Bacterial xenogeneic silencing proteins selectively bind to and silence expression from many AT rich regions of the chromosome. They serve as master regulators of horizontally acquired DNA, including a large number of virulence genes. To date, three distinct families of xenogeneic silencers have been identified: H-NS of Proteobacteria, Lsr2 of the Actinomycetes, and MvaT of Pseudomonas sp. Although H-NS and Lsr2 family proteins are structurally different, they all recognize the AT-rich DNA minor groove through a common AT-hook-like motif, which is absent in the MvaT family. Thus, the DNA binding mechanism of MvaT has not been determined. Here, we report the characteristics of DNA sequences targeted by MvaT with protein binding microarrays, which indicates that MvaT prefers binding flexible DNA sequences with multiple TpA steps. We demonstrate that there are clear differences in sequence preferences between MvaT and the other two xenogeneic silencer families. We also determined the structure of the DNA-binding domain of MvaT in complex with a high affinity DNA dodecamer using solution NMR. This is the first experimental structure of a xenogeneic silencer in complex with DNA, which reveals that MvaT recognizes the AT-rich DNA both through base readout by an “AT-pincer” motif inserted into the minor groove and through shape readout by multiple lysine side chains interacting with the DNA sugar-phosphate backbone. Mutations of key MvaT residues for DNA binding confirm their importance with both in vitro and in vivo assays. This novel DNA binding mode enables MvaT to better tolerate GC-base pair interruptions in the binding site and less prefer A tract DNA when compared to H-NS and Lsr2. Comparison of MvaT with other bacterial xenogeneic silencers provides a clear picture that nature has evolved unique solutions for different bacterial genera to distinguish foreign from self DNA.
Bacterial xenogeneic silencers play important roles in bacterial evolution by recognizing and inhibiting expression from foreign genes acquired through horizontal gene transfer, thereby buffering against potential fitness consequences of their misregulated expression. Here, the detailed DNA binding properties of Rok, a xenogeneic silencer in Bacillus subtilis, was studied using protein binding microarray, and the solution structure of its C-terminal DNA binding domain was determined in complex with DNA. The C-terminal domain of Rok adopts a typical winged helix fold, with a novel DNA recognition mechanism different from other winged helix proteins or xenogeneic silencers. Rok binds the DNA minor groove by forming hydrogen bonds to bases through N154, T156 at the N-terminal of α3 helix and R174 of wing W1, assisted by four lysine residues interacting electrostatically with DNA backbone phosphate groups. These structural features endow Rok with preference towards DNA sequences harboring AACTA, TACTA, and flexible multiple TpA steps, while rigid A-tracts are disfavored. Correspondingly, the Bacillus genomes containing Rok are rich in A-tracts and show a dramatic underrepresentation of AACTA and TACTA, which are significantly enriched in Rok binding regions. These observations suggest that the xenogeneic silencing protein and its resident genome may have evolved cooperatively.
5-Methylcytosine (m5C) is a RNA modification that exists in tRNAs and rRNAs and was recently found in mRNAs. Although it has been suggested to regulate diverse biological functions, whether m5C RNA modification influences adult stem cell development remains undetermined. In this study, we show that Ypsilon schachtel (YPS), a homolog of human Y box binding protein 1 (YBX1), promotes germ line stem cell (GSC) maintenance, proliferation, and differentiation in the Drosophila ovary by preferentially binding to m5C-containing RNAs. YPS is genetically demonstrated to function intrinsically for GSC maintenance, proliferation, and progeny differentiation in the Drosophila ovary, and human YBX1 can functionally replace YPS to support normal GSC development. Highly conserved cold-shock domains (CSDs) of YPS and YBX1 preferentially bind to m5C RNA in vitro. Moreover, YPS also preferentially binds to m5C-containing RNAs, including mRNAs, in germ cells. The crystal structure of the YBX1 CSD-RNA complex reveals that both hydrophobic stacking and hydrogen bonds are critical for m5C binding. Overexpression of RNA-binding–defective YPS and YBX1 proteins disrupts GSC development. Taken together, our findings show that m5C RNA modification plays an important role in adult stem cell development.
Edited by Xiao-Fan WangAutophagy is typically a prosurvival cellular process that promotes the turnover of long-lived proteins and damaged organelles, but it can also induce cell death. We have previously reported that the small molecule Z36 induces autophagy along with autophagic cell death in HeLa cells. In this study, we analyzed differential gene expression in Z36-treated HeLa cells and found that Z36-induced endoplasmic reticulum-specific autophagy (ER-phagy) results in ER stress and the unfolded protein response (UPR). This result is in contrast to the common notion that autophagy is generally activated in response to ER stress and the UPR. We demonstrate that Z36 up-regulates the expression levels of FAM134B, LC3, and Atg9, which together mediate excessive ER-phagy, characterized by forming increased numbers of autophagosomes with larger sizes. We noted that the excessive ER-phagy accelerates ER degradation and impairs ER homeostasis and thereby triggers ER stress and the UPR as well as ER-phagy-dependent cell death. Interestingly, overexpression of FAM134B alone in HeLa cells is sufficient to impair ER homeostasis and cause ER stress and cell death. These findings suggest a mechanism involving FAM134B activity for ER-phagy to promote cell death.Autophagy is a highly conserved physiological process, playing important roles in development, differentiation, immune defense, suppression of tumorigenesis and the prevention of neuronal degeneration in multicellular organisms (1-5). It is characterized by the formation of double-membrane autophagosomes, which then fuse with lysosomes for the degradation of components inside. During starvation, autophagy is initiated nonselectively to degrade substrates and thus provide nutrients and energy for survival. Meanwhile, autophagy can function selectively to remove damaged organelles or aggregated proteins, as a quality control mechanism (6). A growing number of subcellular components are found to be cleared by selective autophagy; each is named after its specific target, such as mitochondria (mitophagy) (7), aggregated proteins (aggrephagy) This work was supported by Ministry of Science and Technology of ChinaGrants 2016YFA0501200 and 2012CB910703 (to B. X.) and National Natural Science Foundation of China Grant 91013011 (to B. X.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S5 and Sheets S1-S4. The RNA-Seq data have been deposited in NCBI Gene Expression Omnibus (GEO) under the accession number GSE130006.
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