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C Chiaruttini

Publications and source records attributed to C Chiaruttini.

12 recordsLinked to original sources
PubMed2001

Gas6 induces growth, beta-catenin stabilization, and T-cell factor transcriptional activation in contact-inhibited C57 mammary cells.

Gas6 is a growth factor related to protein S that was identified as the ligand for the Axl receptor tyrosine kinase (RTK) family. In this study, we show that Gas6 induces a growth response in a cultured mammalian mammary cell line, C57MG. The presence of Gas6 in the medium induces growth after confluence and similarly causes cell cycle reentry of density-inhibited C57MG cells. We show that Axl RTK but not Rse is efficiently activated by Gas6 in density-inhibited C57MG cells. We have analyzed the signaling required for the Gas6 proliferative effect and found a requirement for PI3K-, S6K-, and Ras-activated pathways. We also demonstrate that Gas6 activates Akt and concomitantly inhibits GSK3 activity in a wortmannin-dependent manner. Interestingly, Gas6 induces up-regulation of cytosolic beta-catenin, while membrane-associated beta-catenin remains unaffected. Stabilization of beta-catenin in C57MG cells is correlated with activation of a T-cell factor (TCF)-responsive transcriptional element. We thus provide evidence that Gas6 is mitogenic and induces beta-catenin proto-oncogene stabilization and subsequent TCF/Lef transcriptional activation in a mammary system. These results suggest that Gas6-Axl interaction, through stabilization of beta-catenin, may have a role in mammary development and/or be involved in the progression of mammary tumors.

3T3 Cells↗
PubMed1997

Translational coupling by modulation of feedback repression in the IF3 operon of Escherichia coli.

A pseudoknot formed by a long-range interaction in the mRNA of the initiation factor 3 (IF3) operon is involved in the translational repression of the gene encoding ribosomal protein L35 by another ribosomal protein, L20. The nucleotides forming the 5' strand of the key stem of the pseudoknot are located within the gene for IF3, whereas those forming the 3' strand are located 280 nt downstream, immediately upstream of the Shine-Dalgarno sequence of the gene for L35. Here we show that premature termination of IF3 translation at a nonsense codon introduced upstream of the pseudoknot results in a substantial enhancement of L20-mediated repression of L35 expression. Conversely, an increase of IF3 translation decreases repression. These results, in addition to an analysis of the effect of mutations in sequences forming the pseudoknot, indicate that IF3 translation decreases L20-mediated repression of L35 expression. We propose that ribosomes translating IF3 disrupt the pseudoknot and thereby attenuate repression. The result is a novel type of translational coupling, where unfolding of the pseudoknot by ribosomes translating IF3 does not increase expression of L35 directly, but alleviates its repression by L20.

Bacterial Proteins↗
PubMed1996

A long-range RNA-RNA interaction forms a pseudoknot required for translational control of the IF3-L35-L20 ribosomal protein operon in Escherichia coli.

In the IF3-L35-L20 operon encoding translation initiation factor 3 (IF3) and the two ribosomal proteins L35 and L20, the expression of the genes that code for the two ribosomal proteins is negatively regulated at the translational level by the cellular concentration of L20. This translational repressor directly regulates the expression of the gene encoding L35 and, via translational coupling, that of its own gene. Mutations that affect the control of the L35 gene were found exclusively at two sites: the first is located approximately 300 nucleotides upstream, and the second immediately 5' of the translation initiation site of the L35 gene. Mutations that fall between these two sites have little or no effect on the control, and the lack of effect of a deletion in the intervening region confirms this finding. RNA structure mapping in vitro suggests that the first site pairs with the second. We show that this pairing is also likely to occur in vivo because single mutations in either of these sites affect control, but base pair compensatory mutations re-establish control. We propose that these two distant sites can base-pair to form a long-range pseudoknot which is required for the control of the expression of the L35 gene.

Bacterial Proteins↗
PubMed1996

Translational coupling in the Escherichia coli operon encoding translation initiation factor IF3 and ribosomal proteins L20 and L35.

The Escherichia coli IF3-L35-L20 operon encodes translation initiation factor IF3 and the ribosomal proteins L35 and L20, respectively. The expression of the genes encoding the two ribosomal proteins is negatively regulated at the translational level by L20, which acts at an operator located within the IF3 gene and just upstream of the L35 gene. We have previously shown that L20 directly represses the expression of the L35 gene, and indirectly that of the L20 gene, via translational coupling. On the basis of mutational analysis and in vitro RNA structure probing experiments, we proposed that a large secondary structure in which the translation initiation site of the L20 gene is sequestered by base-pairing, is responsible for coupling. The ribosome binding site of the L20 gene becomes available when the secondary structure is melted by ribosomes translating the L35 mRNA. Here we describe that this secondary structure forms in vivo by showing that single mutations in either strand reduce coupling and that compensatory mutations that re-establish pairing also re-establish coupling. In vitro 'toeprinting' analysis enabled us to show that the wild-type inhibitory secondary structure directly blocks ribosome binding to the ribosome binding site of rpIT.

Bacterial Proteins↗
PubMed1996

The role of the AUU initiation codon in the negative feedback regulation of the gene for translation initiation factor IF3 in Escherichia coli.

The expression of the infC gene encoding translation initiation factor IF3 is negatively autoregulated at the level of translation, i.e. the expression of the gene is derepressed in a mutant infC background where the IF3 activity is lower than that of the wild type. The special initiation codon of infC, AUU, has previously been shown to be essential for derepression in vivo. In the present work, we provide evidence that the AUU initiation codon causes derepression by itself, because if the initiation codon of the thrS gene, encoding threonyl-tRNA synthetase, is changed from AUG to AUU, its expression is also derepressed in an infC mutant background. The same result was obtained with the rpsO gene encoding ribosomal protein S15. We also show that derepression of infC, thrS, and rpsO is obtained with other 'abnormal' initiation codons such as AUA, AUC, and CUG which initiate with the same low efficiency as AUU, and also with ACG which initiates with an even lower efficiency. Under conditions of IF3 excess, the expression of infC is repressed in the presence of the AUU or other 'abnormal' initiation codons. Under the same conditions and with the same set of 'abnormal' initiation codons, the repression of thrS and rpsO expression is weaker. This result suggests that the infC message has specific features that render its expression particularly sensitive to excess of IF3. We also studied another peculiarity of the infC message, namely the role of a GC-rich sequence located immediately downstream of the initiation codon and conserved through evolution. This sequence was proposed to interact with a conserved region in 16S RNA and enhance translation initiation. Unexpectedly, mutating this GC-rich sequence increases infC expression, indicating that this sequence has no enhancing role. Chemical and enzymatic probing of infC RNA synthesized in vitro indicates that this GC-rich sequence might pair with another region of the mRNA. On the basis of our in vivo results we propose, as suspected from earlier in vitro results, that IF3 regulates the expression of its own gene by using its ability to differentiate between 'normal' and 'abnormal' initiation codons.

Bacterial Proteins↗
PubMed1993

Gene organization, primary structure and RNA processing analysis of a ribosomal RNA operon in Lactococcus lactis.

Southern blot analysis of genomic DNA of the mesophilic lactic bacterium Lactococcus lactis subsp. lactis strain IL1403, illuminated six rRNA gene clusters. Each cluster contains one copy each of three rRNA genes, displaying the typical eubacterial organization of physically linked 16 S, 23 S and 5 S rRNA genes. Five of the six rRNA clusters were cloned into plasmid pBR322. One recombinant plasmid, pSLCM6, containing a 6500 base-pair genomic DNA fragment, was characterized by physical mapping and the sequences encoding rRNAs and tRNAs were localized by Southern hybridization. This fragment contains a single operon composed of one promoter, a leader sequence, a 16 S rRNA gene, a tRNA(Ala) gene, a 23 S rRNA gene, a 5 S rRNA gene and a tRNA(Asn) gene. S1 nuclease mapping and primer extension analysis of in vivo transcripts localized one transcriptional initiation site 150 base-pairs upstream from the start of the 16 S rRNA gene. These procedures also suggest that this transcript is processed by an RNAse III-like activity similar to Bacillus subtilis; i.e. the L. lactis nuclease might be sequence-specific. The chronology of specific cleavages occurring during the maturation process of the precursor transcript is described. One interesting observation is that the regions flanking the 16 S and 23 S rRNAs containing the primary processing sites are identical and contain sequences that could be involved in transcriptional antitermination. S1 mapping of the 3' ends of in vivo transcripts indicate that a terminator-like sequence a few base-pairs downstream from the distal tRNA(Asn) gene is inefficient in arresting transcription.

Base Sequence↗
PubMed1992

Messenger RNA secondary structure and translational coupling in the Escherichia coli operon encoding translation initiation factor IF3 and the ribosomal proteins, L35 and L20.

The Escherichia coli infC-rpmI-rplT operon encodes translation initiation factor IF3 and the ribosomal proteins, L35 and L20, respectively. The expression of the last cistron (rplT) has been shown to be negatively regulated at a post-transcriptional level by its own product, L20, which acts at an internal operator located within infC. The present work shows that L20 directly represses the expression of rpmI, and indirectly that of rplT, via translational coupling with rpmI. Deletions and an inversion of the coding region of rpmI, suggest an mRNA secondary structure forming between sequences within rpmI and the translation initiation site of rplT. To verify the existence of this structure, detailed analyses were performed using chemical and enzymatic probes. Also, mutants that uncoupled rplT expression from that of rpmI, were isolated. The mutations fall at positions that would base-pair in the secondary structure. Our model is that L20 binds to its operator within infC and represses the translation of rpmI. When the rpmI mRNA is not translated, it can base-pair with the ribosomal binding site of rplT, sequestering it, and abolishing rplT expression. If the rpmI mRNA is translated, i.e. covered by ribosomes, the inhibitory structure cannot form leaving the translation initiation site of rplT free for ribosomal binding and for full expression. Although translational coupling in ribosomal protein operons has been suspected to be due to the formation of secondary structures that sequester internal ribosomal binding sites, this is the first time that such a structure has been shown to exist.

Bacterial Proteins↗
PubMed1989

Crosslinking of ribosomal proteins S4, S5, S7, S8, S11, S12 and S18 to domains 1 and 2 of 16S rRNA in the Escherichia coli 30S particle.

RNA-protein crosslinks were introduced into Escherichia coli 30S ribosomal subunits by treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC). Complexes of 16S rRNA cross-linked to 30S ribosomal proteins were isolated and hybridized with a series of single-stranded bacteriophage M13-rDNA probes. These probes, each carrying an inserted rDNA fragment, were used to select contiguous 16S rRNA sections covering all of domain 1 and the major part of domain 2 (starting at the 5'-P terminus and ending at nucleotide 869) and the proteins covalently linked to each of these sections were identified by 2-dimensional polyacrylamide gel electrophoresis. This procedure identified proteins S4, S5, S7, S8, S11, S12, and S18 as the species most efficiently crosslinked to domains 1 and 2 of 16S rRNA. These results are discussed in the light of current knowledge of the tertiary structure of 16S rRNA in the E. coli 30S ribosomal subunit.

Cross-Linking Reagents↗
PubMed1986

Multiple crosslinks of proteins S7 and S9 to domains 3 and 4 of 16S ribosomal RNA in the Escherichia coli 30S particle.

RNA-protein cross-links were introduced into Escherichia coli 30S subunits by treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. 16S rRNA, cross-linked to 30S ribosomal proteins, was isolated and hybridized with seven single-stranded bacteriophage M13-DNA probes. These probes, each carrying an inserted rDNA fragment, were used to select contiguous RNA sections covering domains 3 and 4 (starting at nucleotide 868 and ending at the 3'OH terminus) of the 16S rRNA. The proteins covalently linked to each selected RNA section were identified by two-dimensional polyacrylamide gel electrophoresis. Proteins S7 and S9 were shown to be efficiently cross-linked to multiple sites belonging to both domains.

Bacterial Proteins↗
PubMed1982

Protein-RNA crosslinking in Escherichia coli 30S ribosomal subunits. Identification of a 16S rRNA fragment crosslinked to protein S12 by the use of the chemical crosslinking reagent 1-ethyl-3-dimethyl-aminopropylcarbodiimide.

1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC) was used to cross-link 30S ribosomal proteins to 16S rRNA within the E. coli 3OS ribosomal subunit. Covalently linked complexes containing 30S proteins and 16S rRNA, isolated by sedimentation of dissociated crosslinked 30S subunits through SDS containing sucrose gradients, were digested with RNase T1, and the resulting oligonucleotide-protein complexes were fractionated on SDS containing polyacrylamide gels. Eluted complexes containing 30S proteins S9 and S12 linked to oligonucleotides were obtained in pure form. Oligonucleotide 5'terminal labelling was successful in the case of S12 containing but not of the S9 containing complex and led to identification of the S12 bound oligonucleotide as CAACUCG which is located at positions 1316-1322 in the 16S rRNA sequence. Protein S12 is crosslinked to the terminal G of this heptanucleotide.

Bacterial Proteins↗