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Genomic Run On (GRO): determination of the nascent transcriptional rates and mRNA levels in several yeast mutants.

ABSTRACT: In order to maintain the appropriate level of mRNA it is necessary coordinate simultaneously all the steps along the mRNA life cycle. It has been shown that several factors act in the regulation of gene expression as global coordinators. Thus, some kind of information is transferred from the nucleus to the cytoplasm, imprinted in the mRNA. In this way, it is conceivable the existence of mechanisms that ensure the balance between mRNA synthesis and degradation through the information flow from the cytoplasm to the nucleus and vice versa, as a crosstalk among both process to ensure the proper mRNA homeostasis in the cell. In this study, we have used null mutants related with transcription and mRNA degradation and we find that the lack of those factors impacts both over transcription and RNA stability at genome wide levels to compensate the impaired effect produce by the loss of each studied factor.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE57467 | GEO | 2015/12/31

SECONDARY ACCESSION(S): PRJNA246528

REPOSITORIES: GEO

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Genomic Run On (GRO): determination of the nascent transcriptional rates and mRNA levels in several yeast mutants.

Project description:In order to maintain the appropriate level of mRNA it is necessary coordinate simultaneously all the steps along the mRNA life cycle. It has been shown that several factors act in the regulation of gene expression as global coordinators. Thus, some kind of information is transferred from the nucleus to the cytoplasm, imprinted in the mRNA. In this way, it is conceivable the existence of mechanisms that ensure the balance between mRNA synthesis and degradation through the information flow from the cytoplasm to the nucleus and vice versa, as a crosstalk among both process to ensure the proper mRNA homeostasis in the cell. In this study, we have used null mutants related with transcription and mRNA degradation and we find that the lack of those factors impacts both over transcription and RNA stability at genome wide levels to compensate the impaired effect produce by the loss of each studied factor. This study focus on the transcriptional activity of RNA pol II in yeast genes. S. cerevisiae cells grown in YPD or YPGal to exponential phase were subjected to Genomic Run On. Data were normalized by the ArrayStat software. Home-made macroarrays containing 6035 ORF were used.

2015-12-31 | E-GEOD-57467 | biostudies-arrayexpress

mRNA-Seq of neuronal compartments with perturbed mRNA degradation machinery (overexpression of dnCaf1) in mouse primary cortical neurons

Project description:To study the correlation between mRNA stability and subcellular mRNA localisation we globally interferred with mRNA degradation in primary cortical neurons by overexpressing a catalytic mutant of deadenylase CAF1 that functions as a dominant negative form. Neurons were separated into subcellular compartments (neurite, soma-cytoplasm and nucleus) and sequenced in parallel with respective compartments from GFP- expressing control neuons.

2023-07-12 | E-MTAB-12887 | biostudies-arrayexpress

Rpb4 functions mainly in mRNA synthesis by RNA polymerase II

Project description:RNA polymerase II (Pol II) is the central enzyme that carries out eukaryotic mRNA transcription and consists of a 10-subunit catalytic core and a heterodimeric subcomplex of subunits Rpb4 and Rpb7 (Rpb4/7). Rpb4/7 has been proposed to shuttle from the nucleus to the cytoplasm, and to function there in mRNA translation and degradation. Here we provide evidence that Rpb4 mainly functions in nuclear mRNA synthesis by Pol II, and evidence arguing against an important cytoplasmic role. We used metabolic RNA labeling and comparative Dynamic Transcriptome Analysis (cDTA) to show that Rpb4 deletion in Saccharomyces cerevisiae causes a drastic defect in mRNA synthesis that is compensated by down-regulation of mRNA degradation, resulting in mRNA level buffering. Deletion of Rpb4 can be rescued by covalent fusion of Rpb4 to the Pol II core subunit Rpb2, which largely restores mRNA synthesis and degradation defects caused by Rpb4 deletion. Thus Rpb4 is a bona fide Pol II core subunit which functions mainly in mRNA synthesis.

2014-05-09 | E-MTAB-2539 | biostudies-arrayexpress

Structural basis for pre-miRNA-31 biogenesis

Project description:MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate protein gene expression post-transcriptionally. By base pairing with target mRNAs, miRNAs triggers mRNA degradation or translational suppression[1-4]. Abnormal miRNAs levels are associated with cancers, diabetes, neurological and other diseases[5-8]. RNA polymerase II transcribes primary miRNA (pri-miR) in the nucleus and pri-miRs are subsequently processed by Microprocessor, which is composed of Drosha and DiGeorge syndrome critical region 8 (DGCR8) proteins, to generate precursor miRNAs (pre-miRs). Pre-miRs are exported from the nucleus to the cytoplasm in a GTP-dependent manner by Exportin-5. In the cytoplasm, a pre-miRs are further processed by Dicer to generate 21-22 nt mature miRNA[4, 9]. Argonaute (Ago) protein loads one of mature miRNAs into protein and forms miRNA-induced silencing complex, which is responsible for mRNA degradation or translational suppression[2, 4].

2023-04-24 | GSE230167 | GEO

A compensatory link between cleavage/polyadenylation and mRNA turnover regulates steady-state mRNA levels in yeast

Project description:Cells have compensatory mechanisms to coordinate the rates of major biological processes, thereby permitting growth in a wide variety of conditions. Here, we uncover a compensatory link between cleavage/polyadenylation in the nucleus and mRNA turnover in the cytoplasm. On a global basis, same-gene 3’ mRNA isoforms with > two-fold differences in half-lives have steady-state mRNA levels that differ by significantly less than a factor of two. In addition, increased efficiency of cleavage/polyadenylation at a specific site is associated with reduced stability of the corresponding 3’ mRNA isoform. This inverse relationship between cleavage/polyadenylation and mRNA isoform half-life reduces the variability in the steady-state levels of mRNA isoforms, and it occurs in all four growth conditions tested. These observations suggest that during cleavage/polyadenylation in the nucleus, mRNA isoforms are marked in a manner that persists upon translocation to the cytoplasm and affects the activity of mRNA degradation machinery, thus influencing mRNA stability.

2022-02-02 | GSE191091 | GEO

Double-stranded RNA formation leads to preferential nuclear export and gene expression

Project description:mRNAs are transcribed and processed in the nucleus before they are exported into the cytoplasm for translation. Export is mediated by the export receptor heterodimer Mex67-Mtr2 in yeast (TAP-p10 in humans). Interestingly, also long non-coding RNAs (lncRNAs) leave the nucleus and so-called XUTs (Xrn1 sensitive unstable transcripts) accumulate in the cytoplasm of the degradation defective xrn1∆ mutant. Curiously, it is currently unclear why so many lncRNAs travel into the cytoplasm. Here we show that such ncRNAs accelerate mRNA export by annealing with their sense counterparts through the helicase Dbp2. These double stranded (ds) RNAs dominate export, because Mex67 has a higher affinity to dsRNAs and more molecules bind. Cells benefit from this mechanism as it quickly regulates gene expression, which is particularly important upon expression program changes. Consequently, prevention of the dsRNA formation or destroying dsRNAs is lethal to cells. This mechanism could explain the general cellular occurrence of antisense RNAs.

2024-04-30 | GSE188455 | GEO

A herpesvirus protein selectively inhibits cellular mRNA nuclear export

Project description:Nuclear export of mRNA is essential for eukaryotic cells to establish the flow of genetic information in the nucleus to protein synthesis in the cytoplasm. This transport process is highly regulated to ensure efficient and accurate gene expression. Viruses are well known for their ability to manipulate host gene expression. Here, we report that ORF10 of Kaposi’s sarcoma associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. This export inhibitory effect of ORF10 requires the interaction with an RNA export factor, Rae1. Genome-wide analysis by RNA sequencing revealed the subset of cellular mRNAs whose nuclear export is blocked by ORF10. The 3’ untranslated regions (3’ UTRs) of ORF10-targeted transcripts confer their sensitivity to nuclear export inhibition by ORF10. In the context of KSHV replication, the interaction of ORF10 with Rae1 is important for the virus to express viral genes and produce infectious virions. Our results suggest that a nuclear replicating DNA virus can selectively interfere with RNA export through Rae1 to restrict host gene expression for optimal viral replication.

2016-10-18 | GSE82241 | GEO

SlamSeq of subcellular compartments in mouse primary cortical neurons

Project description:We performed SlamSeq (thiol(SH)-linked alkylation for metabolic sequencing) to estimate mRNA half-lives in subcellular compartments (neurites, soma-cytoplasm and nucleus) of primary cortical neurons.

2023-07-12 | E-MTAB-12888 | biostudies-arrayexpress

The DExH-box RNA helicase RHAU is a Nuclear Protein Involved in Transcription and mRNA Decay

Project description:RHAU (RNA helicase-associated with AU-rich element) is a DExH protein that was originally identified as a factor accelerating AU-rich element-mediated mRNA degradation. The finding that RHAU is predominantly localized in the nucleus, despite that mRNA degradation occurs in cytoplasm, prompted us to consider nuclear functions of RHAU. In HeLa cells, RHAU was localized throughout the nucleoplasm with some concentration in nuclear speckles in a manner dependent on ATPase activity. Transcriptional arrest altered its localization to nucleolar caps where it was colocalized with other RNA helicases, p68 and p72, suggesting that RHAU is involved in transcription-related RNA metabolism in the nucleus. To see whether RHAU affects global gene expression either transcriptionally or posttranscriptionally, we performed microarray analysis using total RNA prepared from RHAU-depleted HeLa cell lines, measuring both steady-state mRNA levels and mRNA half-lives by ActinomycinD-chase. We found that most transcripts whose steady-state levels were affected by RHAU knockdown did not show changes in their half-lives, suggesting the involvement of transcriptional regulation for these transcripts. We propose that RHAU has dual functions involved in synthesis and degradation of mRNA in different subcellular compartments. Keywords: mRNA decay study using HeLa cells expressing shRNA

2008-01-01 | GSE8192 | GEO

Double stranded RNA formation leads to preferential nuclear export and gene expression

Project description:mRNAs are transcribed and processed in the nucleus before they are exported into the cytoplasm for translation. Export is mediated by the export receptor heterodimer Mex67-Mtr2 in yeast (TAP-p15 in humans). Interestingly, also many lncRNAs leave the nucleus but it is currently unclear why they travel into the cytoplasm. Here we show that antisense (as)RNAs accelerate mRNA export by annealing with their sense counterparts through the helicase Dbp2. These double-stranded (ds)RNAs dominate export compared to single-stranded (ss)RNA, as they have a higher capacity and affinity for the export receptor Mex67. In this way, asRNAs boost gene expression, which is beneficial for cells. This is particularly important upon expression program changes. Consequently, the degradation or prevention of the formation of dsRNA is toxic for cells. This mechanism illuminates the general cellular occurrence of asRNAs and explains their nuclear export.

2024-04-30 | GSE252951 | GEO

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