Posted comment on ´The function of RNA-binding proteins at the synapse: implications for neurodegeneration` by C.F. Sephton and G.Yu and published in Cell. Mol. Life Sci. 2015 vol 72 page 3621 doi 10.1007/s00018-015-1943-x
Sephton and Yu began their article by describing the important role of protein translation taking place locally in the synapse. This type of translation was explained as necessary to accommodate the changes in this area of the neuron brought about by the constantly changing micro-environment due to neuronal activation. In addition, localisation of mRNA in the synapse and protein synthesis has been shown to be critical for synaptic plasticity and memory formation. Control of nuclear translation can depend on different mechanisms such as upstream open reading frames, secondary structures or regulatory protein binding sites, but in their article, Sephton and Yu concentrate on the specificity of the translation of the mRNA by the RNA binding proteins present. This is the main group of proteins regulating mRNA transport and translation in this area and attention to this group has been brought about because of the assumed link between known genetic mutations (or gene deletions) of particular examples and certain neurodegenerative diseases. For example, sufferers of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) exhibit cognitive impairments and loss of motor neuron function and research has showed that there are strong associations between several RNA binding proteins eg. Fused in Sarcoma binding protein (FUS) and Transactive Response DNA binding protein (TDP-43) in both diseases resulting in dysregulation of synaptic function and initiation of abnormal neurodegeneration.
Local translation in neuronal cells is said to occur because of the distances between the soma (the cell body) and the dendrites which creates, what the authors termed, a ´supply and demand challenge` for proteins required in the dendrites for them to function, for example responding to neuronal firing and signal transmission. Therefore, local translation means that all requirements for the neurochemical process have to be located within the dendrites in addition to those in the vicinity of the cell nucleus. The components required include mRNA, ribosomes and translation factors. In the synapse, the mRNA is found to exist within granules. It is associated with the RNA binding proteins in the untranslated regions (5UTR or 3UTR) or coding regions to form messenger ribonucleoprotein complexes (mRNP) which can then assemble into more complex structures known as RNA (or RNP) granules. There are many different types and classification is based on composition, location, response to stimuli and function, eg. regulation of, distribution of, translation of and degradation of mRNA transcripts. The granules are constantly changing, forming different interactions and exchanging mRNPs, cytosolic proteins and polysomes, but research has shown that their presence is responsible for synaptic plasticity and communication in the functioning synapse.
Sephton and Yu continued their article with descriptions of particular examples of multi-mRNP complexes such as transport RNPs (tRNPs), stress granules or processing bodies. The transport tRNP granules contain mRNA and are said to be involved in the storage and transport of mRNA. They can also contain microRNA (miRNA). RNA binding proteins found associated with the tRNP type of granule are, for example Staufen 1 and 2, FUS and TDP-43. However, they are also said to contain at least 40 other proteins including motor transport proteins such as kinesin. The transport of mRNA to the dendrite appears to occur with the mRNA in a translationally dormant state, but this can undergo alteration in certain circumstances. The authors describe the example of stress where mRNPs are exchanged within the tRNP with stress granules and processing bodies. The mRNAs are protected and once stress removed then the stress granules disassemble and mRNAs are repacked into translationally competent forms and their proteins are synthesised. Alternatively, they are selectively exported to another type of granule, the processing bodies which are responsible for degradation, translational repression and recycling.
The role of gene mutations of RNA binding proteins and the resulting neuropathological diseases were described by Sephton and Yu with reference to two mutations in particular, that of FUS mutations and TDP-43 mutations. The mutations of the specific RNA binding proteins in both cases resulted in negative changes in the cell`s mRNA population and protein translation causing adverse effects on synaptic structure and function and neurodegeneration. In both neuropathological diseases, no changes of the processing bodies were observed, but there were changes in tRNP and stress granule populations. FUS-mutations were found to cause an increase in number and size of the tRNP and stress granules. This was suggested as for example, resulting in more spontaneous assembly of tRNP granules that could have an impact on translation, or that it could make both tRNP and stress granules more insoluble. As a result pathological inclusions would be ´seeded` and this is reported as being the case in both ALS and FTD. Regarding TDP-43 disease mutations, larger, but fewer tRNP were observed and larger, but greater numbers of stress granules. Both were assumed to cause the same physiological effects as that seen with the FUS mutations.
In their article, Sephton and Yu then went on to describe how RNA binding proteins regulate local translation. RNA binding proteins are said to be a component of RNA granules and these transport mRNA to the dendrites in a translationally dormant state. This is supported by the presence of nucleic translation initiation factor 4AIII (eIF4AIII) with dendritic mRNA. The mechanism by which translation is inhibited by the RNA granules was said not to be known since it has been shown that some granules contain translation components such as ribosomes and endoplasmic reticulum (ER). For example tRNP granules with the largest number of Staufen 1 and 2 pools contain ribosomes and ER and those containing the smaller number had kinesin, but no ribosomes and ER. It was assumed that this was the group of tRNP where translation was repressed. In this case, neural activity could therefore cause either release of mRNA from the tRNP granule to the polyribosomes where active translation would occur, or in some circumstances repression of translation would continue. The protein composition of the granule particularly the presence of RNA binding proteins appeared to dictate which path would be followed. For example, ZBP1 associates with and transports beta-actin mRNA to synapses. Src phosphorylation causes dissociation between ZBP1 and mRNA, hence synthesis of beta-actin, which is required for both cell migration and neurite growth, is allowed. However, ZBP1 can also repress the joining of ribosomal subunits in the cytoplasm so translational initiation regulation can occur. Other examples described by the authors are the CPEB family of RNA-binding proteins where CPEB1 can act as both repressor and activator of translation and the binding protein, FMRP. FMRP mutations are associated with Fragile X syndrome and studies have suggested that FMRP plays a role in the linking between tRNP granules and polyribosomes. The phosphorylated form of FMRP is associated with stalled ribosomal translocation and the non-phosphorylated form is associated with actively translating ones. FMRP can also regulate transmission when in its phosphorylated form by an association with miRNAs eg. miR-125a and RNA-induced silencing complex (RISC) to repress synthesis of proteins such as PSD-95. Dephosphorylated FRMP can lead to the release of RISC from PSD-95 mRNA so translation can take place.
This link between RNA binding protein deficiency and neurodegenerative diseases was continued with Sephton and Yu looking at how the deficiencies are associated with changes in dendritic synaptic degradation and dendrite morphology, for example as seen in ALS and FTD sufferers. For example Staufen-1 knockout mice are observed to have reduced numbers of dendrites and synapses in the hippocampus and have a defective dendritic delivery system of beta-actin tRNP granules. Sephton and Yu in their article looked at the cases of deficiencies of the FUS and TDP-43 binding proteins in particular.
FUS is a RNA binding protein responsible for the translation of proteins required for neuronal development and synaptic transmission. It is known to bind to thousands of cellular RNAs via two RNA recognition motifs (a zinc-finger domain and 3 arginine-glycine-glycine boxes). As it appears to exist in different ribonucleoprotein complexes it is thought to be involved in all types of translation processes eg. mRNA stability and miRNA biosynthesis. When the neuronal cell is in a steady state, the majority of FUS is localised in the nucleus of the cell where it exists bound with TDP-43 and SMN along the whole length of the nascent RNA. All three binding proteins in this case function in the maintenance of the spliceosome. It is also thought that FUS is involved in transcriptional elongation as it is closely associated with RNA polymerase II, binds to pre-RNA and mRNA at introns and coding sequences etc. Therefore, FUS mutated cells and FUS-null cells demonstrate dysregulation in mRNA and pre-mRNA splicing.
However, FUS can also be localised to cellular compartments other than the nucleus. It has been found in RNA granules and this localisation is facilitated by the non-classical proline-tyrosine nuclear localisation sequence (PY-NLS) and the nuclear export sequence (NES). The localisation results as a response to various stimuli and Sephton and Yu give in their article the example of hippocampal or cortical slices that had been stimulated by treatment with mGluR1/5 agonists. In the response to the agonist presence and hence, neuronal firing, FUS was found in the tRNP granules in the dendrites and also at the synapses where FUS was associated with NMDA receptors themselves. The role of FUS in the tRNP localised in the dendrites was described as being either repression or facilitation of the required translation and there appears to be examples of both. FUS can also associate with cytoplasmic stress granules, but in this case, this only leads to translation repression.
As expected with a promotion or repression role in local translation, FUS is linked with neurodegeneration under some circumstances. The majority of familial ALS mutations are associated with the FUS encoding gene at its PY-NLS terminal which affects the localisation of the protein and its aggregation. For example, abnormal accumulation of FUS in the cytoplasm or nucleus of motor neurons in ALS patients is observed. The extent of the mutations appears to correlate to disease severity. Some mutations eg. those at the 3 prime end, can cause increased FUS expression. Mutations at the RGG1 and N-terminal region can also occur with FUS although this type of mutation is normally linked to FTD. In this case, the localisation of FUS is not as prominent as normally observed and abnormal aggregation can occur in some cases. Animal models expressing the various FUS mutations and their effects on neurodegeneration are available to support the research, eg. ALS-FUS mutations in D.melanogaster and C.elegans and examples of transgenic mice (eg. ALS-FUS R521G) which demonstrate motor deficits and motor neuron abnormalities. However, results from the animal models appear not to always follow expectations, eg. FUS mutations in transgenic mice appear to be regulated differently to wild type mice in response to neuronal stimulation, or could have altered dendrites. This is also supported by neuropathological studies which have shown as an example that pathological FUS aggregation does not occur in any animal model.
Animal models, however, are said by the authors to be of more value in studies on the reactions of stress granules to FUS mutations. ALS-FUS PY-NLS mutations show a correlation between disease severity and localisation in the cytoplasm and in the presence of these mutations larger granules are formed independent of the presence of stress. It has also been observed that FUS mutations may delay the assembly of stress granules and irreversibly sequester a variety of RNA binding proteins and mRNAs. No effect on the processing bodies or their associations has been observed. Therefore, suggestions have been made that the ability to bind mRNAs and sequester them into RNA granules may be one factor in disease neuropathology. Researchers have suggested that FUS mutations ´seed` protein aggregates which sequester more FUS and other proteins, hence depleting the cell of essential proteins and leading to cell death. This view however, requires according to the authors further research support.
The other example of RNA binding protein described in detail by Sephton and Yu in their article is TDP-43 which like FUS regulates gene expression. This binding protein can bind to over 4500 species of RNAs via 2 highly conserved motif regions: the RRM1 which is a major domain for binding of RNA and DNA and the region, RRM2. Like FUS, TDP-43 regulates transcription and multiple RNA processing mechanisms and hence, deletion leads to dysregulation of mRNA and pre-mRNA splicing. TDP-43 is expressed and located primarily in the nucleus, but it too can localise to different cellular compartments and RNA granules via classical NLS and NES binding. Various stimuli cause localisation to these other areas. For example, under basal conditions TDP-43 can be found in hippocampal dendrites and is co-located with RNA granules such as processing bodies. It can also co-localise with the post synaptic protein, PSD-95. The RNA granules containing TDP-43 contain RNAs including mRNA for beta-actin and CaMK11alpha, an important enzyme in neuronal cell plasticity. However, on depolarisation then RNA granules with TDP-43 co-localise FMRP and Staufen-1 in tRNP granules in the dendrites. In oxidative stress, TDP-43 localises in the cytoplasm and into stress granules. Again, no association with processing bodies has been reported even though reports of FMRP, Staufen-1 have been reported in these bodies in some circumstances. There are also reports of TDP-43 being an integral component of different complexes eg. Dicer, Drosha and it is believed to be involved in miRNA biogenesis responsible for neuronal outgrowth. Therefore, it is thought to act like FMRP in that its presence leads to translational repression via miRNA regulation.
Just like FUS mutations, TDP-43 mutations are also associated with neurodegeneration and neuropathology of some diseases. There are more than 40 TDP-43 familial ALS mutations documented (mostly with mutations at the C terminal) and TDP-43 mutations have been observed in approx. 50% of FTD patients. Mutations do not normally lie in the NLS or NES regions, but instead mainly lie in the 3 prime end of the TDP-43 mRNA and these lead to increased levels of TDP-43. No impact on localisation occurs, but because the mutations lie in the glycine rich C terminal region then protein interactions are adversely affected. Animal models where the endogenous levels of wild type TDP-43 or expressed ALS-TDP-43 mutations exhibit drastic neuronal effects, aspects of ALS and FTD diseases are produced. For example, the depletion of TDP-43 in D. Melanogaster causes a decreased life span and locomotor defects due to alterations in dendritic branching and synapses, whereas over- expression can cause a loss of motor function and a reduction in the number of dendrites and synapses. Transgenic TDP-43 mice expressing either wild-type or ALS associated mutations can also exhibit motor defects, which can be reversed if expression is inhibited. Mutations appear to be associated with abnormal neurites and decreased cell viability where depletion of TDP-43 leads to an increase in number of mature spines in hippocampal neurons, an increase in clustering of AMPA R at the dendritic membrane and an increase in neuronal firing. A link to Rac1 was established suggesting that TDP-43 could be an upstream suppressor of this spinogenesis regulator.
TDP-43 and ALS-TDP-43 mutations are shown to be actively recruited in response to stress to the other type of RNA granule that of the stress granule found in the cytoplasm. Both the C terminal glycine rich domain and the N terminal RRM1 region appear to be important for this association and therefore, both RNA binding and protein-protein interactions are required. Large stress granules are formed and like FUS, pathological aggregates of these stress granules occur suggesting a role in seeding for the TDP-43. TDP-43 mutations can also affect tRNP granule formation and migration and TDP-43 has been shown to be associated with RNA granules throughout the dendrites. Aggregation of TDP-43 is assumed to be important for the formation of neuronal tRNP granules and mutations found in ALS-TDP-43 models are shown to increase the size of the tRNP granules in rat hippocampal dendrites under basal conditions. Depolarisation with potassium chloride stimulates TDP43 granule migration into the dendrites with mutated forms demonstrating reduced density as well as slower speed of movement and shorter distances covered than the wild-type. Therefore, TDP-43 mutations can produce effects by eliciting the absence of important RNAs at sites of local translation or by producing dysregulated mRNA products.
Sephton and Yu concluded their article by looking at future perspectives regarding local translation, RNA-binding proteins and neurodegeneration. They state that the topic is gaining interest because of several reasons. There has been an increase in the identification of genetic mutations in genes encoding proteins involved in RNA regulation and associating these to the neuropathology of certain diseases. There also appears to be anomalies between in vivo disease models and in vitro ones and lastly that synaptic dysfunction precedes neurodegeneration and therefore, tRNP granule formation, localisation and protein translation dynamics, affected by RNA binding protein mutations could be the trigger to the neurodegeneration process. In order to understand how this occurs, the authors investigated how RNA granules and RNA binding proteins are involved in the maintenance of the synapse and gave explanations to their function. They suggested that there is evidence that RNA binding proteins are important in transporting mRNA into granules which remain dormant until the mRNA transcripts are required to be locally translated on polyribosomes. The authors also hypothesised that the RNA binding proteins are also important for the presence of dendritic branches and spines since genetic deletions cause alterations in both. Therefore, Sephton and Yu concluded that the role of RNA binding proteins and granules should be researched more intently especially linked to the topic of local translation and downstream consequences at the synapse. This role was said to be especially important in light of evidence that linked RNA binding proteins and RNA granules with neurodegeneration and the pathology of particular neurological diseases such as ALS and FTD. However, the authors did admit that this area of localised mRNA translation would be difficult to study because RNA binding proteins like FUS and TDP-43 are associated with the regulation of thousands of cellular RNAs. Focussing on the area of neuronal stimulation and synaptic responses would they suggest make study of the topic slightly easier.
What makes this article interesting is that is looks at another site of protein synthesis in the neuron other than the common sites close to the cell nucleus. Researchers have proposed that local translation in neurons can take place and the authors of the article commented on in this Blog post describe the local site as being in the neuronal dendrites. The authors also link in their article the idea of genetic mutations of some of this process`s components to certain neurodegenerative and neuropathological diseases. We will concentrate in the comment here on the process of local translation in the dendrites and we begin with a short summary of the local translation process. This then leads on to questioning whether or not RNA granules indicative of this local translation mechanism are ´true components` and not an artefact of the experimental procedure used in cell component separation and if they are ´true components` whether or not the neuronal cell actually needs local translation.
In order to ascertain whether or not RNA granules are ´true components` or not, we should begin with a brief simplified summary of protein synthesis and how local translation fits in with the general process in neuronal cells. The general process of protein synthesis in neuronal cells begins with the nuclear DNA which contains the information required for the protein`s structure and final location. DNA is transcripted into mRNA and we assume that this transcription process is the same whether the protein will be finally synthesised close to the nucleus or will be localised elsewhere. The initial transcripts (the pre-mRNA) still within the nucleus contains introns (codes not translated into proteins) and exons (codes giving rise to the protein) and the introns, which can be regarded as ´guiding/regulating` signals can be spliced out. (Continued inclusion of these at this point could give rise to different end-of-process proteins.) The transcript of the message occurs in the mRNA form and this passes from the nuclear DNA site, through the nuclear membrane to sites where translation of that message into the designated proteins themselves can occur. These sites of translation are either close to the nucleus, or as suggested for neurons at sites well away from the nucleus such as the axons and dendrites. The site of protein synthesis if close to the nucleus is the rough endoplasmic reticulum (ER) which consists of stacks of membranes with ribosomes, the protein synthesis machinery, attached. The mRNA transcripts carrying the protein-building information binds to the ER and is translated in a 5 prime end to 3 prime end direction. Translation means that the ribosomes bring the necessary amino acids of the final protein in the form of tRNA together to allow biochemical binding to occur to make the designated protein. Alternatively proteins, normally those dictated to finally reside in the cytosol, can be synthesised by free ribosomes which attach to the mRNA ´thread` itself to form complexes called polyribosomes. The proteins are synthesised in the same manner as those formed from ribosomes attached to the ER. Folding of the newly formed proteins occurs in the smooth ER followed by post-translational processing which can occur in the Golgi Apparatus (GA) which also is located close by. This is the site where proteins are sorted and processed accordingly to their ultimate destination.
Other researchers and Sephton and Yu in their article described one stage of the protein synthesis mechanism that of translation which was said could occur in the case of the neuron away from the nuclear site and nearer to where the proteins would actually be needed eg. the neuronal dendrites (or axons). This is termed local translation. In local translation, mRNA transcripts of the DNA material are formed just like with nuclear translation and are transported outside the nucleus into the cytoplasm. Although it is not known how long they exist as free threads or where, the next time they are observed is in the synaptic areas of the axons and dendrites where they appear associated with RNA binding proteins in the untranslated regions (5UTR or 3UTR) or coding regions to form messenger ribonucleoprotein complexes (mRNP). These can then assemble into more complex structures known as RNA (or RNP) granules. The complexity of the RNA granules and the range of locations, response to stimuli and function appear to be due to the RNA binding proteins and other components that make up these granules.
Sephton and Yu described in their article particular examples of RNA granules eg. transport RNPs (tRNPs), stress granules and processing bodies. Each has a specific function, but the granules are constantly changing, forming different interactions and exchanging mRNPs, cytosolic proteins and polyribosomes. In each case the transcript exists primarily in the mRNA form and not in the pre-mRNA form and therefore, the presence of the polyadenyl translational signal would be absent having been spliced from the DNA transcript before leaving the nucleus. Instead, it is possible that RNA binding proteins take over this role and this will be commented on later. This is not uncommon and can been seen in other systems, eg. prokaryotes have promotor regions for every gene. It is also likely that the RNA transcript is in this stage of protein synthesis rather than for example the later elongation or termination stages since to have this level of control at these later stages would be a waste of resources and time. It is also unlikely that the granules are some manifestation of clumped microRNA (miRNA), which is formed from sections of mRNA folding back on itself and being spliced out. This is ruled out since the RNA content has been sequenced and found not to be complimentary not just to the 3 end of DNA as in the case of microRNA, but also that a small amount of microRNA is actually observed in the granules themselves. Other supporting evidence for the mRNA form in the granule and not the miRNA is that the latter can cause DNA modification, eg. histone modification, DNA methylation which RNA granules have not been reported to do. The mRNA in the granule dictates the proteins to be translated but as said before they are associated with other constituents into the granule form. What can definitively be said is that the functions of the granules are related to their molecular constituents eg. the RNA binding proteins and their presence is responsible for synaptic plasticity and communication in the functioning synapse. This explains why the contents of the granules are constantly changing and the content dictates the different outcomes eg. neurogenesis in hippocampus or memory consolidation in Aplysia.
So, now we have our mRNA transcripts in an area away from the nucleus and these are associated in granules whose function is dependent on their biochemical constituents. Probably from a neuronal firing point of view the most common form of granule is the most important, that of the transport tRNP granule type, which is said to be involved in the storage and transport of the mRNA. This type of granule can contain mRNA and also can contain microRNA (miRNA). RNA binding proteins found associated with the tRNP type of granule are for example, Staufen 1 and 2, FUS and TDP-43. However, the granules are also said to contain at least 40 other proteins and can be divided into two groups: those containing a low pool of Staufen 1 and 2, but contain the motor transport proteins such as kinesin; and those containing a larger pool of Staufen 1 and 2 and can contain ribosomes and ER. The transport of mRNA to the dendrite appears to occur with the mRNA in a translationally dormant state. In response to neuronal stimulation for example, the mRNA is released from the tRNP and forms polyribosomes so that translation can occur. This leads to local production of the necessary proteins in the synaptic area. It is thought that the mRNA that is translated is associated with the tRNP granules with the larger pools of Staufen 1 and 2 and the translationally dormant form is linked to the kinesin containing ones. It is also believed that the binding protein FMRP may provide a link between the tRNP and polyribosomes and its phosphorylative state may dictate whether translocation occurs or not, eg. the non-phosphorylated form of FMRP is linked to active translocation, the phosphorylated form with stalled. Following translation, the mRNPs can assemble back into the tRNP state, can be degraded or assembled into another form of RNA granule, the processing bodies. These are responsible for mRNA degradation, translational repression, and recycling or decay and hence, contain many proteins involved in these processes. Alternatively, neural stimulation can lead to repressed translation. Again the nature of the granule components eg. the RNA binding proteins determine the action ie. if protein translation occurs or whether is is repressed.Under stress conditions, another form of RNA granule is formed, that of stress granules. In this case, mRNPs are exchanged within the tRNP with stress granules. The mRNA transcripts are therefore protected and once the stress is removed then the stress granules disassemble and mRNAs are repacked into translationally competent forms and their proteins are synthesised. Or, alternatively, they are selectively exported to the associated processing bodies and the mRNA is degraded and hence, translation is repressed.
Although researchers are certain that RNA granules exist, the changeable physical nature of the system must lead to the question as to whether RNA granules actually exist in the natural neuronal environment or whether they are in fact just an artefact or a product of the experimental preparation techniques employed to investigate them or other neuronal components. It could be that otherwise free mRNA observed closer to the nucleus is bound with RNA binding proteins released due to cell separation techniques and the mediums employed. For example, the biochemical components may take the form of a granule due to the pH or ionic composition of the medium which could cause unnatural folding and bonds being formed. Aggregation of the granules is observed naturally and is believed to be related to greater degradation, but is not seen in some neuropathological cases or animal models. Alternatively, the experimental preparation process could remove blocking proteins and fatty acids and allow unnatural ribosomal binding to occur whether it is at the stage of the initial 30S subunit or later where the subunits actively combine. The result of this could be that unnatural translation would occur. The evidence however, does lead to the conclusion that although dynamic and dependent on the role of the cell at that time, the granules are genuine physical structures and the system is a justified mechanism for RNA translation.
Now that we have established that the RNA granule and local translation are real biochemical components and part of a genuine biochemical process we have to look why they are necessary when established mechanisms are already in play. One advantage of this type of translation as discussed by Sephton and Yu is that local translation would provide a quick protein production and transport mechanism to the sites where the proteins are actually needed, eg. the synaptic membrane so that the cell can respond quickly to the functional demands placed on it due to signal transmission. However, it must be said that in some cases of neuronal functioning this is not applicable and there is no difference in length between neuronal cells and other cells. Also, quick responses are believed to be required only in the transmission of the firing signal and longer term responses like memory occur over an extended period of time. Therefore, it is not necessary to have a separate translating system from the more standard 2 nuclear systems. However, another advantage of such a system is that there would also be a reduced set of needs on such as system such as post-translational processing by the GA which dictates the proteins location since the mRNA is already there in the vicinity where it functions and hence, speed of synthesis and complexity of the protein support mechanism would not be required. It should be noted however, that this implies that the protein structure itself of a protein translated locally may have constraints. It may either have to be functional alone with minimal post-translational processing, or it will be part of a ´super complex` with other molecules required to form the viable active protein.
Another possible advantage of the local translation process to that of the nuclear process is that that RNA granules provide the equivalent of a ´protection system` for free mRNA in a location away from either the smooth ER and GA. The cytosol is rich in ions which can be detrimental to protein structure and therefore, it is biochemically not advantageous to have mRNA floating around in it for any length of time. Also neuronal cytoplasm due to the mechanism of firing suffers extreme changes in ionic composition and electrical charge during the course of depolarisation and re-establishment of electrical normality. Therefore, mRNA would have to be protected if it has to travel any distance in this environment from its nuclear source. Binding it with other components eg. RNA binding proteins to form granules would protect the mRNA thread from the possibly destructive biochemical environment of the synapse.
Therefore, although localised translation in areas well away from the nuclear site can be advantageous in terms of speed of reaction and protection, is it necessary to have another separate system in order to carry out this function? The cell already has two types of translation in place: that on the ER and then with GA modification; and that using free mRNA with the ribosomes attaching themselves in the cytoplasm onto the mRNA thread. Local translation is a separate system and involves the mRNA being physically associated with many other components into ´granules` (eg. RNA granules) in for example, the dendrites. It is likely that this ´granule` system is based on a system already in place to transfer proteins and molecules in other cells. This transport system uses vesicles and the molecular motor systems associated with microtubules and microfilaments. Vesicles can hold enzymes for synthesis and degradation of molecules and is a quick and efficient intercellular transport system. Therefore, it can be considered that the RNA granules described in the dendrites are the mRNA equivalent to the normal vesicle system observed for other biochemical compounds. However, there are some differences and a problem. Whereas most vesicles are considered ´active` eg. lysosomes, the mRNA carried in RNA granules may not be. In tRNP granules, mRNA appears to be transported in a translationally dormant state and the granule acts as a carrier and provider of conditions for a change to occur when the mRNA is required to become translationally active. This presents a problem because mRNA becomes active when a signal is received, but because of the constant breakdown of the proteins and other components in the granule which is normal for any biological molecule or system, the mRNA cannot be held in a ´ready` state too long. Therefore, there is a constant need for renewal if the aim of having this type of system is to maintain the cell in a continued state of readiness and this requires the biochemical machinery and energy to do this. Also it appears that the RNA granule transport is one directional and that is away from the nucleus unlike the vesicular system which is bi-directional. This implies that the mRNA is not brought back to the nucleus in order to influence the DNA as is observed in the case of some viruses.
Now that we have established that local translation is a viable mechanism and is dependent on RNA granules of different types we will conclude this comment by taking a quick look at one of the components of the granules which is said to be important for neuronal function and cellular plasticity. Sephton and Yu showed in their article the RNA binding proteins content of the RNA granules present at any one time can dictate whether protein translation is repressed or initiated and the type of granule observed eg. tRNP has binding proteins, Staufen 1 and 2, FUS and TDP-43 plus mRNA in transitionally dormant state. RNA binding proteins can exist at the untranslated end regions or in coding regions of the mRNA and as expected such binding will affect maintenance and translation. The presence of either RNA polymerase I or II makes a difference as to whether the region is translated or not and therefore, RNA binding proteins could dictate which polymerase is allowed to bind. (This of course assumes that RNA granules require the same conditions for mRNA translation as that closer to the nucleus.) The RNA binding proteins could also revert the mRNA form back into a local pre-mRNA form so that the initiation of translation is prevented. Pre-mRNA has specific 5 and 3 ends (5 end – cap structure added, 3 end – poly A tail) which are required for aiding binding of the mRNA to the ribosome, protecting from premature destruction by ribonucleotidases and as a signal for transport to the cytoplasm. Therefore, specific RNA proteins could take on these roles. In nuclear translation splicing occurs to remove these additions and to remove introns so that exons become joined together. This is also likely to be the case in RNA granules when the signal is given to translate the transcripts. This implies that the signal is in some form that can remove RNA binding proteins from the relevant sites. Other RNA binding proteins determine how mRNA interacts with its environment eg. the binding protein FMRP and mRNA and ribosomes. This implies that the binding of RNA binding proteins promotes conformational changes to the mRNA transcript that opens the attachment sites of perhaps the ribosomes. Phosphorylation of FMRP means no translation whereas removal of the phosphate group means that translation occurs. This mechanism is also seen with miRNA and is a common mechanism in other molecules since addition or removal of molecular groups eg. phosphate groups, or disulphide bridges will create changes in tertiary and quartenary molecular shapes that promote or decrease function.
The number of RNA binding proteins and the number of different roles that they play make them ideal targets for manipulation whether natural eg. gene mutations leading to decreased numbers of the binding proteins or experimental, eg. FUS knock-out transgenic mice. Sephton and Yu described in their article some of the RNA binding proteins and their links to neurodegenerative diseases eg. FUS mutations observed in ALS, TDP-43 mutations in frontotemporal dementia. It is clear that anything that affects mRNA formation, transport and translation will have an effect on the functioning of the neuronal synapse. These effects may be short-term or long-term. The question that has to be answered is whether long-term changes in neuronal function and plasticity are caused only by permanent changes in local translation of the nature described above (ie. caused only by gene mutations), or not. It is possible that temporary ´blips` in protein synthesis in the area of synapse will have immediate effects that alter current neuronal firing, but may not be sufficient to cause permanent effects which are only elicited through the system located closer to the nucleus. Only further research will answer this question, but it is an interesting topic and again indicates how complicated neuronal cell mechanisms are.
Since we`re talking about the topic……………………….
…….if we could carry out RNA sequencing at the 5 prime end region of mRNA from free mRNA, ER-bound mRNA or RNA granule-bound mRNA would we see differences in genetic code dependent on the source or will they all have the same promotor sequences and would these differ in content as expected to the promoter regions of prokaryote mRNA?
…….if we use cortical slices and subject the cells to different conditions eg oxidative stress, drugs, then separate out the mRNA from the RNA granules of those cells and subject them to Northern Blots will we be able to see alterations in the mRNA transcripts necessary for the cell adaptations required to cope with each condition?
……can we assume that the mRNA transcript length as measured by agarose gel electrophoresis is the same whether the mRNA transcript undergoes ER/GA translation close to the nucleus or at a site away from it?
……since mutations of some RNA binding proteins are associated with increased neurodegradation can we investigate whether the apopteric enzymes necessary for degradation eg. annexin V are actually translated from mRNA associated close to the nucleus or are they locally translated? If RNA granules are removed would the absence of annexin V labelled with fluoresceinisothiocyanate (FITC) be proof that local translation is responsible for the production of apopteric enzymes?