Differential Regulation of Two Arms of mTORC1 Pathway Fine-Tunes Global Protein Synthesis in Resting B Lymphocytes.

Protein synthesis is tightly regulated by both gene-specific and global mechanisms to match the metabolic and proliferative demands of the cell. While the regulation of global protein synthesis in response to mitogen or stress signals is relatively well understood in multiple experimental systems, how different cell types fine-tune their basal protein synthesis rate is not known. In a previous study, we showed that resting B and T lymphocytes exhibit dramatic differences in their metabolic profile, with implications for their post-activation function. Here, we show that resting B cells, despite being quiescent, exhibit increased protein synthesis in vivo as well as ex vivo. The increased protein synthesis in B cells is driven by mTORC1, which exhibits an intermediate level of activation in these cells when compared with resting T cells and activated B cells. A comparative analysis of the transcriptome and translatome of these cells indicates that the genes encoding the MHC Class II molecules and their chaperone CD74 are highly translated in B cells. These data suggest that the translatome of B cells shows enrichment for genes associated with antigen processing and presentation. Even though the B cells exhibit higher mTORC1 levels, they prevent the translational activation of TOP mRNAs, which are mostly constituted by ribosomal proteins and other translation factors, by upregulating 4EBP1 levels. This mechanism may keep the protein synthesis machinery under check while enabling higher levels of translation in B cells.

Codon optimality has minimal effect on determining translation efficiency in mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is a slow-growing, intracellular pathogen that exhibits a high GC-rich genome. Several factors, including the GC content of the genome, influence the evolution of specific codon usage biases in genomes. As a result, the Mtb genome exhibits strong biases for amino acid usage and codon usage. Codon usage of mRNAs affects several aspects of translation, including accuracy, efficiency, and protein folding. Here we address the effect of codon usage biases in determining the translation efficiency of mRNAs in Mtb. Unlike most commonly studied organisms, Mtb carries a single copy of each tRNA gene. However, we show that the relative levels of tRNAs in the Mtb tRNA pool vary by an order of magnitude. Our results show that the codons decoded by the abundant tRNAs indeed show higher adaptability. Moreover, there is a general positive correlation between genomic codon usage and the tRNA adaptability of codons (TAc). We further estimated the optimality of the codon and mRNAs by considering both the TAc and the tRNA demand. These measures did not show any correlation with mRNA abundance and translation efficiency. There was no correlation between tRNA adaptability and ribosome pausing as well. Taken together, we conclude that the translation machinery, and the tRNA pool of an organism, co-evolve with the codon usage to optimize the translation efficiency of an organism. Thus the deleterious effect of maladapted codons is not pronounced.

Mycobacterium tuberculosis Transcription Factor EmbR Regulates the Expression of Key Virulence Factors That Aid in Ex Vivo and In Vivo Survival.

Mycobacterium tuberculosis encodes ~200 transcription factors that modulate gene expression under different microenvironments in the host. Even though high-throughput chromatin immunoprecipitation sequencing and transcriptome sequencing (RNA-seq) studies have identified the regulatory network for ~80% of transcription factors, many transcription factors remain uncharacterized. EmbR is one such transcription factor whose in vivo regulon and biological function are yet to be elucidated. Previous in vitro studies suggested that phosphorylation of EmbR by PknH upregulates the embCAB operon. Using a gene replacement mutant of embR, we investigated its role in modulating cellular morphology, antibiotic resistance, and survival in the host. Contrary to the prevailing hypothesis, under normal growth conditions, EmbR is neither phosphorylated nor impacted by ethambutol resistance through the regulation of the embCAB operon. The embR deletion mutant displayed attenuated M. tuberculosis survival in vivo. RNA-seq analysis suggested that EmbR regulates operons involved in the secretion pathway, lipid metabolism, virulence, and hypoxia, including well-known hypoxia-inducible genes devS and hspX. Lipidome analysis revealed that EmbR modulates levels of all lysophospholipids, several phospholipids, and M. tuberculosis-specific lipids, which is more pronounced under hypoxic conditions. We found that the EmbR mutant is hypersusceptible to hypoxic stress, and RNA sequencing performed under hypoxic conditions indicated that EmbR majorly regulates genes involved in response to acidic pH, hypoxia, and fatty acid metabolism. We observed condition-specific phosphorylation of EmbR, which contributes to EmbR-mediated transcription of several essential genes, ensuring enhanced survival. Collectively, the study establishes EmbR as a key modulator of hypoxic response that facilitates mycobacterial survival in the host. IMPORTANCE Mycobacterium tuberculosis modulates its transcriptional machinery in response to dynamic microenvironments encountered within the host. In this study, we identified that EmbR, a transcription factor, plays important roles in modulating cellular morphology, antibiotic resistance, and survival in the host. We found that EmbR undergoes condition-specific phosphorylation for its activation. Together, the study establishes a key role of EmbR as a transcriptional activator of genes belonging to multiple pathways, viz., virulence, secretion, or polyketide synthesis, that aid in mycobacterial survival during hypoxia and within the host.

Umbilical cord tissue is a robust source for mesenchymal stem cells with enhanced myogenic differentiation potential compared to cord blood.

Differentiation of mesenchymal stem cells (MSCs) derived from two different sources of fetal tissues such as umbilical cord blood (UCB) and tissue (UCT) into skeletal muscle have remained underexplored. Here, we present a comparative analysis of UCB and UCT MSCs, in terms of surface markers, proliferation and senescence marker expression. We find that CD45-CD34- MSCs obtained from UCT and UCB of term births display differences in the combinatorial expression of key MSC markers CD105 and CD90. Importantly, UCT MSCs display greater yield, higher purity, shorter culture time, and lower rates of senescence in culture compared to UCB MSCs. Using a robust myogenic differentiation protocol, we show that UCT MSCs differentiate more robustly into muscle than UCB MSCs by transcriptomic sequencing and specific myogenic markers. Functional assays reveal that CD90, and not CD105 expression promotes myogenic differentiation in MSCs and could explain the enhanced myogenic potential of UCT MSCs. These results suggest that in comparison to large volumes of UCB that are routinely used to obtain MSCs and with limited success, UCT is a more reliable, robust, and convenient source of MSCs to derive cells of the myogenic lineage for both therapeutic purposes and increasing our understanding of developmental processes.

Role of NF-kappaB2-p100 in regulatory T cell homeostasis and activation.

The immunological roles of the nuclear factor-kappaB (NF-κB) pathway are mediated via the canonical components in immune responses and via non-canonical components in immune organogenesis and homeostasis, although the two components are capable of crosstalk. Regulatory CD4 T cells (Tregs) are homeostatically functional and represent an interesting potential meeting point of these two NF-κB components. We show that mice deficient in the non-canonical NF-κB component gene Nfkb2 (p100) had normal thymic development and suppressive function of Tregs. However, they had enhanced frequencies of peripheral 'effector-phenotype' Tregs (eTregs). In bi-parental chimeras of wild-type (WT) and Nfkb2-/- mice, the Nfkb2-/- genotype was over-represented in Tregs, with a further increase in the relative prominence of eTregs. Consistent with distinct properties of eTregs, the Nfkb2-/- genotype was more prominent in Tregs in extra-lymphoid tissues such as liver in the bi-parental chimeras. The Nfkb2-/- Tregs also displayed greater survival, activation and proliferation in vivo. These Nfkb2-/- Tregs showed higher nuclear NF-κB activity mainly comprising of RelB-containing dimers, in contrast to the prominence of cRel- and RelA-containing dimers in WT Tregs. Since p100 is an inhibitor of RelB activation as well as a participant as cleaved p52 in RelB nuclear activity, we tested bi-parental chimeras of WT and Relb-/- mice, and found normal frequencies of Relb-/- Tregs and eTregs in these chimeric mice. Our findings confirm and extend recent data, and indicate that p100 normally restrains RelB-mediated Treg activation, and in the absence of p100, p50-RelB dimers can contribute to Treg activation.

Interactions between RNAP III transcription machinery and tRNA processing factors.

Eukaryotes have at least three nuclear RNA polymerases to carry out transcription. While RNA polymerases I and II are responsible for ribosomal RNA transcription and messenger RNA transcription, respectively, RNA Polymerase III transcribes approximately up to 300 nt long noncoding RNAs, including tRNA. For all three RNAPs, the nascent transcripts generated undergo extensive post-transcriptional processing. Transcription of mRNAs by RNAP II and their processing are coupled with the aid of the C-terminal domain of the RNAP II. RNAP I transcription and the processing of its transcripts are co-localized to the nucleolus and to some extent, rRNA processing occurs co-transcriptionally. Here, I review the current evidence for the interaction between tRNA processing factors and RNA polymerase III. These interactions include the moonlighting functions of tRNA processing factors in RNAP III transcription and the indirect effect of tRNA transcription levels on tRNA modification machinery.