Irina A. Eliseeva

N⁶-methyladenosine (m⁶A) plays an important role in translation control and, particularly, in cap-independent initiation. The impact of m⁶A is usually studied in the context of its location in the transcripts, but the global impact of m⁶A along mRNAs remains unclear. Here, we combined ribosome profiling under conditions of mTOR inhibition and m⁶A mapping to identify distinct subsets of mRNAs that differ in their sensitivity to the suppression of cap-dependent initiation. We found that the sensitivity strongly correlated with the total m⁶A methylation of the transcripts. Further, upon decreased mTOR activity, m⁶A methylation facilitated the enhanced association of mRNAs with components of the eIF4F complex. Thus, the efficiency of cap-independent translation initiation is primarily defined not by precise localization but by the total level of m⁶A methylation, and m⁶A has a compensatory role in maintaining translation when the canonical cap-dependent pathway is impaired. Our findings underscore the significance of contemplating global m⁶A methylation status as a pivotal element in translational control, particularly under stress or signaling perturbations.

Isolation of various groups of protein preparations is a crucial task in many areas of biochemical research. Creating an effective, simple and economically feasible system for recombinant protein production is highly valuable for both basic research and applied biotechnology. Most laboratory studies focus on bacterial systems for heterologous gene expression. However, such systems can encounter major difficulties with target proteins that have specific properties. Some proteins can aggregate, form insoluble inclusion bodies or undergo proteolytic cleavage during overproduction. Moreover, they can be toxic, and thus adversely affect the host cell when overproduced. These issues can be circumvented by using cellular secretion systems to export the target protein from the cell during production. The present study compares three established systems for exporting target proteins to the bacterial cell surface. Three model proteins were selected: the cytokine IL-17A, the catalytic domain of TEV protease, and the calcium-binding domain of nucleobindin 1. Three protein export systems were used: the first was based on the E. coli outer membrane OmpA protein, the second on the E. coli Ag43 autotransporter of the AIDA-I family, and the third on the INP of Pseudomonas syringae. Recombinant proteins were cleaved by site-directed chemical proteolysis.

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca ²⁺ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gαi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

More than 600 distinct skeletal muscles constitute up to 40% of the total mass of the human body. Human skeletal muscles differ in anatomical position, morphology, origin, and function, but the diversity of their molecular phenotypes, the gene expression and protein abundance profiles, remains poorly explored. Here, we report the large-scale CAGE-Seq promoterome profiling of 75 human skeletal muscles, complemented by 22 matched proteomes obtained with mass spectrometry. We identified 37001 transcribed regulatory elements and 1804 protein groups encompassing 1895 proteins, 80% of which demonstrated non-uniform expression across different muscles. The skeletal muscles of the eye, tongue, and diaphragm had the most distinctive molecular phenotypes, while the overall diversity was driven by hundreds of transcription factors with tissue-specific activity. By analyzing the allelic imbalance of CAGE-Seq reads, we discovered 6653 allele-specific single-nucleotide variants often coinciding with muscle-related GWAS SNPs, including muscle volume. Finally, we provide an interactive online atlas of transcriptomic and proteomic molecular phenotypes, facilitating further studies of gene regulation and heritable pathologies of skeletal muscles.

The function of the highly conserved GTPase LepA, a homolog of elongation factor EF-G, remains unknown in translation. However, there is biochemical data that it implicates in the 30S ribosomal subunit biogenesis. Here, using cryo-electron microscopy, we characterized 30S subunits isolated from an Escherichia coli strain with a deleted lepA gene. The cryo-EM maps for ∆lepA 30S particles were divided into classes corresponding to consecutive assembly intermediates: from particles characterized by unformed helices h44/h45 of the central decoding center (CDR) and highly flexible head, through intermediates with a distorted CDR and a partial stabilization of the head, to near-mature 30S subunits with correctly docked h44 in the CDR, accessible 3′ end of 16S rRNA for translation but significant flexibility in head domain. Cryo-EM analysis of ΔlepA 30S intermediates revealed that they predominantly proceed to nearly mature functional state and exhibit suboptimal flexibility in the head domain. This finding suggests that LepA likely contributes to the final proper stabilization of the 3′ domain of the 30S subunit during ribosome assembly.

N ⁶ -methyladenosine (m ⁶ A) role in translation control and, particularly, in cap-independent initiation has attracted major attention. A common approach is to study the impact of m ⁶ A depending on its particular location in the transcripts, but the global impact of m ⁶ A along mRNAs remains unclear. Here, we combined ribosome profiling under conditions of mTOR inhibition and m ⁶ A mapping to identify distinct subsets of mRNAs that differ in their sensitivity to the suppression of cap-dependent initiation. The sensitivity was strongly correlated with the total m ⁶ A methylation of the transcripts. Further, we demonstrated that upon decreased mTOR activity, m ⁶ A methylation facilitated the enhanced association of mRNAs with components of the eIF4F complex. Thus, the efficiency of cap-independent translation initiation is primarily defined not by precise localization but by the total level of m ⁶ A methylation, and m ⁶ A has a compensatory role in maintaining translation when the canonical cap-dependent pathway is impaired. All in all, our findings underscore the significance of contemplating global m ⁶ A methylation status as a pivotal element in translational control, particularly under stress or signaling perturbations. GRAPHICAL

Inorganic polyphosphates and respective metabolic pathways and enzymes are important factors for yeast active growth in unfavorable conditions. However, particular proteins of polyphosphate metabolism remain poorly explored in this context. Here we report biochemical and transcriptomic characterization of the CRN/PPN2 yeast strain (derived from Ppn1-lacking CRN strain) overexpressing poorly studied Ppn2 polyphosphatase. We showed that Ppn2 overexpression significantly reduced lag phase in the alkaline medium presumably due to the ability of Ppn2 to efficiently hydrolyze inorganic polyphosphates and thus neutralize hydroxide ions in the cell. With RNA-Seq, we compared the molecular phenotypes of CRN/PPN2 and its parent CRN strain grown in YPD or alkaline medium and detected transcriptomic changes induced by Ppn2 overexpression and reflecting the adaptation to alkaline conditions. The core set of upregulated genes included several genes with a previously unknown function. Respective knockout strains (∆ecm8, ∆yol160w, ∆cpp3, ∆ycr099c) exhibited defects of growth or cell morphology in the alkaline medium, proving the functional involvement of the respective proteins in sustaining growth in alkaline conditions.

Y-box-binding proteins (YB proteins) are multifunctional DNA- and RNA-binding proteins that play an important role in the regulation of gene expression. The high homology of their cold shock domains and the similarity between their long, unstructured C-terminal domains suggest that Y-box-binding proteins may have similar functions in a cell. Here, we consider the functional interchangeability of the somatic YB proteins YB-1 and YB-3. RNA-seq and Ribo-seq are used to track changes in the mRNA abundance or mRNA translation in HEK293T cells solely expressing YB-1, YB-3, or neither of them. We show that YB proteins have a dual effect on translation. Although the expression of YB proteins stimulates global translation, YB-1 and YB-3 inhibit the translation of their direct CLIP-identified mRNA targets. The impact of YB-1 and YB-3 on the translation of their mRNA targets is similar, which suggests that they can substitute each other in inhibiting the translation of their mRNA targets in HEK293T cells.

YB proteins are DNA/RNA binding proteins, members of the family of proteins with cold shock domain. Role of YB proteins in the life of cells, tissues, and whole organisms is extremely important. They are involved in transcription regulation, pre-mRNA splicing, mRNA translation and stability, mRNA packaging into mRNPs, including stress granules, DNA repair, and many other cellular events. Many processes, from embryonic development to aging, depend on when and how much of these proteins have been synthesized. Here we discuss regulation of the levels of YB-1 and, in part, of its homologs in the cell. Because the amount of YB-1 is immediately associated with its functioning, understanding the mechanisms of regulation of the protein amount invariably reveals the events where YB-1 is involved. Control over the YB-1 abundance may allow using this gene/protein as a therapeutic target in cancers, where an increased expression of the YBX1 gene often correlates with the disease severity and poor prognosis.