A comparison of Polysome Profiling, RNC-Seq and Ribo-Seq (Part One)

Posted March 24, 2020 by Bonnibelle

In all areas of life, proteins are synthesized by ribosomes during translation.

In all areas of life, proteins are synthesized by ribosomes during translation. The magnitude of translation regulation exceeds the sum of transcription, mRNA degradation, and protein degradation. Therefore, it is necessary to study translation on a general scale. Like other "omics" methods, translationomics studies all components of the translation process, including but not limited to translation of mRNAs, ribosomes, tRNAs, regulatory RNA, and nascent polypeptide chains.
Recent advances in technology have made breakthroughs in the study of these components worldwide, including their composition and dynamics. These methods have been applied in more and more studies to reveal multiple aspects of translation control. The process of translation is not limited to the conversion of mRNA coding sequences into polypeptide chains. It also controls the composition of the proteome in a delicate and sensitive way. Therefore, translationomics has unique innovative capabilities in the fields of proteomics, cancer research, bacterial stress response, biorhythm, and plant biology. Proper design in translation can increase the yield of recombinant proteins by thousands of times. This article reviews the main research methods of translationomics, highlights the latest discoveries in the field, and introduces the application of translationomics in basic biology and biomedical research.
Proteins perform various biological functions in life, so they are under subtle control. According to the central law of molecular biology (to describe the flow of genetic information from DNA through mRNA to proteins), the generation of the entire proteome includes four main regulatory steps: RNA synthesis (including epigenetic and transcriptional regulation), RNA degradation, protein synthesis (ie, translational regulation) and protein degradation. With the development of the current omics technology, mathematical models and omics measurements show that translation regulation accounts for more than half of all adjustments, exceeding the sum of all other adjustments. Therefore, translation regulation is the most important regulatory step in an organism. Since the 1970s, a lot of research has been done on the translation process, but the research on a global scale has been achieved in recent years. Like genome-wide "genomics", "transcriptomics" of all transcripts, and "proteomics" of all proteins, the term "translationomics" is used to study all elements related to translation.
Due to technical difficulties, translation omics has received little attention for a period of time. First, both nucleic acids and proteins are involved in the regulation of translation, increasing complexity and the diversity of biological macromolecules involved in the translation process. Translation omics research requires multiple omics tools and skills. Second, the translation mechanism is very complex. The response to environmental and physiological changes requires rapid and specific adaptation of the translation mechanism within minutes, which makes the experiment challenging. The lack of research on translationomics indicates that there is a large gap in understanding the most important regulatory steps in the flow of genetic information. In recent years, continuous development in the field of translation regulation has enabled us to study the characteristics of translation in a comprehensive manner.
Translationomics research methods
The broad definition of a translation body includes all elements directly involved in the translation process, such as mRNAs (also known as RNC-mRNAs) to be translated, ribosomes, tRNAs, some regulatory RNAs (miRNA, lncRNA), etc. Please note that not all regulatory RNAs are involved in translation, nascent polypeptide chains, and various translation factors. Generally the term "Translatome" represents the whole of the translated mRNA. RNA and protein are two major macromolecules in translationomics research. Their static components and dynamics are regulated by a complex and complex system. For each element involved in translation, specific methods have been developed for research on a global scale.
Methods for mRNAs being translated
mRNA provides a blueprint for protein synthesis. Studying the mRNA that is being translated is a top priority for translationomics. Due to the non-covalent binding of ribosomes and mRNA, the ribosomal nascent strand complex (RNC) is very fragile and is prone to dissociation or degradation after cell lysis. Several important and classic methods have been developed to analyze the different characteristics of mRNAs being translated: polysome profiling, full-length mRNA analysis (RNC-seq) being translated, ribosomal affinity purification (TRAP-seq) and ribosome analysis (Ribo-seq).
1. Polysome Profiling
In the 1960s, Polysome Profiling was developed based on sucrose gradient ultracentrifugation. Ribosomes are the largest polymer machinery in most high-density cells. mRNA molecules that bind more ribosomes deposit faster in the sucrose gradient. Therefore, after centrifugation of the sucrose density gradient, free RNA and protein floated on top of the sucrose gradient due to different concentrations. By slowly pumping the sucrose solution from the bottom, mRNAs that bind to different numbers of ribosomes can be isolated. Then Northern analysis, microarray or RT-PCR were used to analyze the mRNA in each component to reflect the distribution of transcript translation. This technique is often used to detect large changes in translation. For example, under hyperosmotic pressure, the composition of a single ribosome increased significantly, and under oxidative stress, the number of ribosomes bound to a single mRNA increased significantly.
Notably, researchers believe that active translational mRNAs often bind multiple ribosomes. However, recent studies have shown that translation is active for those mRNAs that bind a single ribosome. For example, in HEK293 cells and exponentially growing E. coli, monomeric components (70S of prokaryotes and 80S of eukaryotes) that are considered to be very active in translation dominate. When the monomeric portion is isolated and transferred to a cell-free translation system, the ribosome can resume translation and produce proteins. In Saccharomyces cerevisiae, the monomer is extended, not original. Short open reading frames (ORFs), fast translation genes, and low-abundance mRNAs tend to be enriched in the monomeric portion. These results demonstrate that translational activity is not directly proportional to the number of ribosomes on the mRNA.
The main disadvantage of polysome profiling is that it is difficult to perform in-depth analysis of all translated mRNAs (RNC-mRNA). Due to the large volume of the sucrose gradient, the concentration of RNC-mRNA in each component is very low, and high concentrations of sucrose inhibit some further enzymatic reactions. The total amount of RNC-mRNA recovered from the sucrose gradient is generally only sufficient for RT-PCR quantification, but unless a large amount of starting material is used, it is difficult to obtain sufficient mRNA for full-spectrum analysis, such as microarray or RNA sequencing.
To be continued in Part Two…
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Issued By https://www.cd-genomics.com/
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Categories Biotech
Last Updated March 24, 2020