The use of ribosome-nascent chain complex-seq to reveal the translated mRNA profile and the role of ASN1 in resistance to Verticillium wilt in cotton.

We combined traditional mRNA-seq and RNC-seq together to reveal post-transcriptional regulation events impacting gene expression and interactions between the serious fungal pathogen Verticillium dahliae and a susceptible host, Gossypium hirsutum TM-1. After screening the differentially expressed and translated genes, V. dahliae infection was observed to influence gene transcription and translation in its host. Interestingly, the asparagine synthase (ASN1) gene transcripts increased significantly with the increase of infection time, while the rate of ASN1 protein accumulation in host TM-1 was distinctly lower than that in resistant hosts. We knocked down the ASN1 gene in resistant plants (ZZM2), and found that Verticillium-resistance was significantly reduced upon knockdown of ASN1. Our study revealed both transcriptional and post-transcriptional regulation of gene expression in TM-1 cotton plants infected by V. dahliae, and showed that ASN1 functions in the V. dahliae resistance process. These insights support breeding of disease resistance in cotton.

Translatome analysis reveals the regulatory role of betaine in high fat diet (HFD)-induced hepatic steatosis.

Non-alcoholic fatty liver disease (NAFLD) is a common disease with a multitude of complications. Increasing evidence shows that the dietary supplement with betaine, a natural chemical molecule, can effectively reduce the fat accumulation in the liver. Translational regulation is considered to play a vital role in gene expression, but whether betaine functions through the regulation of gene translational level is still unclear. To this end, RNC-seq (mRNAs bound to ribosome-nascent chain complex sequencing) and RNA-seq co-analyses were performed to identify betaine target genes by using the liver samples from high-fat diet adding betaine treated and high-fat diet treated mice. The results showed that betaine does play a lipid-lowering role by regulating the expression of gene translation levels; some NAFLD- and lipid metabolism-associated genes were differentially expressed at translational level, for example. And the translation ratio (TR) of gene significantly increased after betaine treatment. Finally, we identified a novel function gene, Gpc1, which may mediate the lipid-lowering effect of betaine in the liver. To sum up, this study depicted the molecular portrait of mice liver with or without betaine treatment from the angel of translatome and transcriptome, giving insights into the molecular mechanism of betaine-mediated lipid-lowering effect and also providing new clues for understanding and prevention of NAFLD.

Comprehensive analysis of the translatome reveals the relationship between the translational and transcriptional control in high fat diet-induced liver steatosis.

Translational regulation plays a critical role in gene expression. However, there are few genome-wide studies on translational regulation in non-alcoholic fatty liver disease (NAFLD), which is a severe non-communicable epidemic worldwide. In this study, we performed RNC-mRNA (mRNAs bound to ribosome-nascent chain complex) sequencing and mRNA sequencing to probe the translation status of high-fat-diet (HFD) induced mouse fatty liver. Generally, in the HFD group compared to the control group, changes of translation ratios and changes in mRNA abundance had a negative correlation. The relative abundance of RNC-mRNAs and mRNAs were positively correlated, yet the former changed more slowly than the latter. However, the rate of change became more balanced when it came to the livers of mice that were fed the HFD plus lycopene, an antioxidant. This indicated relatively independent roles of translational modulation and transcriptional regulation. Furthermore, many genes were differentially regulated at the transcriptional or translational levels, suggesting a new screening strategy for functional genes. In conclusion, our analysis revealed the different and correlated role of translational control with transcriptional regulation in the HFD-induced mouse fatty liver relative to the control, which indicates critical roles of translational control for liver steatosis; thus, adding a new dimension towards a better understanding and improvement of treatment for NAFLD.

Translatomics: The Global View of Translation.

In all kingdoms of life, proteins are synthesized by ribosomes in a process referred to as translation. The amplitude of translational regulation exceeds the sum of transcription, mRNA degradation and protein degradation. Therefore, it is essential to investigate translation in a global scale. Like the other "omics"-methods, translatomics investigates the totality of the components in the translation process, including but not limited to translating mRNAs, ribosomes, tRNAs, regulatory RNAs and nascent polypeptide chains. Technical advances in recent years have brought breakthroughs in the investigation of these components at global scale, both for their composition and dynamics. These methods have been applied in a rapidly increasing number of studies to reveal multifaceted aspects of translation control. The process of translation is not restricted to the conversion of mRNA coding sequences into polypeptide chains, it also controls the composition of the proteome in a delicate and responsive way. Therefore, translatomics has extended its unique and innovative power to many fields including proteomics, cancer research, bacterial stress response, biological rhythmicity and plant biology. Rational design in translation can enhance recombinant protein production for thousands of times. This brief review summarizes the main state-of-the-art methods of translatomics, highlights recent discoveries made in this field and introduces applications of translatomics on basic biological and biomedical research.

Protein-Level Integration Strategy of Multiengine MS Spectra Search Results for Higher Confidence and Sequence Coverage.

Multiple search engines based on various models have been developed to search MS/MS spectra against a reference database, providing different results for the same data set. How to integrate these results efficiently with minimal compromise on false discoveries is an open question due to the lack of an independent, reliable, and highly sensitive standard. We took the advantage of the translating mRNA sequencing (RNC-seq) result as a standard to evaluate the integration strategies of the protein identifications from various search engines. We used seven mainstream search engines (Andromeda, Mascot, OMSSA, X!Tandem, pFind, InsPecT, and ProVerB) to search the same label-free MS data sets of human cell lines Hep3B, MHCCLM3, and MHCC97H from the Chinese C-HPP Consortium for Chromosomes 1, 8, and 20. As expected, the union of seven engines resulted in a boosted false identification, whereas the intersection of seven engines remarkably decreased the identification power. We found that identifications of at least two out of seven engines resulted in maximizing the protein identification power while minimizing the ratio of suspicious/translation-supported identifications (STR), as monitored by our STR index, based on RNC-Seq. Furthermore, this strategy also significantly improves the peptides coverage of the protein amino acid sequence. In summary, we demonstrated a simple strategy to significantly improve the performance for shotgun mass spectrometry by protein-level integrating multiple search engines, maximizing the utilization of the current MS spectra without additional experimental work.

High expression circRALGPS2 in atretic follicle induces chicken granulosa cell apoptosis and autophagy via encoding a new protein.

BACKGROUND: The reproductive performance of chickens mainly depends on the development of follicles. Abnormal follicle development can lead to decreased reproductive performance and even ovarian disease among chickens. Chicken is the only non-human animal with a high incidence of spontaneous ovarian cancer. In recent years, the involvement of circRNAs in follicle development and atresia regulation has been confirmed. RESULTS: In the present study, we used healthy and atretic chicken follicles for circRNA RNC-seq. The results showed differential expression of circRALGPS2. It was then confirmed that circRALGPS2 can translate into a protein, named circRALGPS2-212aa, which has IRES activity. Next, we found that circRALGPS2-212aa promotes apoptosis and autophagy in chicken granulosa cells by forming a complex with PARP1 and HMGB1. CONCLUSIONS: Our results revealed that circRALGPS2 can regulate chicken granulosa cell apoptosis and autophagy through the circRALGPS2-212aa/PARP1/HMGB1 axis.

Understanding the proteome encoded by "non-coding RNAs": new insights into human genome.

A great number of non-coding RNAs (ncRNAs) account for the majority of the genome. The translation of these ncRNAs has been noted but seriously underestimated due to both technological and theoretical limitations. Based on the development of ribosome profiling (Ribo-seq), full length translating RNA analysis (RNC-seq) and mass spectrometry technology, more and more ncRNAs are being found to be translated in different organism, and some of them can produce functional peptides. While recently, not only individual new functional proteins, but also a new proteome have been experimentally discovered to be encoded by endogenous lncRNAs and circRNAs. These new proteins are of biological significance, suggesting the connection of the translation of ncRNAs to human physiology and diseases. Therefore, an in-depth and systematic understanding of the coding capabilities of ncRNAs is necessary for basic biology and medicine. In this review, we summarize the advances in the field of discovering this new proteome, i.e. "ncRNA-coded" proteins.