Over the last three decades, those long-held beliefs have been shattered. For this, they earned the Nobel Prize in ! What is RNA?
Deoxyribonucleic acid DNA is a molecule you may already be familiar with; it contains our genetic code, the blueprint of life. The order of these bases determines the genetic blueprint, similar to the way the order of letters in the alphabet are used to form words.
However, RNA has been known since the late s, and research on its function has been recognized with some 30 Nobel Prizes over the years. The central dogma of biology, formulated in the 20th century after the discovery of DNA, postulates that genes provide instructions for the cell to build proteins, or functional molecules needed to perform the different jobs in the cell, and that RNA serves as an intermediate messenger to transmit the flow of genetic information from DNA to its encoded protein products.
This process involves two main steps: transcription and translation. While this still holds true, we now know that RNA is much more than just a messenger, and scientists have started to appreciate the complexity and versatility of functions of the different RNA populations.
The vast majority of the mammalian genome is transcribed to RNA, yet less than three percent of it encodes for proteins. Scientists have found that the majority of the RNA transcripts play regulatory roles in gene expression and genome architecture, controlling when and how the genetic information is expressed and adding a whole new level of complexity to our understanding of life. Newly synthesized proteins on the other hand are imported into the nucleus and rapidly disperse throughout its volume, implying effective intranuclear trafficking.
Furthermore, proteins rapidly relocalize within the nucleus upon experimental or physiological changes in conditions. For example, inhibition of protein synthesis leads to rapid and dramatic relocalization of a large number of nucleolar proteins Andersen et al. How proteins and RNAs move within the nucleus and how they find their targets, however, was unknown for long. It was not clear whether movements are energy dependent, whether they occur by directed transport or what the speed of intranuclear trafficking is.
These fundamental questions have recently been answered Misteli The investigation of intranuclear protein and RNA dynamics was made possible by the development of techniques to visualize and measure the motion of these molecules by time-lapse microscopy. Tracking of proteins by photobleaching methods revealed a surprising degree and speed of intranuclear trafficking. As expected for diffusion, this rapid motion is energy-independent and non-directional.
RNA motion within the nucleus is similarly rapid and non-directional. Several methodological approaches involving fluorescently labeled, microinjected, engineered or endogenous RNAs demonstrate that ribosomal RNAs as well as polyA-RNAs move freely in a non-directional manner with a diffusion coefficient of 0.
A diffusion coefficient of this magnitude is sufficient to ensure transport of an RNA particle from deep within the nucleus to the cytoplasm within a few minutes, consistent with biochemical observations on kinetics of RNA maturation and transport. Thus energy-independent, diffusion-based movement of RNA particles alone can account for the observed kinetics of RNA export, and no active mechanisms are required to ensure rapid export. An impressive demonstration of the non-directional motion of mRNA comes from studies in which a nascent RNA is visualized at its site of transcription and its export to the cytoplasm measured in living cells Ritland-Politz et al.
These studies show that RNAs synthesized from genes positioned in proximity to the nuclear envelope diffuse away from their site of synthesis in all directions rather than follow a direct path to the nearest nuclear pore. This observation powerfully demonstrates the non-directed, diffusion-based motion of RNAs in the nucleus. One of the conceptually most challenging problems in cell biology is the question of how molecules find their specific targets within a cell or within an organelle.
This problem is particularly complex in the cell nucleus where transcriptional regulators need to find their specific target genes amongst the myriad of potential binding sites within the genome. Somewhat counter-intuitively, the non-directional, but rapid, motion of proteins within the cell nucleus provides a means to ensure targeting of proteins to specific genome locations Misteli The power of targeting by random diffusion within the nucleus is best illustrated when considering how a transcription factor finds its target genes but it also applies to targeting of proteins to nuclear compartments or any other nuclear site.
Proteins a priori do not know where their targets are and we do know of any directed transport systems, such as a molecular motor-based mechanism, that would bring a factor to a specific gene or a specific location within the nucleus. Thus, the only way for a transcription factor to find its target is to scan the genome. The intrinsic ability of proteins to rapidly move within the nucleus by diffusion-based mechanisms permits such genome scanning Misteli This occurs by a transcription factor freely diffusing within the nucleoplasm until it interacts by chance with chromatin.
The molecule will now probe whether the sequence it has encountered is a specific binding site such as in the promoter of one of its target genes. If it is, the transcription factor will be captured and stably associated with its specific target site. If the sequence is not a binding site, the molecule, after a short interaction, will dissociate from chromatin and continue its diffusional journey through the nucleus. It might appear at first glance that such a stop-and-go model for genome scanning would be insufficiently effective.
However, the observed dynamic properties of transcription factors are entirely consistent with this model. We know that the residence time of most transcription factors even on specific DNA binding sites is in the order of a few seconds and that their interaction with non-specific sites is even faster, most likely in the order of tens of milliseconds Gorski et al.
Assuming these time scales for binding, one can calculate that it takes a single transcription factor molecule only a few minutes to search the entire genome space. Considering that most transcription factors exist in several thousand copies and have multiple target genes, their random diffusional motion is entirely sufficient to ensure a steady supply at their target genes. This assumption is further supported by direct measurement of transcription factor flux on an endogenous rRNA promoter demonstrating the collision of several hundred molecules per second Dundr et al.
Since proteins similarly move by diffusional motion within the cytoplasm, it stands to reason that the random scanning is also a key mechanism of protein targeting in the cytoplasm and represents a universal mechanism for how proteins find their targets. A key feature, and a requirement, in a genome-scanning model of targeting is that the interactions of proteins with chromatin are transient.
This has been confirmed by photobleaching methods on a large number of DNA binding proteins Gorski et al. The transient nature of protein—chromatin interactions is important for three reasons. First, it allows proteins to maintain a high rate of motion and thus allows faster scanning. Were protein—chromatin interactions static, they would get stuck at non-specific or incorrect binding sites which would slow down their overall motion.
Second, the short-life of protein—chromatin interactions continuously makes available binding sites which can then be scanned by diffusing transcription factors. If proteins interacted for extended periods of time on chromatin, non-specific or improper binding would block access of the correct factors.
Third, the dynamic dissociation allows for change. Were protein complexes permanently bound to their target sites, changes in transcriptional activity such as in response to physiological stimuli could only occur after active removal of the bound complex, presumably by dedicated and specialized molecular machinery. In contrast, in a dynamic binding model the natural flux of proteins provides a window of opportunity for association of a distinct regulator each time a bound protein or complex dissociates as part of its normal binding cycle.
A hallmark of the mammalian cell nucleus is the presence of distinct subnuclear compartments and domains in which particular functions occur Handwerger and Gall ; Hernandez-Verdun Fig. The prototypical nuclear compartment is the nucleolus, a distinct intranuclear compartment in which ribosomal RNAs are synthesized and partially processed.
Other prominent nuclear domains include splicing factor compartments which serve as storage and assembly sites for spliceosomal components, and the Cajal bodies which are possibly involved in maturation of small nuclear RNPs. The structure of intranuclear bodies is not determined by a membrane, and the principles underlying their biogenesis are extremely poorly understood.
The recently revealed dynamic properties of proteins in nuclear compartments give a hint as to the principles involved in subnuclear compartment assembly. Intranuclear compartments. The mammalian cell nucleus contains a larger number of distinct intranuclear compartments. The nucleolus is the site of ribosomal RNA synthesis and is a prototypical nuclear body. The complex organization of the nucleolus is revealed by multi-color staining of distinct nucleolar components.
The key property in understanding the formation of nuclear compartment is the remarkable fact that the association of proteins with their compartments is highly dynamic Dundr et al.
0コメント