Transcriptional Repressor NIR Functions in the Ribosome RNA Processing of Both 40S and 60S Subunits

BACKGROUND: NIR was identified as an inhibitor of histone acetyltransferase and it represses transcriptional activation of p53. NIR is predominantly localized in the nucleolus and known as Noc2p, which is involved in the maturation of the 60S ribosomal subunit. However, how NIR functions in the nucleolus remains undetermined. In the nucleolus, a 47S ribosomal RNA precursor (pre-rRNA) is transcribed and processed to produce 18S, 5.8S and 28S rRNAs. The 18S rRNA is incorporated into the 40S ribosomal subunit, whereas the 28S and 5.8S rRNAs are incorporated into the 60S subunit. U3 small nucleolar RNA (snoRNA) directs 18S rRNA processing and U8 snoRNA mediates processing of 28S and 5.8 S rRNAs. Functional disruption of nucleolus often causes p53 activation to inhibit cell proliferation. METHODOLOGY/PRINCIPAL FINDINGS: Western blotting showed that NIR is ubiquitously expressed in different human cell lines. Knock-down of NIR by siRNA led to inhibition of the 18S, 28S and 5.8S rRNAs evaluated by pulse-chase experiment. Pre-rRNA particles (pre-rRNPs) were fractionated from the nucleus by sucrose gradient centrifugation and analysis of the pre-RNPs components showed that NIR existed in the pre-RNPs of both the 60S and 40S subunits and co-fractionated with 32S and 12S pre-rRNAs in the 60S pre-rRNP. Protein-RNA binding experiments demonstrated that NIR is associated with the 32S pre-rRNA and U8 snoRNA. In addition, NIR bound U3 snoRNA. It is a novel finding that depletion of NIR did not affect p53 protein level but de-repressed acetylation of p53 and activated p21. CONCLUSIONS: We provide the first evidence for a transcriptional repressor to function in the rRNA biogenesis of both the 40S and 60S subunits. Our findings also suggested that a nucleolar protein may alternatively signal to p53 by affecting the p53 modification rather than affecting p53 protein level

Bathymetry predicted from vertical gravity gradient anomalies and ship soundings

In this paper, the admittance function between seafloor undulations and vertical gravity gradient anomalies was derived. Based on this admittance function, the bathymetry model of 1 minute resolution was predicted from vertical gravity gradient anomalies and ship soundings in the experimental area from the northwest Pacific. The accuracy of the model is evaluated using ship soundings and existing models, including ETOPOl, GEBCO, DTU10 and V15. 1 from SIO. The model's STD is 69.481m, comparable with V15. 1 which is generally believed to have the highest accuracy

The Protective Roles of ROS-Mediated Mitophagy on 125I Seeds Radiation Induced Cell Death in HCT116 Cells

For many unresectable carcinomas and locally recurrent cancers (LRC), 125I seeds brachytherapy is a feasible, effective, and safe treatment. Several studies have shown that 125I seeds radiation exerts anticancer activity by triggering DNA damage. However, recent evidence shows mitochondrial quality to be another crucial determinant of cell fate, with mitophagy playing a central role in this control mechanism. Herein, we found that 125I seeds irradiation injured mitochondria, leading to significantly elevated mitochondrial and intracellular ROS (reactive oxygen species) levels in HCT116 cells. The accumulation of mitochondrial ROS increased the expression of HIF-1α and its target genes BINP3 and NIX (BINP3L), which subsequently triggered mitophagy. Importantly, 125I seeds radiation induced mitophagy promoted cells survival and protected HCT116 cells from apoptosis. These results collectively indicated that 125I seeds radiation triggered mitophagy by upregulating the level of ROS to promote cellular homeostasis and survival. The present study uncovered the critical role of mitophagy in modulating the sensitivity of tumor cells to radiation therapy and suggested that chemotherapy targeting on mitophagy might improve the efficiency of 125I seeds radiation treatment, which might be of clinical significance in tumor therapy

Screening for Virulence-Related Genes via a Transposon Mutant Library of Streptococcus suis Serotype 2 Using a Galleria mellonella Larvae Infection Model

Streptococcus suis (S. suis) is a zoonotic bacterial pathogen causing lethal infections in pigs and humans. Identification of virulence-related genes (VRGs) is of great importance in understanding the pathobiology of a bacterial pathogen. To identify novel VRGs, a transposon (Tn) mutant library of S. suis strain SC19 was constructed in this study. The insertion sites of approximately 1700 mutants were identified by Tn-seq, which involved 417 different genes. A total of 32 attenuated strains were identified from the library by using a Galleria mellonella larvae infection model, and 30 novel VRGs were discovered, including transcription regulators, transporters, hypothetical proteins, etc. An isogenic deletion mutant of hxtR gene (ΔhxtR) and its complementary strain (CΔhxtR) were constructed, and their virulence was compared with the wild-type strain in G. mellonella larvae and mice, which showed that disruption of hxtR significantly attenuated the virulence. Moreover, the ΔhxtR strain displayed a reduced survival ability in whole blood, increased sensitivity to phagocytosis, increased chain length, and growth defect. Taken together, this study performed a high throughput screening for VRGs of S. suis using a G. mellonella larvae model and further characterized a novel critical virulence factor

NIR is expressed in different human cell lines and mainly localized to the nucleolus.

<p>A. Cytosolic and nuclear extracts were fractionized from multiple cancer cell lines as indicated at the top of blot. Same amount of protein was separated on SDS-PAGE, and transferred onto PVDF membranes. Blots were probed with anti-NIR. Fractionation was controlled by using nuclear marker protein topoisomerase I (Topo I) and a cytosolic marker protein RhoA. N represents nuclear extract and C represents cytoplasmic extract. B. Indirect immunofluorescence was performed with anti-NIR polyclonal antibody. NIR specific signal was recognized with FITC-conjugated goat anti-rabbit IgG. As a nucleolar protein, 1A6/DRIM was detected with anti-1A6/DRIM monoclonal antibody. 1A6/DRIM specific signal was recognized with TRITC-conjugated goat anti-mouse IgG. Nucleus was stained with DAPI. The image was obtained with confocal microscopy. C. Cytoplasmic, nucleoplasmic and nucleolar lysates were fractionized from U2OS and HeLa cells respectively. Same amount of protein from the above lysates was separated on SDS-PAGE, and transferred onto PVDF membranes. Blots were probed with anti-NIR. Rho A, lamin A/C and fibrillarin were used as controls for cytoplasmic, nucleoplasmic and nucleolar fractions respectively. Beta-actin was used as a loading control.</p

NIR was associated with U3 snoRNA <i>in vivo</i> and knock down of NIR didn't affect U3 snoRNA level.

<p>A. Immunoprecipitation was performed with anti-NIR antibody and whole cell lysates from U2OS cells. Proteins from NIR immunoprecipitates were separated on a SDS-PAGE and blotted onto a PVDF membrane. Blot was probed with anti-NIR antibody. Ten percent of the lysates were loaded as input control (upper panel). RNA was extracted from the above immunoprecipitation, resolved on a 7% polyacrylamide–8.3 M urea gel and blotted onto a nylon membrane. The blot was probed with biotin-labeled U3 snoRNA-specific probe or U1-specific RNA probe (lower panel). B. U2OS cells were transfected with NIR specific siRNA (siNIR) or a siRNA targeting luciferase (siLC) as a control. Seventy-two hours post transfection, total RNA was extracted. Equal amount RNA was resolved on a 7% polyacrylamide–8.3 M urea gel and transferred onto a nylon membrane. Blot was hybridized with U3 snoRNA specific probe.</p

Knockdown of 1A6/DRIM results in a decrease in 47S rRNA.

<p>A. Cartoon illustrating the location of the probe on the 47S rRNA used for Northern blotting to detect 47S rRNA transcript. B. A 1A6/DRIM specific siRNA (si2-8) was transfected into U2OS cells. RNA was extracted 72 hours posttransfection, resolved on a 1% glyoxal-agarose gel and transferred onto a positively charged nylon membrane. Blots were probed with a Biotin-labeled DNA oligonucleotide (as shown in A) to detect 47S rRNA (left upper panel). Ethidium Bromide (EB) staining of the 28S rRNA on the agarose gel was used as a loading control. Cell lysates were prepared 72 hours after siRNA transfection and proteins from the lysates were separated on SDS-PAGE and transferred onto a PVDF membrane. Blots were probed with anti-1A6/DRIM antibody. Topoisomerase I (Topo I) was used as a loading control (left lower). The experiment was repeated three times and the difference in the densitometry scanning of the 47S rRNA bands is displayed in the right panel. C. A 1A6/DRIM specific siRNA (si2-8) was transfected into U2OS cells. RNA was extracted 72 hours posttransfection and reverse transcription was carried out. Real time PCR was performed with primers amplifying the 5′-extreme end fragment of the 47S rRNA. The experiment was repeated three times in duplicate and the statistical significance of the difference is shown. Statistical analyses were performed with two-tailed unpaired <i>t</i> test.</p

NIR is present in both of the 40S pre-rRNP and 60S pre-rRNP and co-sediment with 32S and 12S rRNA precursors in the nucleolus.

<p>A. Nuclear extracts were prepared from U2OS cells and fractionized on 10% to 40% sucrose density gradient. The absorbance at 254 nm (A₂₅₄) of each fraction was profiled and the position of pre-ribosomal subunits was indicated. B. Proteins from fractions described in A were separated on a SDS-PAGE and subjected to immunoblotting analysis using anti-NIR antibody. Fibrillarin was probed as a control. C. RNA from each fraction was resolved on a 1% agarose-glyoxal gel and transferred onto nylon membrane after stained with EB (lower panel). Blot was probed with biotin-labeled ITS-2 oligonucleotide (upper panel). In, un-fractionized nuclear extract.</p