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Characterization of Arabidopsis thaliana promoter bidirectionality and antisense RNAs by depletion of nuclear RNA decay pathways [RNA-seq]

ABSTRACT: In animals, transcription by RNA polymerase II initiates bidirectionally from gene promoters to produce pre-mRNAs on the forward strand and promoter upstream transcripts (PROMPTs) on the reverse strand. PROMPTs are rapidly degraded by the nuclear exosome. Previous studies based on nascent RNA approaches concluded that Arabidopsis thaliana does not produce PROMPTs. Here, we used steady-state RNA sequencing methods in mutants defective in nuclear RNA decay, including by the exosome, to reassess the existence of PROMPTs in A. thaliana. While PROMPTs are overall rare in A. thaliana, about 100 clear cases of exosome-sensitive PROMPTs were identified. We also found that PROMPTs become sources of 21-22 nt small interfering RNAs in exosome-deficient mutants, perhaps explaining why plants have evolved mechanisms to suppress PROMPT production. In addition, we found ~200 transcription start sites within 3’-UTR-encoding regions that produce long unspliced, exosome-sensitive antisense RNAs. The previously characterized non-coding (nc) RNA that regulates expression of the key seed dormancy regulator, DELAY OF GERMINATION1, is a typical representative of this class of RNAs. Transcription factor genes are overrepresented among loci with exosome-sensitive antisense RNAs, suggesting a potential for widespread control of gene expression via this class of ncRNAs. Lastly, we assess the use of alternative promoters in A. thaliana and compare the accuracy of existing TSS annotations.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE136362 | GEO | 2020/02/17

REPOSITORIES: GEO

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Characterization of Arabidopsis thaliana promoter bidirectionality and antisense RNAs by depletion of nuclear RNA decay pathways

Project description:In animals, transcription by RNA polymerase II initiates bidirectionally from gene promoters to produce pre-mRNAs on the forward strand and promoter upstream transcripts (PROMPTs) on the reverse strand. PROMPTs are rapidly degraded by the nuclear exosome. Previous studies based on nascent RNA approaches concluded that Arabidopsis thaliana does not produce PROMPTs. Here, we used steady-state RNA sequencing methods in mutants defective in nuclear RNA decay, including by the exosome, to reassess the existence of PROMPTs in A. thaliana. While PROMPTs are overall rare in A. thaliana, about 100 clear cases of exosome-sensitive PROMPTs were identified. We also found that PROMPTs become sources of 21-22 nt small interfering RNAs in exosome-deficient mutants, perhaps explaining why plants have evolved mechanisms to suppress PROMPT production. In addition, we found ~200 transcription start sites within 3’-UTR-encoding regions that produce long unspliced, exosome-sensitive antisense RNAs. The previously characterized non-coding (nc) RNA that regulates expression of the key seed dormancy regulator, DELAY OF GERMINATION1, is a typical representative of this class of RNAs. Transcription factor genes are overrepresented among loci with exosome-sensitive antisense RNAs, suggesting a potential for widespread control of gene expression via this class of ncRNAs. Lastly, we assess the use of alternative promoters in A. thaliana and compare the accuracy of existing TSS annotations.

2020-02-17 | GSE142555 | GEO

Characterization of Arabidopsis thaliana promoter bidirectionality and antisense RNAs by depletion of nuclear RNA decay pathways [CAGE-seq]

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The RNA exosome shapes the expression of key protein-coding genes

Project description:The ribonucleolytic exosome complex is central for nuclear RNA degradation, primarily targeting non-coding RNAs, while the majority of mRNAs are protected through their 3’ polyadenylation and nuclear export. Still, the nuclear exosome could have protein-coding (pc) gene-specific regulatory activities. By depleting an exosome core component, or key components of nucleoplasmic exosome adaptor complexes, we identify a number of transcription start sites (TSSs) from within pc genes that produce exosome-sensitive transcripts and two classes of pc-genes, which both employ a single, annotated TSS across cells and tissues, but the former class primarily produces full-length, exosome-sensitive transcripts, whereas the latter primarily produces prematurely terminated transcripts. Genes within the former class often belong to immediate early response transcription factors, while genes within the latter are likely transcribed as a consequence of their proximity to strong upstream TSSs on the opposite strand. We also show that when genes have multiple active TSSs, alternative TSSs that produce exosome sensitive transcripts typically do not contribute substantially to overall gene expression, and most such exosome sensitive transcripts are prematurely terminated. Our results display a complex landscape of sense transcription within pc-gene regions and imply a direct role for nuclear RNA turnover in the expression regulation of a subset of pc- genes.

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Total RNA-Seq in wild-type (WT), dcr1, xrn1 and xrn1 dcr1 strains of N. castellii

Project description:Antisense (as)lncRNAs are extensively degraded by the nuclear exosome and the cytoplasmic exoribonuclease Xrn1 in the budding yeast Saccharomyces cerevisiae, lacking RNA interference (RNAi). Whether the ribonuclease III Dicer affects aslncRNAs in close RNAi-capable relatives remains unknown. Using genome-wide RNA profiling, here we show that aslncRNAs are primarily targeted by the exosome and Xrn1 in the RNAi-capable budding yeast Naumovozyma castellii, Dicer only affecting Xrn1-sensitive lncRNAs (XUTs) levels in Xrn1-deficient cells. The dcr1 and xrn1 mutants display synergic growth defects, indicating that Dicer becomes critical in absence of Xrn1. Small RNA sequencing showed that Dicer processes aslncRNAs into small RNAs, with a preference for asXUTs. Consistently, Dicer localizes into the cytoplasm. Finally, we observed an expansion of the exosome-sensitive antisense transcriptome in N. castellii compared to S. cerevisiae, suggesting that the presence of cytoplasmic RNAi has reinforced the nuclear RNA surveillance machinery to temper aslncRNAs expression. Our data provide fundamental insights into aslncRNAs metabolism and open perspectives into the possible evolutionary contribution of RNAi in shaping the aslncRNAs transcriptome.

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Dcr1-GFP is functional for 22-23 nt small RNAs production

Project description:Antisense (as)lncRNAs are extensively degraded by the nuclear exosome and the cytoplasmic exoribonuclease Xrn1 in the budding yeast Saccharomyces cerevisiae, lacking RNA interference (RNAi). Whether the ribonuclease III Dicer affects aslncRNAs in close RNAi-capable relatives remains unknown. Using genome-wide RNA profiling, here we show that aslncRNAs are primarily targeted by the exosome and Xrn1 in the RNAi-capable budding yeast Naumovozyma castellii, Dicer only affecting Xrn1-sensitive lncRNAs (XUTs) levels in Xrn1-deficient cells. The dcr1 and xrn1 mutants display synergic growth defects, indicating that Dicer becomes critical in the absence of Xrn1. Small RNA sequencing showed that Dicer processes aslncRNAs into small RNAs, with a preference for asXUTs. Consistently, Dicer localizes into the cytoplasm. Finally, we observed an expansion of the exosome-sensitive antisense transcriptome in N. castellii compared to S. cerevisiae, suggesting that the presence of cytoplasmic RNAi has reinforced the nuclear RNA surveillance machinery to temper aslncRNAs expression. Our data provide fundamental insights into aslncRNAs metabolism and open perspectives into the possible evolutionary contribution of RNAi in shaping the aslncRNAs transcriptome.

2019-08-21 | GSE129269 | GEO

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PAP-gamma associates with PAXT nuclear exosome to control the abundance of PROMPT ncRNAs

Project description:Pervasive transcription of the human genome generates an abundance of RNAs that must be processed and degraded. The nuclear RNA exosome is the main RNA degradation machinery in the nucleus. However, nuclear exosome must be recruited to its substrates by targeting complexes, such as NEXT or PAXT. By proteomic analysis, we have identified additional subunits of PAXT, including many orthologs of MTREC found in S. pombe. In particular, we show that polyA polymerase gamma was associated with PAXT. Genome-wide mapping of the binding sites of ZFC3H1, RBM26 and PAPgamma, showed that PAXT is recruited to the TSS of hundreds of genes. Loss of ZFC3H1 abolished recruitment of PAXT subunits including PAPgamma to TSSs and concomitantly increased the abundance of PROMPTs at the same sites. Moreover, PAPgamma polyadenylated PROMPTs and modulated their stability. Our results thus provide key insights into the direct targeting and degradation of PROMPT ncRNAs by PAXT, at their genomic sites and depending on polyadenylation of RNAs.

2023-10-02 | GSE189157 | GEO

Yeast exosome: immunoprecipitation 1

Project description:Nuclear RNA degradation pathways are highly conserved across eukaryotes and play important roles in RNA quality control. Key substrates for exosomal degradation include aberrant functional RNAs and cryptic unstable transcripts (CUTs). It has recently been reported that the nuclear exosome is inactivated during meiosis in budding yeast through degradation of the subunit Rrp6, leading to the stabilisation of a subset of meiotic unstable transcripts (MUTs) of unknown function. We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis which are undetectable in wild-type cells, suggesting that the nuclear exosome remains functional, and we further show that the meiotic exosome complex still contains Rrp6. Indeed Rrp6 over-expression is insufficient to suppress MUT transcripts, showing that the reduced amount of Rrp6 in meiotic cells does not directly cause MUT accumulation. Lack of TRAMP activity stabilises ~1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC). CBC mutants display defects in the formation of meiotic double strand breaks (DSBs), and we see similar defects in TRAMP mutants, suggesting that a key function of the nuclear exosome is to prevent saturation of the CBC complex by CUTs. Together, our results show that the nuclear exosome remains active in meiosis and has an important role in facilitating meiotic recombination.

2015-05-15 | PXD001223 | Pride

Yeast exosome: immunoprecipitation 2

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