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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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XRN2 Autoregulation and Control of Polycistronic Gene Expression in Caenorhabditis elegans

ABSTRACT: XRN2 is a conserved 5â??-->3â?? exoribonuclease that complexes with XTB-domain containing proteins. Thus, in Caenorhabditis elegans (C. elegans), the XTBD-protein PAXT-1 stabilizes XRN2 to retain its activity. XRN2 activity is also promoted by 3'(2'),5'-bisphosphate nucleotidase 1 (BPNT1) through its hydrolysis of 3â??-phosphoadenosine-5'-bisphosphate (PAP), an endogenous XRN inhibitor. Here, we find through unbiased screening that loss of bpnt-1 function suppresses lethality caused by paxt-1 deletion. This unexpected finding is explained by XRN2 autoregulation, which occurs through repression of a cryptic promoter activity and destabilization of the xrn-2 transcript. Autoregulation appears to be triggered at different thresholds of XRN2 inactivation, such that more robust XRN2 perturbation, by elimination of both PAXT-1 and BPNT1, is less detrimental to worm viability than absence of PAXT-1 alone. Like more than 15% of C. elegans genes, xrn-2 occurs in an operon, and we identify additional operons under its control, consistent with a broader function of XRN2 in polycistronic gene regulation. Regulation occurs through intercistronic regions that link genes in an operon, but similar mechanisms may allow XRN2 to operate on monocistronic genes in organisms lacking operons. Wild-type C. elegans worms were subjected to mock or xrn-2 RNAi from L1 to L4 stage at 20Â°C. Total RNA was extracted from the worms, and polyadenylated RNA was analyzed.

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: Sarah Carl

PROVIDER: E-GEOD-79994 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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XRN2 Autoregulation and Control of Polycistronic Gene Expression in Caenorhabditis elegans

Project description:XRN2 is a conserved 5’-->3’ exoribonuclease that complexes with XTB-domain containing proteins. Thus, in Caenorhabditis elegans (C. elegans), the XTBD-protein PAXT-1 stabilizes XRN2 to retain its activity. XRN2 activity is also promoted by 3'(2'),5'-bisphosphate nucleotidase 1 (BPNT1) through its hydrolysis of 3’-phosphoadenosine-5'-bisphosphate (PAP), an endogenous XRN inhibitor. Here, we find through unbiased screening that loss of bpnt-1 function suppresses lethality caused by paxt-1 deletion. This unexpected finding is explained by XRN2 autoregulation, which occurs through repression of a cryptic promoter activity and destabilization of the xrn-2 transcript. Autoregulation appears to be triggered at different thresholds of XRN2 inactivation, such that more robust XRN2 perturbation, by elimination of both PAXT-1 and BPNT1, is less detrimental to worm viability than absence of PAXT-1 alone. Like more than 15% of C. elegans genes, xrn-2 occurs in an operon, and we identify additional operons under its control, consistent with a broader function of XRN2 in polycistronic gene regulation. Regulation occurs through intercistronic regions that link genes in an operon, but similar mechanisms may allow XRN2 to operate on monocistronic genes in organisms lacking operons.

2016-08-25 | GSE79994 | GEO

Localization of RNAPII and 3â end formation factor CstF subunits on C. elegans genes and operons [ChIP-seq]

Project description:Transcription termination is mechanistically coupled to pre-mRNA 3â end formation to prevent transcription much beyond the gene 3â end. C. elegans, however, engages in polycistronic transcription of operons in which 3â end formation between genes is not accompanied by termination. We have performed RNA polymerase II (RNAPII) and CstF ChIP-seq experiments to investigate at a genome-wide level how RNAPII can transcribe through multiple poly-A signals without causing termination. Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Total RNAPII shows a small peak at the promoter of the gene cluster and a much larger peak at 3â ends. These 3â peaks coincide with maximal phosphorylation of Ser2 within the C-terminal domain (CTD) of RNAPII and maximal localization of the 3â end formation factor CstF. This pattern occurs at all 3â ends including those at internal sites in operons where termination does not occur. Thus the normal mechanism of 3â end formation does not always result in transcription termination. Furthermore, reduction of CstF50 by RNAi did not substantially alter the pattern of CstF64, total RNAPII, or Ser2 phosphorylation at either internal or terminal 3â ends. However, CstF50 RNAi did result in a subtle reduction of CstF64 binding upstream of the site of 3â cleavage, suggesting that the CstF50/CTD interaction may facilitate bringing the 3â end machinery to the transcription complex. ChIP-seq examination of RNAPII and CstF subunits using C. elegans in duplicates.

2016-04-02 | E-GEOD-79821 | biostudies-arrayexpress

Localization of RNAPII and 3â end formation factor CstF subunits on C. elegans genes and operons [RNA-seq]

Project description:Transcription termination is mechanistically coupled to pre-mRNA 3â end formation to prevent transcription much beyond the gene 3â end. C. elegans, however, engages in polycistronic transcription of operons in which 3â end formation between genes is not accompanied by termination. We have performed RNA polymerase II (RNAPII) and CstF ChIP-seq experiments to investigate at a genome-wide level how RNAPII can transcribe through multiple poly-A signals without causing termination. Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Total RNAPII shows a small peak at the promoter of the gene cluster and a much larger peak at 3â ends. These 3â peaks coincide with maximal phosphorylation of Ser2 within the C-terminal domain (CTD) of RNAPII and maximal localization of the 3â end formation factor CstF. This pattern occurs at all 3â ends including those at internal sites in operons where termination does not occur. Thus the normal mechanism of 3â end formation does not always result in transcription termination. Furthermore, reduction of CstF50 by RNAi did not substantially alter the pattern of CstF64, total RNAPII, or Ser2 phosphorylation at either internal or terminal 3â ends. However, CstF50 RNAi did result in a subtle reduction of CstF64 binding upstream of the site of 3â cleavage, suggesting that the CstF50/CTD interaction may facilitate bringing the 3â end machinery to the transcription complex. RNA-seq analysis of wild type (empty vector) and RNAi CstF50 mRNA (polyA-selected) and rRNA-depleted RNA (total RNA).

2016-04-02 | E-GEOD-79822 | biostudies-arrayexpress

Caenorhabditis elegans

Project description:XRN2 Autoregulation and Control of Polycistronic Gene Expression in Caenorhabditis elegans

| PRJNA317589 | ENA

Transcription profiling of C. elegans operons

Project description:Caenorhabditis elegans and its relatives are unique among animals, possibly even among eukaryotes, in having operons1. In these regulated multigene transcription units, a polycistronic pre-mRNA is processed to monocistronic mRNAs by 3' end formation and trans-splicing utilizing a special snRNP, the SL2 snRNP2, for downstream mRNAs1. Previously, the correlation between downstream location in an operon and SL2 trans-splicing has been strong, but anecdotal3. Although only 28 operons have been reported previously, the complete sequence of the genome reveals numerous gene clusters4. To determine how many represent operons, we probed full-genome microarrays for SL2-containing mRNAs. We found significant enrichment for about 1200 genes including most of a group of several hundred genes represented by cDNAs that contain SL2 sequence. Analysis of their genomic arrangements indicates that >90% are downstream genes, falling in 790 distinct operons. We conclude that the genome contains at least 1000 operons, 2- 8 genes in length, that contain ~15% of C. elegans genes. Most of the operons have not been reported previously, and numerous examples of co-transcription of genes encoding functionally related proteins are evident. Inspection of the operon list should reveal heretofore unknown functional relationships.

2005-07-13 | E-SMDB-1388 | biostudies-arrayexpress

Global analysis of Caenorhabditis elegans operons

Project description:Caenorhabditis elegans and its relatives are unique among animals, possibly even among eukaryotes, in having operons. In these regulated multigene transcription units, a polycistronic pre-mRNA is processed to monocistronic mRNAs by 3' end formation and trans-splicing utilizing a special snRNP, the SL2 snRNP, for downstream mRNAs1. Previously, the correlation between downstream location in an operon and SL2 trans-splicing has been strong, but anecdotal. Although only 28 operons have been reported previously, the complete sequence of the genome reveals numerous gene clusters. To determine how many represent operons, we probed full-genome microarrays for SL2-containing mRNAs. We found significant enrichment for about 1200 genes including most of a group of several hundred genes represented by cDNAs that contain SL2 sequence. Analysis of their genomic arrangements indicates that >90% are downstream genes, falling in 790 distinct operons. We conclude that the genome contains at least 1000 operons, 2- 8 genes in length, that contain ~15% of C. elegans genes. Most of the operons have not been reported previously, and numerous examples of co-transcription of genes encoding functionally related proteins are evident. Inspection of the operon list should reveal heretofore unknown functional relationships. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set Computed

2005-07-22 | E-GEOD-2975 | biostudies-arrayexpress

Global analysis of Caenorhabditis elegans operons

2005-07-22 | GSE2975 | GEO

High-resolution analysis of the m-xylene/toluene biodegradation subtranscriptome of Pseudomonas putida mt-2 [RNA-seq]

Project description:Pseudomonas putida mt-2 metabolizes m-xylene and other aromatic compounds through the enzymes encoded by the xyl operons of the TOL plasmid pWW0 along with other chromosomally-encoded activities. Tiling arrays of densely overlapping oligonucleotides were designed to cover every gene involved in this process, allowing dissection of operon structures and exposing the interplay of plasmid and chromosomal functions. All xyl sequences were transcribed in response to aromatic substrates and the 3'-termini of both upper and lower mRNA operons extended beyond the ir coding regions, i.e., the 3â-end of the lower operon mRNA penetrated into the convergent xylS regulatory gene. Furthermore, xylR mRNA for the master m-xylene responsive regulator of the system was decreased by aromatic substrates, while the cognate upper operon mRNA was evenly stable throughout its full length. RNA-seq confirmed these data at a single-nucleotide level and refined the formerly misannotated xylL sequence. The chromosomal ortho route for degradation of benzoate (the ben, cat clusters and some pca genes) was activated by this aromatic, but not by the TOL substrates, toluene or m-xylene. We advocate this scenario as a testbed of natural 16 retroactivity between a pre-existing metabolic network and a new biochemical pathway implanted through gene transfer. Genome Analyzer IIx system (1x75bp) was used for sequencing total RNA extracted from P. putida (mt-2) containing the plasmid pWW0, in two different physiological states (treated with m-xylene and in C-runout condition).

2016-07-07 | E-GEOD-71852 | biostudies-arrayexpress

Multifaceted stoichiometry control of bacterial operons revealed by data-independent acquisition mass spectrometry

Project description:More than half of the protein-coding genes in bacteria are organized in polycistronic operons composed of two or more genes. Whether the operon organization maintains the stoichiometric expression of the genes within an operon remain under debate. In this study, we performed a label-free data-independent acquisition hyper reaction monitoring mass-spectrometry (HRM-MS) experiment to quantify the Escherichia coli proteome in exponential phase and quantified 93.6% of the cytosolic proteins, covering 67.9% and 56.0% of the translating polycistronic operons in BW25113 and MG1655 strains, respectively. We found the shorter operons tend to be more tightly controlled for stoichiometry than longer operons, and those operons for metabolic pathways are less controlled for stoichiometry compared with operons for protein complexes, illustrating the multifaceted nature of the operon-wise regulation: the operon-wise unified transcriptional level and gene-specific translational level. This multi-level regulation benefits the host by optimizing the efficiency of the productivity of metabolic pathways and maintenance of different types of protein complexes.

2019-07-01 | GSE122971 | GEO

RNA polymerase II CTD S2P is dispensable during embryogenesis but regulates the developmental diapause in C. elegans [RNA-seq]

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2020-10-20 | GSE145457 | GEO

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