Parallel sequencing: A new era in proteomics

Reading molecular fingerprints
Pavel Natalyin, "Biomolecule"

The amazing possibilities of next-generation sequencing technologies have a huge impact on the most diverse areas of modern molecular biology [1, 2]. For example, with the complete decoding of new genomes or re-reading (resequencing) the speed and relative cheapness of new technologies already decoded genomes make it possible to conduct experiments on a scale that could not have been imagined before. In the case when the object of research is RNA (DNA copies of transcripts are sequenced - the so–called cDNA), parallel sequencing makes it possible to conduct a more accurate quantitative analysis of gene expression with a wider dynamic range than traditional methods. Finally, when profiling mRNAs involved in translation (the process of protein synthesis), the use of new sequencing technologies allows not only to efficiently and very accurately study this stage of gene expression, but also provides a lot of qualitatively new information. Recently published in the journal Science, an article from the laboratory of Jonathan Weissman from the University of California, San Francisco, reports the first results of this approach.

The ribosome, "sitting" on the mRNA and leading its translation, closes a segment of 30 nucleotides long, making them inaccessible to the effects of ribonuclease, which destroys the rest (unprotected) RNA. Since the length of the average mRNA molecule significantly exceeds the number of nucleotides occupied by the ribosome on RNA, one RNA molecule is translated by a polysome, or polyribosome – a complex of several ribosomes. The number of ribosomes in a polysome depends on the rate of initiation, elongation and termination on a particular RNA. Currently, a model has been adopted in which in eukaryotes, the beginning of the mRNA (5' untranslated site) and its end (3' untranslated site) are located close to each other due to the interaction of one of the translation initiation factors (IF4G/F) with a poly(A)-binding protein (PUB) associated with 3' an untranslated section.

The figure shows one of the first polysome preparations under an electron microscope [6] and the scheme of operation of the polyribosome.Parallel sequencing of cDNA libraries obtained from all protected RNA fragments remaining in the cell allowed Weissman and his colleagues to obtain an exhaustive picture of protein synthesis in the cell [3, 4].

On the right is a diagram of an experiment on ribosomal profiling (reading ribosome prints on mRNA) or sequencing of randomly fragmented mRNA molecules [3].This approach can be used to solve a variety of different tasks.

Firstly, it is very likely that this approach will be used for a detailed study of the proteome [5], which will make it possible to compile a complete "catalog" of all polypeptides synthesized by the cell. According to Weissman himself: "For complex genomes (such as human), it is almost impossible to annotate all expressed proteins. However, our approach essentially allows you to do that." The article published by Weissman and his co-authors reports on the results obtained on baker's yeast, but in principle such studies can be carried out on any organism. Moreover, the use of ribosomes containing artificially introduced epitopes (an epitope is a portion of a molecule specifically recognized by an antibody) will make it possible to study translation in individual groups of cells (tissues). "I think that for some areas [of research], such as molecular neuroanatomy, this will serve as the beginning of a new era," Weissman says with exemplary modesty.

Secondly, the construction of polyribosomal profiles is a more accurate way to study the production of proteins in a cell than simply measuring the amount of a particular mRNA. Scientists from Weissman's lab used ribosome profiling in combination with parallel sequencing to map the density of ribosome "fingerprints" on thousands of different mRNAs. These measurements were then used to calculate the translation rates of protein molecules. The researchers claim that the translation rate calculated in this way makes it possible to predict the amount of synthesized protein more accurately than measuring the amount of the corresponding mRNA. "What is attractive about quantitative proteomics," Weissman says, "is that it allows us to assess how well we are progressing." Indeed, by correcting for the increased density of ribosomes at the 5’ end of the mRNA, the scientists found a significant relationship between the level of translation and the amount of synthesized protein (correlation coefficient ≈0.6).

The third area in which ribosomal profiles will undoubtedly find application is the study of translation control. In an article by California scientists, this method was used to study the effect of amino acid starvation on translation in yeast. There is no doubt that this method will also be used to study the regulation of protein synthesis in various diseases and stress conditions in the cells of higher organisms.

Finally, it should be noted that the described method has a high resolution (up to one nucleotide), which makes it possible to uniquely determine the open reading frame of the mRNA involved in translation. Therefore, the method can be used to study programmed shifts of the reading frame and "slips" of stop codons. Or, as was the case in the work of Weissman's laboratory, the method turns out to be useful for mapping non-canonical translation initiation sites in 5’-untranslated regions of mRNA molecules.

According to Weissman's own conclusion, "today it has become possible to directly carry out high-precision measurements of the level of protein translation. This approach will be used to find out exactly which proteins and in what quantity are produced in the cell. In addition, this method in itself is an excellent analytical tool for studying the translation process as such."

Literature

1. 454-sequencing (high-performance DNA pyrosequencing);
2. Sk true anecdote: a Negro, a Chinese and Craig Venter ...;
3. Ingolia N.T., Ghaemmaghami S., Newman J.R., Weissman J.S. (2009). Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223;
4. de Souza N. (2009). Deep sequencing of ribosome footprints. Nature Methods 6, 244;
5. A billion for proteomics;
6. Warner J.R., Knopf P.M., Rich A. (1963). A multiple ribosomal structure in protein synthesis. Proc. Natl. Acad. Sci. U.S.A. 49,122–129.

Portal "Eternal youth" www.vechnayamolodost.ru05.05.2009