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Transcriptomic analysis of mutant yeast strains deficient in Xrn1 nuclear import

ABSTRACT: Transcriptomic analysis of mutant yeast strains deficient in Xrn1 nuclear import

PROVIDER: PRJNA664589 | ENA |

REPOSITORIES: ENA

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Transcriptomic analysis of mutant yeast strains deficient in Xrn1 nuclear import

Project description:The goal of the project was to study the transcription rates and mRNA levels, genome-wide, in several mutants in Xrn1 defetive in nuclear import. We used Genomic Run-On (GRO) experiment in wild type and xrn1 mutant strains.

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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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Transcription profiling of yeast wild type, xrn1, xrn1 upf1, xrn1 nmd2, and xrn1 upf3 strains to investigate mRNA decay

Project description:To determine the effects of inactivation of both the nosense-mediated mRNA decay pathway and the general 5' to 3' decay pathway on yeast mRNA decay, we compared the expression profiles of the wild-type, xrn1, xrn1 upf1, xrn1 nmd2, and xrn1 upf3 strains.

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Xrn1-sensitive lncRNAs are insensitive to Dbp2 nuclear depletion

Project description:To investigate whether the action of Dbp2 on Xrn1-sensitive lncRNAs depends on a nuclear or cytoplasmic localization, we used the anchor-away approach to deplete a Dbp2-FRB-GFP fusion from the nucleus upon rapamycin treatment.

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Project description:The main cytoplasmic mRNA decay pathway in yeast uses the 5’-3’ exonuclease Xrn1 (Parker and Song, Nat Struct Mol Biol. 2004 ). This protein shuttles from the cytoplasm to the nucleus, where it has a role as transcription factor (Haimovich et al. Cell 2013). In this work, we find that most of the global phenotypes of an xrn1 mutant are partially complemented by a cytoplasmic version of the paralogous 5’-3’exonuclease Rat1 (cRat1) indicating that this 5’-3’-exonuclease has a similar enzymatic capacity as Xrn1. The lack of a cytoplasmatic 5’-3’-exoribonuclease is the cause of the physiological defects of an xrn1 mutant. The capacity of cRat1 to perform co-translational decay is, however, very limited. The comparison with the strain having a NLS1∆-NLS2∆-Xrn1 version shows that it is slightly deficient in 5’→3’-co-translational decay but much more efficient than cRat1. In both strains, cRat1 and -Xrn1-NLS1&2, the lack of nuclear Xrn1 has a very minor influence on cell growth.

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AKIRIN2 controls the nuclear import of proteasomes in vertebrates

Project description:AKIRIN2 controls the nuclear import of proteasomes in vertebrates

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RNA-seq analysis of starvation and starvation exit

Project description:To find out if Xrn1 nuclear import enhances transcription of certain genes during exit from starvation.

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AKIRIN2 controls the nuclear import of proteasomes in vertebrates

Project description:Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth. Our screens uncover a highly conserved protein, AKIRIN2, that is essentially required for nuclear protein degradation. We found that AKIRIN2 forms homodimers that directly bind to fully assembled 20S proteasomes to mediate their nuclear import. During mitosis, proteasomes are excluded from condensing chromatin and re-imported into newly formed daughter nuclei in a highly dynamic, AKIRIN2-dependent process. Cells undergoing mitosis in the absence of AKIRIN2 become devoid of nuclear proteasomes, rapidly causing accumulation of MYC and other nuclear proteins. Collectively, our study reveals a dedicated pathway controlling the nuclear import of proteasomes in vertebrates and establishes a scalable approach to decipher regulators in essential cellular processes.

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Xrn1-sensitive lncRNAs are insensitive to Dbp2 nuclear depletion

Project description:Xrn1-sensitive lncRNAs are insensitive to Dbp2 nuclear depletion

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Translational landscape of Xrn1-sensitive lncRNAs in yeast

Project description:To define the translational landscape of Xrn1-sensitive lncRNAs in yeast, we performed Ribo-Seq in WT and upf1 mutant cells, in native conditions or upon treatment with translation elongation inhibitor (cycloheximide).

2024-02-20 | GSE203278 | GEO

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