image: Leaderless mRNA is capable of directing protein synthesis in human cells by any of four possible means. view more
Credit: Sergey Dmitriev
Biologists from the Lomonosov Moscow State University in collaboration with colleagues from the Engelhardt Institute of Molecular Biology, Russian Academy of Sciences used RNA transfection and in vitro techniques to show how the same mRNA can direct protein synthesis in a cell by four different means. The research results have been published in a peer reviewed journal Scientific Reports.
Scientists from the Belozersky Institute of Physico-Chemical Biology, a department of the Lomonosov Moscow State University, along with their colleagues, have applied a transfection method to deliver RNA into the cell, that has allowed them to study the impact of cell stress on protein biosynthesis on a short-time scale.
Cell stress and reprogramming of protein synthesis
Sergey Dmitriev, Senior Researcher at the Belozersky Institute of Physico-Chemical Biology, the Lomonosov Moscow State University and the Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, being a leading author of the article, shares the following comments. He says: "Our project is devoted to the studies of protein biosynthesis mechanisms, including the situations of cell stress. The research highlights three aspects. The first one concerns methods as we present a technique, which allows analyzing protein synthesis in a cell with the help of a short-term RNA transfection technique".
Transfection is a method of DNA and RNA delivery to a living cell. Usually DNA is used: after entrance into the nucleus it initiates the processes of new RNA synthesis, and only afterwards the RNAs are exported into cytoplasm and can participate in protein production. Biologists from the Lomonosov Moscow State University propose a methodology of introducing an artificially synthesized RNA into the cell, so it could become a template for protein synthesis immediately. RNA is delivered to cells with the help of a special chemical agent. Once penetrated into cytoplasm, it's released and gets ready to participate in protein production; the only thing left to do for the RNA is to meet a ribosome. So, the way to the final product becomes much shorter. In as little as 1 or 2 hours one could observe protein activity and estimate its quantity.
This technique allows studying the impact of a stress on the cell in a short-time scale. To cell stresses one could refer, for example, heat shock, caused by elevated temperature; oxidative stress, provoked by reactive oxygen species; response to chemical agents, which disrupt homeostasis (including antibiotics and medical drugs). Factors of cell stress compel the cell to suspend protein biosynthesis (or "reprogram" it), until the system redresses the balance.
Sergey clarifies: "Usually these processes last from one to four hours, and our technique of "fast" RNA transfection is the most convenient way to study the effect of these processes. We've conducted our research on cultured cells of human kidney, which serve as a standard model for such studies. Finally, we have elaborated a technique that allows one to obtain artificial RNA, transfer it into cells and obtain the result in a very short period of time. We've named the whole method FLERT (for "fleeting mRNA transfection"), which sounds in Russian a bit like flirtation,"- adds Sergey and smiles.
Why is the sum of 40S and 60S equal to 80S in case of a ribosome?
Messenger RNA (mRNA) is a polymer of nucleotides, coding for a protein. One amino acid is encoded by 3 nucleotides. There is a special molecular machine, called ribosome, existing in the cell for protein production. Moving along mRNA, the ribosome reads information in a triplet by triplet manner.
The structure of the protein synthesis machine is very complicated. It comprises two subunits: a small one (40S) and a large one (60S). A whole ribosome is obtained when they join. However, it's specified not as 100S, but as 80S. The reason is that these figures refer not to the particle mass, but to the sedimentation coefficient, determined during centrifugation. This coefficient depends on several parameters, including the shape of a particle.
In order to start information decoding, first of all it is necessary to find the right starting point - a triplet from which the reading begins. Detection of the starting point is problematic, as there are no marks for triplets in mRNA. However, if you start reading from a wrong nucleotide, the reading frame will be shifted, and everything will go wrong. Special proteins (translation initiation factors) help the ribosome to find the right place in the template to start reading triplets.
Usually there is a distance between the beginning of the mRNA chain and the starting point, called "leader". A ribosome should pass this leader by, without reading. Russian scientists have decide to check what will happen if mRNA will begin right with the start codon - from the "first word". It's interesting that in archaea (single-celled prokaryotic organisms, which have been living on the Earth for billion years and are capable of surviving in extreme conditions) and some other primitive organisms most mRNAs begin right from the start codon. Such RNAs are called leaderless. Leaderless mRNAs are supposed to be an evolutionary prototype of messenger RNAs because ancient ribosomes were not able to find starting points and initiated decoding from the very beginning of the mRNA chain.
A ribosome is to pass through several phases in order to interact with mRNA and start protein synthesis. Normally, at first the 40S subunit of the ribosome binds mRNA, and then the large 60S subunit joins it at the start codon. In contrast, the leaderless mRNA can be loaded directly into the whole ribosome. This discovery was made in the 90-s by Ivan Shatsky, Professor from the Lomonosov Moscow State University.
In the new project scientists have demonstrated that due to their unique properties, leaderless RNAs are resistant to many stress types and continue directing protein synthesis even in such conditions, when common RNAs with leader stop working in the fist minutes after the impact. With the help of FLERT technique scientists have shown this in living cells.
All ways are OK - you can choose whatever you like
The research extension has brought even more interesting results. It has been turned out that unique properties of the leaderless mRNA provide it with high flexibility in the choice of protein synthesis mechanisms.
It has been found out that eukaryotes possess several pathways by which a ribosome could find itself on the start codon. These modes are mediated by distinct sets of specialized proteins - called translation initiation factors - and have been shown to operate on different mRNAs.
The most common pathway, which could be used by any cellular mRNA, is provided by eIF2 protein. However, this factor is very quickly inactivated under conditions of stress. As a result, ribosomes fail to recognize the start codons on all mRNAs, except those that use other initiation factors.
Later on scientists discovered that eIF2 is not the only factor able to do this work. For instance, mRNA of hepatitis C virus is capable of doing without eIF2 and can use other factors, eIF5B or eIF2D, instead (this discovery was also made by scientists from the Lomonosov Moscow State University earlier). This virus was supposed to be unique in this sense - while canonical templates are passively waiting until a ribosome binds them, the hepatitis C virus mRNA "grasps" 40S subunit and "puts" it to the right place in the chain. This unusual property makes possible the usage of alternative ways. And now scientists have proved that the leaderless mRNA is capable of acting in the same way.
It's also interesting that all organisms possess eIF5B factor as it's an evolutionary conserved protein. In contrast, eIF2 exists only in eukaryotes and archaea - so, it's not universal. All the above mentioned results allow to say that the well-studied classical factor eIF2 is needed only if ribosomes recognize mRNA by active searching for the start codon. Such mean of translation initiation is called scanning and requires eIF2. When the start codon is found, eIF2 is replaced by eIF5B and protein synthesis starts. More evolutionary-ancient leaderless mRNA can use a primitive mechanism, immediately recruiting eIF5B factor.
Sergey Dmitriev concludes: "We've got a nice result, which has explained everything. We've found out that a primitive mRNA could use an evolutionary ancient mechanism. Moreover, it is capable of using the other three pathways: through eIF2, eIF2D or direct recruitment of the whole 80S ribosome".
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Journal
Scientific Reports