When a ribosome is affixed to the polypeptide (protein) it is making, the ribosome-nascent chain complex (RNC) is the compilation of molecules that make up the ribosome. One of several methods can be used to halt the synthesis of the nascent polypeptide. RNCs are made and purified in labs to study the dynamics, biochemistry, folding, and interactions that the ribosome and proteins undergoing synthesis.

Figure 1. The generation process of RNA in vitro. (Schaffitzel, 2007)

The ribosome, as a critical part of the central dogma, is a node in the flow of genetic information, modifying both mRNA input and protein output. With poor correlations between mRNA and protein abundances in different species, with R2 spanning from 0.01 to 0.50, it has been theorized for years that the quantity of translating mRNAs (mRNAs bound to the ribosome-nascent chain complex, RNC-mRNA) may better represent protein abundances. However, at least in recent studies involving yeasts, HEK293 cells, and tumor cells, this appeared to be uncertain, with R2 0.37. These findings suggested that translational modulation can occur at any time during the three phases of translation, initiation, elongation, and termination, as well as in the spatial organization of mRNAs. However, studies on translational kinetics have uncovered that translational initiation, which decides the fraction of mRNA molecules subjected to translation, is the most influential variable in this modulation.

Introduction to RNC-mRNA Sequencing

RNCs are enhanced and sequenced with full-length mRNAs bound to numerous ribosomes by sucrose density gradient hyper-centrifugation in the RNC-Seq protocol. The molecular weight of the mRNA increases as it binds to the ribosome to form a complex. The greater the number of ribosomes that bind to mRNAs, the higher the molecular weight of the complex. The mRNA molecule being translated will create polysomes with numerous ribosomes, which will normally suspend in a high-density sucrose solution layer where the ongoing translating mRNAs can be segregated. The next step is very similar to RNA-Seq.

RNC sequencing is a method for profiling the Ribosome Nascent-chain Complex using next-generation sequencing (NGS) (RNC). The ribosome attached to the polypeptide it is synthesizing is known as the RNC, and it is one of the most important components of the translation process. The translation ratio (TR) or translation efficiency is defined as the ratio of RNC mRNA to total mRNA. TR is linked to cell phenotype and is influenced by the UTR region, miRNA, and other factors. The study of gene TR changes in different samples is important for understanding the molecular mechanism of ncRNAs. Meanwhile, it's a useful tool for studying disease occurrence and progression, pharmacological action, bio-developmental molecular mechanisms, and more.

Workflow and Bioinformatic Analysis Pipelines of RNC-mRNA Sequencing

There are four major steps in the RNC-mRNA sequencing workflow: (1) sample preparation, (2) library preparation, (3) sequencing, and (4) data analysis. RNA purification, quality assessment, and quantification are all steps in the sample preparation process. Library preparation, on the other hand, contains RNA fragmentation and cDNA library preparation. The results are analyzed by visualizing and preprocessing them, as well as performing custom bioinformatics analysis.

RNC-mRNA sequencing bioinformatics analysis pipelines involve in-depth analysis of the following: differential expression analysis, pathway analysis, SNV & InDel detection, splicing analysis; data quality control, reference-based mapping, gene fusion detection; differential expression analysis, pathway analysis, SNV & InDel detection, splicing analysis, and prediction of novel mRNAs.

References:

1. Luo Z, Hu H, Liu S, et al. Comprehensive analysis of the translatome reveals the relationship between the translational and transcriptional control in high fat diet-induced liver steatosis. RNA biology. 2021 Jun 3;18(6).
2. Cabrita LD, Hsu ST, Launay H, et al. Probing ribosome-nascent chain complexes produced in vivo by NMR spectroscopy. Proceedings of the National Academy of Sciences. 2009 Dec 29;106(52).
3. Schaffitzel C, Ban N. Generation of ribosome nascent chain complexes for structural and functional studies. Journal of structural biology. 2007 Jun 1;158(3).