Liposome-encapsulated aprotinin biodistribution in mice: Side-by-side comparison with free drug formulation.

Injuries of the respiratory system caused by viral infections (e.g., by influenza virus, respiratory syncytial virus, metapneumovirus, or coronavirus) can lead to long-term complications or even life-threatening conditions.

Deficiency of the ribosomal protein uS10 (RPS20) reorganizes human cells translatome according to the abundance, CDS length and GC content of mRNAs.

Ribosomal protein uS10, a product of the gene, is an essential constituent of the small (40S) subunit of the human ribosome. Disruptive mutations in its gene are associated with a predisposition to hereditary colorectal carcinoma. Here, using HEK293T cells, we show that a deficiency of this protein leads to a decrease in the level of ribosomes (ribosomal shortage).

Mutation at the Site of Hydroxylation in the Ribosomal Protein uL15 (RPL27a) Causes Specific Changes in the Repertoire of mRNAs Translated in Mammalian Cells.

Ribosomal protein uL15 (RPL27a) carries a specific modification, hydroxylation, at the His39 residue, which neighbors the CCA terminus of the E-site-bound tRNA at the mammalian ribosome. Under hypoxia, the level of hydroxylation of this protein decreases. We transiently transfected HEK293T cells with constructs expressing wild-type uL15 or mutated uL15 (His39Ala) incapable of hydroxylation, and demonstrated that ribosomes containing both proteins are competent in translation.

Reorganization of the Landscape of Translated mRNAs in NSUN2-Deficient Cells and Specific Features of NSUN2 Target mRNAs.

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.

Changes in the Transcriptome Caused by Mutations in the Ribosomal Protein uS10 Associated with a Predisposition to Colorectal Cancer.

A number of mutations in the 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.

Deficiency of the Ribosomal Protein uL5 Leads to Significant Rearrangements of the Transcriptional and Translational Landscapes in Mammalian Cells.

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).

Knockdown of the Ribosomal Protein eL38 in HEK293 Cells Changes the Translational Efficiency of Specific Genes.

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.

Knockdown of the mRNA encoding the ribosomal protein eL38 in mammalian cells causes a substantial reorganization of genomic transcription.

The ribosomal protein eL38 is a component of the mammalian translation machine. The deletion of the Rpl38 locus in mice results in the Tail-short (Ts) mutant phenotype characterized by a shortened tail and other defects in the axial skeleton development. Here, using the next-generation sequencing of total RNA from HEK293 cells knocked down of eL38 mRNA by transfection with specific siRNAs, we examined the effect of reduced eL38 content on genomic transcription.

Main Strategies for the Identification of Neoantigens.

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.

The human ribosomal protein eL29 binds in vivo to the cognate mRNA by interacting with its coding sequence, as revealed from in-cell cross-linking data.

The balance of ribosomal proteins is important for the assembly of ribosomal subunits and cell viability. The synthesis of ribosomal proteins in a eukaryotic cell is controlled by various mechanisms, including autoregulation, which so far has been revealed for only a few of these proteins. We applied the photoactivatable 4-thiouridine-enhanced cross-linking and immunoprecipitation assay to HEK293T cells overproducing FLAG-labeled human ribosomal protein eL29 (eL29) to determine which RNAs other than rRNA interact with eL29.

Knockdown of the Ribosomal Protein eL29 in Mammalian Cells Leads to Significant Changes in Gene Expression at the Transcription Level.

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.

The functional role of the C-terminal tail of the human ribosomal protein uS19.

The eukaryotic ribosomal protein uS19 has a C-terminal tail that is absent in its bacterial homologue. This tail has been shown to be involved in the formation of the decoding site of human ribosomes. We studied here the previously unexplored functional significance of the 15 C-terminal amino acid residues of human uS19 for the assembly of ribosomes and translation using HEK293-based cell cultures capable of producing FLAG-labeled uS19 (uS19) or its mutant form deprived of the mentioned amino acid ones.

Degenerate consensus sequences in the 3'-untranslated regions of cellular mRNAs as specific motifs potentially involved in the YB-1-mediated packaging of these mRNAs.

The multifunctional protein YB-1 has previously been shown to be the only protein of the cytoplasmic extract of HEK293 cells, which is able to specifically interact with imperfect RNA hairpins containing motifs that are often found in exosomal (e) RNAs. In addition, it has been revealed that similar hairpins formed by degenerate consensus sequences corresponding to three eRNA-specific motifs are responsible for the cooperative binding of YB-1 to RNA in vitro. Here, using the photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation method applied to HEK293 cells producing FLAG-labeled YB-1, we identified mRNAs cross-linked to YB-1 in vivo and then carried out a search for the aforementioned sequences in the regions of the YB-1 cross-linking sites.

mRNA regions where 80S ribosomes pause during translation elongation in vivo interact with protein uS19, a component of the decoding site.

In eukaryotic ribosomes, the conserved protein uS19, formerly known as S15, extends with its C-terminal tail to the decoding site. The cross-linking of uS19 to the A site codon has been detected using synthetic mRNAs bearing 4-thiouridine (s4U) residues. Here, we showed that the A-site tRNA prevents this cross-linking and that the P site codon does not contact uS19.

Tetrapeptide 60-63 of human ribosomal protein uS3 is crucial for translation initiation.

Conserved ribosomal protein uS3 contains a decapeptide fragment in positions 55-64 (human numbering), which has a very specific ability to cross-link to various RNA derivatives bearing aldehyde groups, likely provided by K62. It has been shown that during translation in the cell-free protein-synthesizing system, uS3 becomes accessible for such cross-linking only after eIF3j leaves the mRNA binding channel of the 40S ribosomal subunit. We studied the functional role of K62 and its nearest neighbors in the ribosomal assembly and translation with the use of HEK293T-derived cell cultures capable of producing FLAG-tagged uS3 (uS3) or its mutant form with amino acid residues at positions 60-63 replaced with alanines.

The eS26 protein is involved in the formation of a nucleophosmin binding site on the human 40S ribosomal subunit.

Human ribosomal protein eS26 is an indispensable component of the small (40S) ribosomal subunit and, along with other ribosomal proteins, is involved in interaction with mRNAs during translation. Here, we explored the behavior of the exogenous ribosomal protein eS26 modified at the C-terminus in the events related to translation in human cells using a doxycycline-inducible HEK293-derived cell line enabling the stable production of C-terminal FLAG-tagged eS26 (eS26). The production of eS26 in cells was accompanied by a decrease in the endogenous eS26 content although its mRNA level did not change.

Structural features of the interaction of the 3'-untranslated region of mRNA containing exosomal RNA-specific motifs with YB-1, a potential mediator of mRNA sorting.

We have previously shown that YB-1 is the only protein of the HEK293 cell cytoplasmic (S100) extract that specifically interacts with RNA hairpins each containing one of the motifs ACCAGCCU (1), CAGUGAGC (2) and UAAUCCCA (3), which had been identified as often found in exosomal RNA and proposed as potential cis-acting elements targeting RNAs into exosomes. Here we explored the interactions of YB-1 with a fragment of the 3'-untranslated region (UTR) of septin 14 mRNA (SEPT14 RNA), which contains all three motifs. We demonstrated the occurrence of YB-1 among proteins pulled down from the HEK293 S100 extract using biotinylated SEPT14 RNA.

Human ribosomal protein eS1 is engaged in cellular events related to processing and functioning of U11 snRNA.

Ribosomal proteins are involved in many cellular processes through interactions with various RNAs. Here, applying the photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation approach to HEK293 cells overproducing ribosomal protein (rp) eS1, we determined the products of RNU5A-1 and RNU11 genes encoding U5 and U11 snRNAs as the RNA partners of ribosome-unbound rp eS1. U11 pre-snRNA-associated rp eS1 was revealed in the cytoplasm and nucleus where rp eS1-bound U11/U12 di-snRNP was also found.

Cytosolic YB-1 and NSUN2 are the only proteins recognizing specific motifs present in mRNAs enriched in exosomes.

Exosomes, membranous vesicles secreted by various cells, are involved in intercellular communication and carry vast repertoires of RNAs and proteins. Processes mediating RNA sorting into exosomes are currently poorly understood. Using bioinformatics approaches, three structural motifs ACCAGCCU, CAGUGAGC and UAAUCCCA have been discovered as enriched in exosomal mRNAs and long noncoding RNAs.

Recognition but no repair of abasic site in single-stranded DNA by human ribosomal uS3 protein residing within intact 40S subunit.

Isolated human ribosomal protein uS3 has extra-ribosomal functions including those related to base excision DNA repair, e.g. AP lyase activity that nicks double-stranded (ds) DNA 3΄ to the abasic (AP) site.

Exploring human 40S ribosomal proteins binding to the 18S rRNA fragment containing major 3'-terminal domain.

Association of ribosomal proteins with rRNA during assembly of ribosomal subunits is an intricate process, which is strictly regulated in vivo. As for the assembly in vitro, it was reported so far only for prokaryotic subunits. Bacterial ribosomal proteins are capable of selective binding to 16S rRNA as well as to its separate morphological domains.