny by holding them in a self-renewing state. Blocking the production of non-coding RNAs leads to nothing less that a complete stop of the developmental program. This phenotype can be used to study their specific role.
Screening of a multitude of microRNAs for their ability to induce differentiation identified Let7 as a microRNA that induced silencing of self-renewal and pluripotency, thus pushing development in Dicer knockout cells. Interestingly, its effect is repressed in wild-type ESc by a family of microRNAs (ESC cell cycle regulating miRNAs or ESSC miRNAs) that have opposite roles. MicroRNAs often work in loops with each other, repressing the mRNA of an antagonist microRNA. This is the case of Let7 and lin28: In ESc the ESSC miRNAs indirectly leads to a predominant activation of lin28 which directly maintains low let7 levels. During differentiation lin28 is switch off and let7 levels increase, silencing, amongst other targets, lin28 (Figure 2).
One of the powerful features of microRNAs is that they can regulate multiple targets at the same time, therefore shifting the cell fate quickly and effectively. This is a very useful ability in development. The post-transcriptional regulation of mRNA is a fundamental process in early development that has to be finely regulated.
MicroRNA research has seen an explosion in recent years. This is due to the potential that small RNAs can offer when attempting to manipulate and differentiate ESc in vitro. This is particularly true for regenerative medicine based on the differentiation of ESc to specific cell types. Expanding pluripotent ESc in vitro is challenging, but has been achieved by systematic control of extrinsic factors and culture conditions that recapitulate the natural environment or “niche” of the cells in the blastocyst. However, to drive these cells to generate what we want from them is another chapter. Here, without permanently modifying the genome, a mixture of factors must be powerful enough to lead the pluripotent cells to a certain differentiation pathway. Supplementing ESc with growth factors or inhibitors and using specific culture media helps, but the possibility of transiently modifying the post-transcriptional profile of the cells is appealing experimentally. It is now documented that microRNAs play a role in defining the adult cell types together with proteins and gene expression. Adding selective microRNAs that have been found to be important in some cell types better supports the differentiation of ESc towards those lineages. For example, mir-1 has been found to play a key role in the development of cardiac progenitor cells. Now, it has been used to improve the differentiation of ESc to cardiac myocytes and test the regeneration ability of transplanted cells in an infarcted mouse heart.
Better understanding of the endogenous function of non-coding RNAs is critical if we aim to translate the knowledge to possible new therapeutic protocols. The unveiled world of non-coding RNAs is going to take up a big space in the developmental biologists agenda and, it is time to say, in many leading field of bio-medicine too.
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