Stem Cell Proliferation Is Kept in Check by the Chromatin Regulators Kismet/CHD7/CHD8 and Trr/MLL3/4.

Chromatin remodeling accompanies differentiation, however, its role in self-renewal is less well understood. We report that in Drosophila, the chromatin remodeler Kismet/CHD7/CHD8 limits intestinal stem cell (ISC) number and proliferation without affecting differentiation. Stem-cell-specific whole-genome profiling of Kismet revealed its enrichment at transcriptionally active regions bound by RNA polymerase II and Brahma, its recruitment to the transcription start site of activated genes and developmental enhancers and its depletion from regions bound by Polycomb, Histone H1, and heterochromatin Protein 1. We demonstrate that the Trithorax-related/MLL3/4 chromatin modifier regulates ISC proliferation, colocalizes extensively with Kismet throughout the ISC genome, and co-regulates genes in ISCs, including Cbl, a negative regulator of Epidermal Growth Factor Receptor (EGFR). Loss of kismet or trr leads to elevated levels of EGFR protein and signaling, thereby promoting ISC self-renewal. We propose that Kismet with Trr establishes a chromatin state that limits EGFR proliferative signaling, preventing tumor-like stem cell overgrowths

Ribosomal protein S1 influences trans-translation in vitro and in vivo

When the bacterial ribosome stalls on a truncated mRNA, transfer–messenger RNA (tmRNA) acts initially as a transfer RNA (tRNA) and then as a messenger RNA (mRNA) to rescue the ribosome and add a peptide tag to the nascent polypeptide that targets it for degradation. Ribosomal protein S1 binds tmRNA but its functional role in this process has remained elusive. In this report, we demonstrate that, in vitro, S1 is dispensable for the tRNA-like role of tmRNA but is essential for its mRNA function. Increasing or decreasing the amount of protein S1 in vivo reduces the overall amount of trans-translated proteins. Also, a truncated S1 protein impaired for ribosome binding can still trigger protein tagging, suggesting that S1 interacts with tmRNA outside the ribosome to keep it in an active state. Overall, these results demonstrate that S1 has a role in tmRNA-mediated tagging that is distinct from its role during canonical translation

Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism

Using an experimental approach, we investigated the RNome of the pathogen Staphylococcus aureus to identify 30 small RNAs (sRNAs) including 14 that are newly confirmed. Among the latter, 10 are encoded in intergenic regions, three are generated by premature transcription termination associated with riboswitch activities, and one is expressed from the complementary strand of a transposase gene. The expression of four sRNAs increases during the transition from exponential to stationary phase. We focused our study on RsaE, an sRNA that is highly conserved in the bacillales order and is deleterious when over-expressed. We show that RsaE interacts in vitro with the 5′ region of opp3A mRNA, encoding an ABC transporter component, to prevent formation of the ribosomal initiation complex. A previous report showed that RsaE targets opp3B which is co-transcribed with opp3A. Thus, our results identify an unusual case of riboregulation where the same sRNA controls an operon mRNA by targeting two of its cistrons. A combination of biocomputational and transcriptional analyses revealed a remarkably coordinated RsaE-dependent downregulation of numerous metabolic enzymes involved in the citrate cycle and the folate-dependent one-carbon metabolism. As we observed that RsaE accumulates transiently in late exponential growth, we propose that RsaE functions to ensure a coordinate downregulation of the central metabolism when carbon sources become scarce

Initiation de la traduction et autorégulation du gène rpsA chez Escherichia coli

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

The Highly Efficient Translation Initiation Region from the Escherichia coli rpsA Gene Lacks a Shine-Dalgarno Element

The translational initiation region (TIR) of the Escherichia coli rpsA gene, which encodes ribosomal protein S1, shows a number of unusual features. It extends far upstream (to position −91) of the initiator AUG, it lacks a canonical Shine-Dalgarno sequence (SD) element, and it can fold into three successive hairpins (I, II, and III) that are essential for high translational activity. Two conserved GGA trinucleotides, present in the loops of hairpins I and II, have been proposed to form a discontinuous SD. Here, we have tested this hypothesis with the “specialized ribosome” approach. Depending upon the constructs used, translation initiation was decreased three- to sevenfold upon changing the conserved GGA to CCU. However, although chemical probing showed that the mutated trinucleotides were accessible, no restoration was observed when the ribosome anti-SD was symmetrically changed from CCUCC to GGAGG. When the same change was introduced in the SD from a conventional TIR as a control, activity was stimulated. This result suggests that the GGA trinucleotides do not form a discontinuous SD. Others hypotheses that may account for their role are discussed. Curiously, we also find that, when expressed at moderate level (30 to 40% of total ribosomes), specialized ribosomes are only twofold disadvantaged over normal ribosomes for the translation of bulk cellular mRNAs. These findings suggest that, under these conditions, the SD-anti-SD interaction plays a significant but not essential role for the synthesis of bulk cellular proteins

The Deleterious Effect of an Insertion Sequence Removing the Last Twenty Percent of the Essential Escherichia coli rpsA Gene Is Due to mRNA Destabilization, Not Protein Truncation▿

Ribosomal protein S1, the product of the essential rpsA gene, consists of six imperfect repeats of the same motif. Besides playing a critical role in translation initiation on most mRNAs, S1 also specifically autoregulates the translation of its own messenger. ssyF29 is a viable rpsA allele that carries an IS10R insertion within the coding sequence, resulting in a protein lacking the last motif (S1ΔC). The growth of ssyF29 cells is slower than that of wild-type cells. Moreover, translation of a reporter rpsA-lacZ fusion is specifically stimulated, suggesting that the last motif is necessary for autoregulation. However, in ssyF29 cells the rpsA mRNA is also strongly destabilized; this destabilization, by causing S1ΔC shortage, might also explain the observed slow-growth and autoregulation defect. To fix this ambiguity, we have introduced an early stop codon in the rpsA chromosomal gene, resulting in the synthesis of the S1ΔC protein without an IS10R insertion (rpsAΔC allele). rpsAΔC cells grow much faster than their ssyF29 counterparts; moreover, in these cells S1 autoregulation and mRNA stability are normal. In vitro, the S1ΔC protein binds mRNAs (including its own) almost as avidly as wild-type S1. These results demonstrate that the last S1 motif is dispensable for translation and autoregulation: the defects seen with ssyF29 cells reflect an IS10R-mediated destabilization of the rpsA mRNA, probably due to facilitated exonucleolytic degradation

Quantification of the -translated proteins in cells expressing varied S1 concentration

<p><b>Copyright information:</b></p><p>Taken from "Ribosomal protein S1 influences -translation and "</p><p></p><p>Nucleic Acids Research 2007;35(7):2368-2376.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874662.</p><p>© 2007 The Author(s)</p> ( Strains depleted for S1 contain less his-tagged proteins. Lanes 1 and 2: control strain grown in the absence (−) or presence (+) of IPTG, respectively. Lanes 3 and 4: deleted strain () grown in the presence (+; complementation condition) or absence of IPTG (−; depletion condition), respectively. IPTG induces expression of the extra plasmid-borne copy present in the strains (section ‘Experimental procedures’). Western blot analysis was performed for samples of the cultures used to purify his-tagged protein. Blot was revealed with S1 antibodies mixed with PNPase antibodies to normalize quantification. The S1/PNPase ratio was determined by quantification of the S1 and PNPase signals on a chemi-smart 5000 (Vilbert-Lourmat). Doubling time of each strain is indicated. Amounts of purified his-tagged protein were measured by absorbance (section ‘Experimental procedures’). Percentage of his-tagged proteins was determined after normalization to the amount of total proteins extracted from each strain and strain (lane 1) was used as reference. ( Excess of free S1 affects the accumulation of -translated proteins. Up: schematic representation of the chromosomal fusion used as translational reporter. Western blot analysis was performed as described earlier. β-Galactosidase activities from translational fusion are given in β-galactosidase units corresponding to nanomoles of ONPG hydrolyzed per min and per mg of total protein. The values shown are averages of three independent assays. Presence of the pS1 plasmid strongly decreases β-galactosidase synthesis (20-fold) due to S1 autogenous control. Doubling time is given for both strains. Percentage of his-tagged protein was determined as in , and strain containing the control plasmid pAC, was used as reference