Genetic instability of tumors leads to the appearance of numerous tumor-specific somatic mutations that could potentially result in the production of mutated peptides that are presented on the cell surface by the MHC molecules. Peptides of this kind are commonly called neoantigens. Their presence on the cell surface specifically distinguishes tumors from healthy tissues. This feature makes neoantigens a promising target for immunotherapy. The rapid evolution of high-throughput genomics and proteomics makes it possible to implement these techniques in clinical practice. In particular, they provide useful tools for the investigation of neoantigens. The most valuable genomic approach to this problem is whole-exome sequencing coupled with RNA-seq. High-throughput mass-spectrometry is another option for direct identification of MHC-bound peptides, which is capable of revealing the entire MHC-bound peptidome. Finally, structure-based predictions could significantly improve the understanding of physicochemical and structural features that affect the immunogenicity of peptides. The development of pipelines combining such tools could improve the accuracy of the peptide selection process and decrease the required time. Here we present a review of the main existing approaches to investigating the neoantigens and suggest a possible ideal pipeline that takes into account all modern trends in the context of neoantigen discovery.

An imbalance in the synthesis of ribosomal proteins can lead to the disruption of various cellular processes. For mammalian cells, it has been shown that the level of the eukaryote-specific ribosomal protein eL29, also known as the one interacting with heparin/heparan sulfate, substantially affects their growth. Moreover, in animals lacking this protein, a number of anatomical abnormalities have been observed. Here, we applied next-generation RNA sequencing to HEK293 cells transfected with siRNAs specific for the mRNA of eL29 to determine what changes occur in the transcriptome profile with a decrease in the level of the target protein. We showed that an approximately 2.5-fold decrease in the content of eL29 leads to statistically significant changes in the expression of more than a thousand genes at the transcription level, without a noticeable effect on cell viability, rRNA level, and global translation. The set of eL29-dependent genes included both up-regulated and down-regulated ones, among which there are those previously identified as targets for proteins implicated in oncogenesis. Thus, our findings demonstrate that an insufficiency of eL29 in mammalian cells causes a significant reorganization of gene expression, thereby highlighting the relationship between the cellular balance of eL29 and the activities of certain genes.

Protein uL5 (formerly called L11) is an integral component of the large (60S) subunit of the human ribosome, and its deficiency in cells leads to the impaired biogenesis of 60S subunits. Using RNA interference, we reduced the level of uL5 in HEK293T cells by three times, which caused an almost proportional decrease in the content of the fraction corresponding to 80S ribosomes, without a noticeable diminution in the level of polysomes. By RNA sequencing of uL5-deficient and control cell samples, which were those of total mRNA and mRNA from the polysome fraction, we identified hundreds of differentially expressed genes (DEGs) at the transcriptome and translatome levels and revealed dozens of genes with altered translational efficiency (GATEs). Transcriptionally up-regulated DEGs were mainly associated with rRNA processing, pre-mRNA splicing, translation and DNA repair, while down-regulated DEGs were genes of membrane proteins; the type of regulation depended on the GC content in the 3′ untranslated regions of DEG mRNAs. The belonging of GATEs to up-regulated and down-regulated ones was determined by the coding sequence length of their mRNAs. Our findings suggest that the effects observed in uL5-deficient cells result from an insufficiency of translationally active ribosomes caused by a deficiency of 60S subunits.

A number of mutations in the RPS20 gene encoding the ribosomal protein uS10 have been found to be associated with a predisposition to hereditary non-polyposis colorectal carcinoma (CRC). We transfected HEK293T cells with constructs carrying the uS10 minigene with mutations identical to those mentioned above and examined the effects of the produced proteins on the cellular transcriptome. We showed that uS10 with mutations p.V50SfsX23 or p.L61EfsX11 cannot be incorporated into 40S ribosomal subunits, while the protein with the missense mutation p.V54L functionally replaces the respective endogenous protein in the 40S subunit assembly and the translation process. The comparison of RNA-seq data obtained from cells producing aberrant forms of uS10 with data for those producing the wild-type protein revealed overlapping sets of upregulated and downregulated differently expressed genes (DEGs) related to several pathways. Among the limited number of upregulated DEGs, there were genes directly associated with the progression of CRC, e.g., PPM1D and PIGN. Our findings indicate that the accumulation of the mutant forms of uS10 triggers a cascade of cellular events, similar to that which is triggered when the cell responds to a large number of erroneous proteins, suggesting that this may increase the risk of cancer.

The protein eL38 is one of the smallest proteins of the mammalian ribosome, which is a component of its large (60S) subunit. The haploinsufficiency of eL38 in mice leads to the Tail-short mutant phenotype characterized by defects in the development of the axial skeleton caused by the poor translation of mRNA subsets of Hox genes. Using the ribosome profiling assay applied to HEK293 cells knocked down of eL38, we examined the effects of the lack of eL38 in 60S subunits on gene expression at the level of translation. A four-fold decrease in the cell content of eL38 was shown to result in significant changes in the translational efficiencies of 150 genes. Among the genes, whose expression at the level of translation was enhanced, there were mainly those associated with basic metabolic processes; namely, translation, protein folding, chromosome organization, splicing, and others. The set of genes with reduced translation efficiencies contained those that are mostly involved in the processes related to the regulation of transcription, including the activation of Hox genes. Thus, we demonstrated that eL38 insufficiency significantly affects the expression of certain genes at the translational level. Our findings facilitate understanding the possible causes of some anomalies in eL38-deficient animals.

The RNA cytosine C5 methyltransferase NSUN2 has a variety of RNA substrates and plays an important role in mRNA metabolism. NSUN2 binds to specific sequences enriched in exosomal mRNAs, suggesting its possible involvement in the sorting of mRNAs into exosomes. We applied the photoactivatable.4-thiouridine-enhanced cross-linking and immunoprecipitation assay involving high-throughput RNA sequencing (RNA-seq) to HEK293T cells to determine NSUN2 mRNA targets. NSUN2 cross-linking sites were found in more than one hundred relatively abundant mRNAs with a high GC content and a pronounced secondary structure. Then, utilizing RNA-seq for the total and polysome-associated mRNA from HEK293T cells with and without the knockdown of NSUN2, we identified differentially expressed genes, as well as genes with altered translational efficiency (GATEs). It turned out that the up-regulated GATE mRNAs were much shorter on average than the down-regulated ones, and their GC content was higher; moreover, they contained motifs with C residues located in GC-rich environments. Our findings reveal the specific features of mRNAs that make them potential targets for NSUN2 and expand our understanding of the role of NSUN2 in controlling translation and, possibly, in mRNA sorting into exosomes implemented through the methylation of cytosine residues.