Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

SUMMARYThe use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes but, strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the ‘alternative’ bacterial proteome. The internal start sites my also play regulatory roles in gene expression.

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Mutagenesis of the Rns regulator of enterotoxigenic Escherichia coli reveals roles for a linker sequence and two helix–turn–helix motifs

Microbiology ◽

10.1099/mic.0.038521-0 ◽

2010 ◽

Vol 156 (9) ◽

pp. 2796-2806 ◽

Cited By ~ 8

Author(s):

Vivienne Mahon ◽

Cyril J. Smyth ◽

Stephen G. J. Smith

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Protein A ◽

Diarrhoeal Disease ◽

Enterotoxigenic Escherichia Coli ◽

Insertion Mutagenesis

The pathogenesis of diarrhoeal disease due to human enterotoxigenic Escherichia coli absolutely requires the expression of fimbriae. The expression of CS1 fimbriae is positively regulated by the AraC-like protein Rns. AraC-like proteins are DNA-binding proteins that typically contain two helix–turn–helix (HTH) motifs. A program of pentapeptide insertion mutagenesis of the Rns protein was performed, and this revealed that both HTH motifs are required by Rns to positively regulate CS1 fimbrial gene expression. Intriguingly, a pentapeptide insertion after amino acid C102 reduced the ability of Rns to transactivate CS1 fimbrial expression. The structure of Rns in this vicinity (NACRS) was predicted to be disordered and thus might act as a flexible linker. This hypothesis was confirmed by deletion of this amino acid sequence from the Rns protein; a truncated protein that lacked this sequence was no longer functional. Strikingly, this sequence could be functionally substituted in vivo and in vitro by a flexible seven amino acid sequence from another E. coli AraC-like protein RhaS. Our data indicate that HTH motifs and a flexible sequence are required by Rns for maximal activation of fimbrial gene expression.

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Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1520560113 ◽

2016 ◽

Vol 113 (7) ◽

pp. E829-E838 ◽

Cited By ~ 20

Author(s):

Yuhei Chadani ◽

Tatsuya Niwa ◽

Shinobu Chiba ◽

Hideki Taguchi ◽

Koreaki Ito

Keyword(s):

Escherichia Coli ◽

Membrane Proteins ◽

Ribosome Profiling ◽

Cytosolic Proteins ◽

Nascent Polypeptide ◽

Nascent Chain ◽

Chemical Trait

Although the importance of the nonuniform progression of elongation in translation is well recognized, there have been few attempts to explore this process by directly profiling nascent polypeptides, the relevant intermediates of translation. Such approaches will be essential to complement other approaches, including ribosome profiling, which is extremely powerful but indirect with respect to the actual translation processes. Here, we use the nascent polypeptide's chemical trait of having a covalently attached tRNA moiety to detect translation intermediates. In a case study,Escherichia coliSecA was shown to undergo nascent polypeptide-dependent translational pauses. We then carried out integrated in vivo and in vitro nascent chain profiling (iNP) to characterize 1,038 proteome members ofE.colithat were encoded by the first quarter of the chromosome with respect to their propensities to accumulate polypeptidyl–tRNA intermediates. A majority of them indeed undergo single or multiple pauses, some occurring only in vitro, some occurring only in vivo, and some occurring both in vivo and in vitro. Thus, translational pausing can be intrinsically robust, subject to in vivo alleviation, or require in vivo reinforcement. Cytosolic and membrane proteins tend to experience different classes of pauses; membrane proteins often pause multiple times in vivo. We also note that the solubility of cytosolic proteins correlates with certain categories of pausing. Translational pausing is widespread and diverse in nature.

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ASXL1 Mutations Promote Myeloid Transformation Through Inhibition of PRC2-Mediated Gene Repression

Blood ◽

10.1182/blood.v118.21.405.405 ◽

2011 ◽

Vol 118 (21) ◽

pp. 405-405 ◽

Cited By ~ 2

Author(s):

Omar Abdel-Wahab ◽

Mazhar Adli ◽

Lindsay Saunders ◽

Jie Gao ◽

Alan H. Shih ◽

...

Keyword(s):

Gene Expression ◽

Research Funding ◽

Hematopoietic Cells ◽

Loss Of Function ◽

Genome Wide ◽

Hoxa Gene Expression ◽

Primary Leukemia ◽

Hoxa Gene

Abstract Abstract 405 Somatic mutations in ASXL1 have been identified in patients with myeloid malignancies and are associated with worsened overall survival in AML and MDS patients. However the mechanisms of myeloid transformation of ASXL1 mutations had not been delineated. We therefore performed extensive in vitro and in vivo studies to assess the functional implications of ASXL1 mutations in the hematopoietic compartment. Transcriptional and Western blot analysis demonstrated loss of ASXL1 protein in primary leukemia samples with endogenous ASXL1 mutations indicating that these mutations are loss-of-function disease alleles. Further, ASXL1 depletion by shRNA in normal and malignant hematopoietic cells leads to robust upregulation of a set of genes including the posterior HOXA cluster (HoxA5-HoxA13). Increased HoxA gene expression was confirmed in human hematopoietic stem progenitor cells targeted with ASXL1 siRNA and in mice with conditional deletion of Asxl1 in the hematopoietic compartment. Previous studies in Drosophila had revealed that Asxl forms the polycomb-repressive deubiquitinase (PR-DUB) complex with BAP1, which normally opposes the function of polycomb repressive complex 1 (PRC1) by removing H2AK119 ubiquitination. We verified that wild-type, but not mutant ASXL1 associates with BAP1 in co-immunoprecipitation studies. However, BAP1 depletion in hematopoietic cells did not result in significant changes in HoxA gene expression, suggesting that ASXL1 regulates gene expression in hematopoietic cells independent of its role in the PR-DUB complex. We therefore performed CHIP sequencing for known activating and repressive chromatin marks and histone mass spectrometry to elucidate the genome-wide effects of ASXL1 loss on chromatin state in hematopoietic cells. This allowed us to show that ASXL1 loss resulted in genome-wide loss of the transcriptionally repressive mark H3K27me3 in hematopoietic cells and primary patient samples with ASXL1 mutations. These data were supported by western blot analysis and histone mass spectrometry demonstrating a significant loss of H3K27 trimethylation in ASXL1-mutant cells. Moreover, ASXL1 mutations in primary leukemia samples are characterized by loss of H3K27 trimethylation at the HoxA locus. These data led us to hypothesize that ASXL1 interacts with the PRC2 complex; co-immunoprecipitation studies revealed that ASXL1 associates with members of the PRC2 complex including EZH2 and SUZ12 but not with the PRC1 repressive complex. Importantly, ASXL1 downregulation resulted in loss of EZH2 recruitment to the HOXA locus indicating a role of ASXL1 in recruiting the PRC2 complex to known leukemogenic loci. We next assessed the effects of ASXL1 loss in vivo by generating a conditional knock-out model of ASXL1 and also by employing shRNA to deplete ASXL1 in hematopoietic cells expressing the NRASG12D oncogene. Consonant with the in vitro data, we observed HOXA9 overexpression with ASXL1 loss/depletion in vivo. Preliminary analysis reveals that conditional, hematopoietic specific ASXL1-knockout (ASXL1fl/fl Vav-Cre) mice are characterized by progressive expansion of LSK and myeloid progenitor cells in mice less than 6 months of age. After 6 months of age a significant proportion of ASXL1fl/fl Vav-Cre mice developed leukocytosis, anemia, thrombocytopenia, and splenomegaly; pathologic analysis of tissues revealed a phenotype consistent with myelodysplasia with myeloproliferative features. Moreover, loss of ASXL1 in cooperation with expression of NRasG12D resulted in impaired survival, increased myeloproliferation, and progressive anemia consistent with MPN/MDS in vivo. Taken together, these results reveal that ASXL1 mutations result in a loss-of-function and suggest a specific role for ASXL1 in epigenetic regulation of gene expression by facilitating PRC2-mediated transcriptional repression of known leukemic oncogenes. Moreover, our in vivo data validate the importance of ASXL1 mutations in the pathogenesis of myeloid malignancies and provide insight into how mutations that inhibit PRC2 function contribute to myeloid transformation through epigenetic dysregulation of specific target genes. Disclosures: Carroll: Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.

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Ribosomal stalling landscapes revealed by high-throughput inverse toeprinting of mRNA libraries

Life Science Alliance ◽

10.26508/lsa.201800148 ◽

2018 ◽

Vol 1 (5) ◽

pp. e201800148 ◽

Cited By ~ 3

Author(s):

Britta Seip ◽

Guénaël Sacheau ◽

Denis Dupuy ◽

C Axel Innis

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

High Throughput ◽

Quantitative Measure ◽

Ribosome Profiling ◽

Coding Region ◽

Versatile Tool

Although it is known that the amino acid sequence of a nascent polypeptide can impact its rate of translation, dedicated tools to systematically investigate this process are lacking. Here, we present high-throughput inverse toeprinting, a method to identify peptide-encoding transcripts that induce ribosomal stalling in vitro. Unlike ribosome profiling, inverse toeprinting protects the entire coding region upstream of a stalled ribosome, making it possible to work with random or focused transcript libraries that efficiently sample the sequence space. We used inverse toeprinting to characterize the stalling landscapes of free and drug-boundEscherichia coliribosomes, obtaining a comprehensive list of arrest motifs that were validated in vivo, along with a quantitative measure of their pause strength. Thanks to the modest sequencing depth and small amounts of material required, inverse toeprinting provides a highly scalable and versatile tool to study sequence-dependent translational processes.

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To New Beginnings: Riboproteogenomics Discovery of N-Terminal Proteoforms in Arabidopsis Thaliana

Frontiers in Plant Science ◽

10.3389/fpls.2021.778804 ◽

2022 ◽

Vol 12 ◽

Author(s):

Patrick Willems ◽

Elvis Ndah ◽

Veronique Jonckheere ◽

Frank Van Breusegem ◽

Petra Van Damme

Keyword(s):

Arabidopsis Thaliana ◽

Translation Initiation ◽

Single Gene ◽

Ribosome Profiling ◽

Common Mechanism ◽

Alternative Translation Initiation ◽

Clear Cut ◽

Alternative Translation ◽

New Beginnings ◽

Chloroplast Stroma

Alternative translation initiation is a widespread event in biology that can shape multiple protein forms or proteoforms from a single gene. However, the respective contribution of alternative translation to protein complexity remains largely enigmatic. By complementary ribosome profiling and N-terminal proteomics (i.e., riboproteogenomics), we provide clear-cut evidence for ~90 N-terminal proteoform pairs shaped by (alternative) translation initiation in Arabidopsis thaliana. Next to several cases additionally confirmed by directed mutagenesis, identified alternative protein N-termini follow the enzymatic rules of co-translational N-terminal protein acetylation and initiator methionine removal. In contrast to other eukaryotic models, N-terminal acetylation in plants cannot generally be considered as a proxy of translation initiation because of its posttranslational occurrence on mature proteolytic neo-termini (N-termini) localized in the chloroplast stroma. Quantification of N-terminal acetylation revealed differing co- vs. posttranslational N-terminal acetylation patterns. Intriguingly, our data additionally hints to alternative translation initiation serving as a common mechanism to supply protein copies in multiple cellular compartments, as alternative translation sites are often in close proximity to cleavage sites of N-terminal transit sequences of nuclear-encoded chloroplastic and mitochondrial proteins. Overall, riboproteogenomics screening enables the identification of (differential localized) N-terminal proteoforms raised upon alternative translation.

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199 IN VITRO-DEVELOPED BOVINE BLASTOCYSTS ARE MARKED WITH ABERRANT HYPER- AND HYPO-METHYLATED GENOMIC REGIONS

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab199 ◽

2015 ◽

Vol 27 (1) ◽

pp. 190

Author(s):

D. Salilew-Wondim ◽

M. Hoelker ◽

U. Besenfelder ◽

V. Havlicek ◽

F. Rings ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Expression Patterns ◽

Proximal Promoter ◽

Altered Gene Expression ◽

Genome Wide ◽

Altered Gene ◽

Genomic Regions

Most often, in vitro produced embryos display poor quality and altered gene expression patterns compared to their in vivo counterparts. Aberrant DNA methylation occurring during in vitro embryo development is believed to be one of the multifaceted factors which may cause altered gene expression and poor embryo quality. Here, we investigated the genome-wide DNA methylation patterns of in vitro derived embryos using the recently developed Bovine EmbryoGENE Methylation Platform (BEGMP) array (Shojaei Saadi et al. BMC Genomics 2014 15, 451. doi: 10.1186/1471-2164-15-451) to unravel the aberrantly methylated genomic region in in vitro developed embryos. For this, in vitro and in vivo produced blastocysts were produced and used for genome-wide DNA methylation analysis. In vitro blastocysts were produced from oocytes retrieved from ovaries collected from the local abattoir and matured, fertilized, and cultured in vitro using SOF media. The in vivo blastocysts were produced by superovulation and AI of Simmental heifers followed by uterine flushing. Genomic DNA (gDNA) was then isolated from four replicates (each 10 blastocysts) of in vivo and in vitro derived blastocysts using Allprep DNA/RNA micro kit (Qiagen, Valencia, CA, USA) and the gDNA was then fragmented using the MseI enzyme. Following this, MseLig21 and MseLig were ligated to the MseI-digested genomic fragments in the presence of Ligase enzyme. Methyl-sensitive enzymes, HpaII, AciI, and Hinp1I, were used to cleave unmethlayted genomic regions within the MseI-MseI region of the fragmented DNA. The gDNA was subjected to two rounds of ligation-mediated polymerase chain reaction (LM-PCR) amplification. After removal of the adapters, the amplified gDNA samples from in vivo or in vitro groups were labelled either Cy-3 or Cy-5 dyes in dye-swap design using ULS Fluorescent gDNA labelling kit (Kreatech Biotechnology BV, Amsterdam, The Netherlands). Hybridization was performed for 40 h at 65°C. Slides were scanned using Agilent's High-Resolution C Scanner (Agilent Technologies Inc., Santa Clara, CA, USA) and features were extracted with Agilent's Feature Extraction software (Agilent Technologies Inc.). The results have shown that from a total of 414 566 probes harboured by the BEGMP array, 248 453 and 253 147 probes were detected in in vitro and in vivo derived blastocysts, respectively. Data analysis using the linear modelling for microarray (LIMMA) package and R software (The R Project for Statistical Computing, Vienna, Austria) revealed a total of 3434 differentially methylated regions (DMRs; Fold change ≥1.5, P-value <0.05), of which 42 and 58% were hyper- and hypo-methylated, respectively, in in vitro derived blastocysts compared to their in vivo counterparts. The DMRs were found to be localised in the intronic, exonic, promoter, proximal promoter, and distal promoter, and some of the probes did not have nearby genes. In addition, 10.8% of the DMRs were found to be stretched in short, long, or intermediate CpG islands. Thus, this study demonstrated genome-wide dysregulation in the epigenome landscape of in vitro-derived embryos by the time they reach to the blastocysts stage.

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Dual Function of Aar, a Member of the New AraC Negative Regulator Family, in Escherichia coli Gene Expression

Infection and Immunity ◽

10.1128/iai.00100-20 ◽

2020 ◽

Vol 88 (6) ◽

Cited By ~ 2

Author(s):

Abigail S. Mickey ◽

James P. Nataro

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Negative Regulator ◽

Dual Function ◽

Content Type ◽

Virulence Gene Expression ◽

E Coli ◽

K 12

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is an E. coli pathotype associated with diarrhea and growth faltering. EAEC virulence gene expression is controlled by the autoactivated AraC family transcriptional regulator, AggR. AggR activates transcription of a large number of virulence genes, including Aar, which in turn acts as a negative regulator of AggR itself. Aar has also been shown to affect expression of E. coli housekeeping genes, including H-NS, a global regulator that acts at multiple promoters and silences AT-rich genes (such as those in the AggR regulon). Although Aar has been shown to bind both AggR and H-NS in vitro, functional significance of these interactions has not been shown in vivo. In order to dissect this regulatory network, we removed the complex interdependence of aggR and aar by placing the genes under the control of titratable promoters. We measured phenotypic and genotypic changes on downstream genes in EAEC strain 042 and E. coli K-12 strain DH5α, which lacks the AggR regulon. In EAEC, we found that low expression of aar increases aafA fimbrial gene expression via H-NS; however, when aar is more highly expressed, it acts as a negative regulator via AggR. In DH5α, aar affected expression of E. coli genes in some cases via H-NS and in some cases independent of H-NS. Our data support the model that Aar interacts in concert with AggR, H-NS, and possibly other regulators and that these interactions are likely to be functionally significant in vivo.

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Genome-Wide Identification of Genes Regulated by the Rcs Phosphorelay System in Erwinia amylovora

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-11-0207 ◽

2012 ◽

Vol 25 (1) ◽

pp. 6-17 ◽

Cited By ~ 31

Author(s):

Dongping Wang ◽

Mingsheng Qi ◽

Bernarda Calla ◽

Schuyler S. Korban ◽

Steven J. Clough ◽

...

Keyword(s):

Gene Expression ◽

Erwinia Amylovora ◽

Virulence Gene ◽

Luria Bertani ◽

Regulatory Genes ◽

Bacterial Cells ◽

Virulence Gene Expression ◽

Genome Wide

The exopolysaccharide amylovoran is one of the major pathogenicity factors in Erwinia amylovora, the causal agent of fire blight of apples and pears. We have previously demonstrated that the RcsBCD phosphorelay system is essential for virulence by controlling amylovoran biosynthesis. We have also found that the hybrid sensor kinase RcsC differentially regulates amylovoran production in vitro and in vivo. To further understand how the Rcs system regulates E. amylovora virulence gene expression, we conducted genome-wide microarray analyses to determine the regulons of RcsB and RcsC in liquid medium and on immature pear fruit. Array analyses identified a total of 648 genes differentially regulated by RcsCB in vitro and in vivo. Consistent with our previous findings, RcsB acts as a positive regulator in both conditions, while RcsC positively controls expression of amylovoran biosynthetic genes in vivo but negatively controls expression in vitro. Besides amylovoran biosynthesis and regulatory genes, cell-wall and cell-envelope (membrane) as well as regulatory genes were identified as the major components of the RcsBC regulon, including many novel genes. We have also demonstrated that transcripts of rcsA, rcsC, and rcsD genes but not the rcsB gene were up-regulated when bacterial cells were grown in minimal medium or following infection of pear fruits compared with those grown in Luria Bertani medium. Furthermore, using the genome of E. amylovora ATCC 49946, a hidden Markov model predicted 60 genes with a candidate RcsB binding site in the intergenic region, 28 of which were identified in the microarray assay. Based on these findings as well as previous reported data, a working model has been proposed to illustrate how the Rcs phosphorelay system regulates virulence gene expression in E. amylovora.

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Human mineralocorticoid receptor A and B protein forms produced by alternative translation sites display different transcriptional activities

Acta Endocrinologica ◽

10.1530/eje.0.1500585 ◽

2004 ◽

pp. 585-590 ◽

Cited By ~ 30

Author(s):

L Pascual-Le Tallec ◽

C Demange ◽

M Lombes

Keyword(s):

Translation Initiation ◽

Mineralocorticoid Receptor ◽

Single Gene ◽

In Vitro Transcription ◽

Targeted Mutagenesis ◽

In Vitro Translation ◽

Terminal Fragment ◽

Initiation Of Translation ◽

Alternative Translation

OBJECTIVE: Aldosterone binds the mineralocorticoid receptor (MR) and is involved in the regulation of ionic transport, mainly sodium retention. MR is encoded by one single gene transcribed into various messengers that are thought to be translated into a unique 107 kDa protein. The aim of this study was to identify and characterize translation initiation variants of the human MR protein. METHODS: For this purpose we analyzed the extreme N-terminal fragment of various MR species, and confronted the predicted pattern of expression with in vitro translation results. Transactivation functions of the identified MR forms obtained by targeted mutagenesis were evaluated by transient transfection assays with different promoter reporter genes. RESULTS: Two major forms of human MR (hMR), named MRA and MRB, generated by alternative initiation of translation from start methionine 1 and 15 respectively, were predicted. Their expected expressions were confirmed by in vitro transcription/translation studies. Interestingly, each form of MR displayed reduced transactivation capacities with wild type (WT)=A+B>MRA>MRB suggesting that the extreme N-terminal fragment of MR has an effect on the transcriptional properties of the receptor. CONCLUSIONS: Altogether these data indicate that MR is expressed, through alternative translation initiation, as distinct protein variants which possess different functional properties. These MR forms could tightly dictate the modulation of aldosterone responsiveness in various tissues or in pathophysiological situations.

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