Comparison of total and cytoplasmic mRNA reveals global regulation by nuclear retention and miRNAs
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE60296 | GEO | 2014/08/12
SECONDARY ACCESSION(S): PRJNA257975
REPOSITORIES: GEO
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Project description:We extracted RNA from whole cells and RNA from the cytoplasm and performed RNA sequening to compare differences in gene expression level and investigate what is the most appropriate estimate of the amount of mRNA present in a given cell population. The study was based on three human cell lines. Analyze of transcriptome in 3 human cell lines (U-2 OS, A-431, U-251MG). Each cell line was prepared with four biological replicates for total RNA and four for cytoplasmic RNA.
2014-08-12 | E-GEOD-60296 | biostudies-arrayexpress
Project description:BackgroundThe majority of published gene-expression studies have used RNA isolated from whole cells, overlooking the potential impact of including nuclear transcriptome in the analyses. In this study, mRNA fractions from the cytoplasm and from whole cells (total RNA) were prepared from three human cell lines and sequenced using massive parallel sequencing.ResultsFor all three cell lines, of about 15000 detected genes approximately 400 to 1400 genes were detected in different amounts in the cytoplasmic and total RNA fractions. Transcripts detected at higher levels in the total RNA fraction had longer coding sequences and higher number of miRNA target sites. Transcripts detected at higher levels in the cytoplasmic fraction were shorter or contained shorter untranslated regions. Nuclear retention of transcripts and mRNA degradation via miRNA pathway might contribute to this differential detection of genes. The consequence of the differential detection was further investigated by comparison to proteomics data. Interestingly, the expression profiles of cytoplasmic and total RNA correlated equally well with protein abundance levels indicating regulation at a higher level.ConclusionsWe conclude that expression levels derived from the total RNA fraction be regarded as an appropriate estimate of the amount of mRNAs present in a given cell population, independent of the coding sequence length or UTRs.
| S-EPMC3495644 | biostudies-literature
Project description:Human histomonocytic U937 cells exhibit macrophage-like properties after phorbol ester stimulation. Accumulating evidence demonstrated that remarkable number of transcripts changed in their amount at total RNA levels. However, post-transcriptional regulation has not been fully elucidated in this model. Thus, we compared expression profiles between polysomal and cytoplasmic RNAs before and after phorbol 12-myristate 13-acetate stimulation (48h, 32nM). Table 1: Detailed description of the expression data of human histomonocytic cell line U937 cells. Data of total cytoplasmic and polysomal fractions before and after PMA stimulation is shown. This table contains all spot data except: 1) saturated spots, 2) undetectable spots, 3) stained spots, and 4) spots only detected in less than one samples. Column A: Gene symbol (âI_xxxxxxâ is corresponding to EST clones); Column B: Genbank accession no. Column C: Description; Column D: Average value of expression levels in total cytoplasmic fraction in the absence of PMA; Column E: Average value of expression levels in total cytoplasmic fraction in the presence of PMA (32nM, 48h); Column F: Average value of expression levels in polysomal fraction in the absence of PMA; Column G: Average value of expression levels in polysomal fraction in the presence of PMA; Column H: Expression ratio of polysome, PMA (-) to cytoplasm, PMA(-); Column I: Expression ratio of polysome, PMA (+) to cytoplasm, PMA(+); Column J: Expression ratio of cytoplasm, PMA (+) to cytoplasm, PMA(-); Column K: Expression ratio of polysome, PMA (+) to polysome, PMA(-); Column L: Different expression between polysome, PMA (-) and cytoplasm, PMA(-): different=1; Column M: Different expression between polysome, PMA (+) and cytoplasm, PMA(+): different=1; Column N: Different expression between cytoplasm, PMA (+) and cytoplasm, PMA(-): different=1; Column O: Different expression between polysome, PMA (+) and polysome, PMA(-): different=1; ; Table 2: Candidates post-transcriptionally regulated by PMA. We extracted 105 transcripts whose expression levels were altered only in either fraction accompanied by changes in polysome/cytoplasm expression ratio. Column A: Gene symbol (âI_xxxxxxâ is corresponding to EST clones); Column B: Genbank accession no. Column C: Description; Column D: Average value of expression levels in total cytoplasmic fraction in the absence of PMA; Column E Average value of expression levels in total cytoplasmic fraction in the presence of PMA; Column F: Average value of expression levels in polysomal fraction in the absence of PMA; Column G: Average value of expression levels in polysomal fraction in the presence of PMA; Column H: Expression ratio of polysome, PMA (-) to cytoplasm, PMA(-); Column I: Expression ratio of polysome, PMA (+) to cytoplasm, PMA(+); Column J: Expression ratio of cytoplasm, PMA (+) to cytoplasm, PMA(-); Column K: Expression ratio of polysome, PMA (+) to polysome, PMA(-)
2008-06-11 | E-GEOD-1783 | biostudies-arrayexpress
Project description:Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and non-neuronal cells. However, in non-neuronal cells, the splicing of 3M-bM-^@M-^Y-terminal introns within these genes is repressed by polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery including the nuclear pore-associated protein Tpr and components of the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context. PTBP1 siRNA, PTBP1+PTBP2 siRNA, or control siRNA
2012-07-12 | E-GEOD-37933 | biostudies-arrayexpress
Project description:Human histomonocytic U937 cells exhibit macrophage-like properties after phorbol ester stimulation. Accumulating evidence demonstrated that remarkable number of transcripts changed in their amount at total RNA levels. However, post-transcriptional regulation has not been fully elucidated in this model. Thus, we compared expression profiles between polysomal and cytoplasmic RNAs before and after phorbol 12-myristate 13-acetate stimulation (48h, 32nM). Table 1: Detailed description of the expression data of human histomonocytic cell line U937 cells. Data of total cytoplasmic and polysomal fractions before and after PMA stimulation is shown. This table contains all spot data except: 1) saturated spots, 2) undetectable spots, 3) stained spots, and 4) spots only detected in less than one samples. Column A: Gene symbol (“I_xxxxxx” is corresponding to EST clones) Column B: Genbank accession no. Column C: Description Column D: Average value of expression levels in total cytoplasmic fraction in the absence of PMA Column E: Average value of expression levels in total cytoplasmic fraction in the presence of PMA (32nM, 48h) Column F: Average value of expression levels in polysomal fraction in the absence of PMA Column G: Average value of expression levels in polysomal fraction in the presence of PMA Column H: Expression ratio of polysome, PMA (-) to cytoplasm, PMA(-) Column I: Expression ratio of polysome, PMA (+) to cytoplasm, PMA(+) Column J: Expression ratio of cytoplasm, PMA (+) to cytoplasm, PMA(-) Column K: Expression ratio of polysome, PMA (+) to polysome, PMA(-) Column L: Different expression between polysome, PMA (-) and cytoplasm, PMA(-): different=1 Column M: Different expression between polysome, PMA (+) and cytoplasm, PMA(+): different=1 Column N: Different expression between cytoplasm, PMA (+) and cytoplasm, PMA(-): different=1 Column O: Different expression between polysome, PMA (+) and polysome, PMA(-): different=1 Table 2: Candidates post-transcriptionally regulated by PMA. We extracted 105 transcripts whose expression levels were altered only in either fraction accompanied by changes in polysome/cytoplasm expression ratio. Column A: Gene symbol (“I_xxxxxx” is corresponding to EST clones) Column B: Genbank accession no. Column C: Description Column D: Average value of expression levels in total cytoplasmic fraction in the absence of PMA Column E Average value of expression levels in total cytoplasmic fraction in the presence of PMA Column F: Average value of expression levels in polysomal fraction in the absence of PMA Column G: Average value of expression levels in polysomal fraction in the presence of PMA Column H: Expression ratio of polysome, PMA (-) to cytoplasm, PMA(-) Column I: Expression ratio of polysome, PMA (+) to cytoplasm, PMA(+) Column J: Expression ratio of cytoplasm, PMA (+) to cytoplasm, PMA(-) Column K: Expression ratio of polysome, PMA (+) to polysome, PMA(-) Keywords = phorbol ester Keywords = macrophage Keywords = polysome Keywords = post-transcriptional regulation Keywords: parallel sample
2004-11-01 | GSE1783 | GEO
Project description:Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and non-neuronal cells. However, in non-neuronal cells, the splicing of 3’-terminal introns within these genes is repressed by polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery including the nuclear pore-associated protein Tpr and components of the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context.
2012-07-12 | GSE37933 | GEO
Project description:Messenger RNA is thought to predominantly reside in the cytoplasm, where it is translated and eventually degraded. Although nuclear retention of mRNA has a regulatory potential it is considered extremely rare in mammals. Here to explore the extent of mRNA retention in metabolic tissues we combine deep sequencing of nuclear and cytoplasmic RNA fractions with single molecule transcript imaging in mouse beta cells, liver and gut. We identify a wide range of protein coding genes for which the levels of spliced polyadenylated mRNA are higher in the nucleus than in the cytoplasm. These include genes such as the transcription factor ChREBP, Nlrp6, Glucokinase and Glucagon receptor. We demonstrate that nuclear retention of mRNA can efficiently buffer cytoplasmic transcript levels from noise that emanates from transcriptional bursts. Our study challenges the view that transcripts predominantly reside in the cytoplasm and reveals a role of the nucleus in dampening gene expression noise.
2015-11-13 | GSE73977 | GEO
Project description:Background: MicroRNAs (miRNAs) are 22-nt small non-coding regulatory RNAs that have generally been considered to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in the nucleus. Methodology/Principal Findings: To determine the number of miRNAs localized to the nucleus, we systematically investigated the subcellular distribution of small RNAs (sRNAs) by independent deep sequencing the nuclear and cytoplasmic pools of 18- to 30-nucleotide sRNAs from human cells. We identified 339 nuclear and 324 cytoplasmic known miRNAs, 300 of which overlap, suggesting that the majority of miRNAs are imported into the nucleus. With the exception of a few miRNAs evidently enriched in the nuclear pool, such as the mir-29b, the ratio of miRNA abundances in the nuclear fraction versus in the cytoplasmic fraction vary to some extent. Moreover, our results revealed that a large number of tRNA 3' trailers are exported from the nucleus and accumulate in the cytoplasm. These tRNA 3' trailers accumulate in a variety of cell types, implying that the biogenesis of tRNA 3' trailers is conserved and that they have a potential functional role in vertebrate cells. Conclusion/Significance: Our results provide the first comprehensive view of the subcellular distribution of diverse sRNAs and new insights into the roles of miRNAs and tRNA 3' trailers in the cell Discovery and characterization of small RNA species through deep sequencing of nuclear and cytoplasmic small RNA fractions from 5-8F cells, a nasopharyngeal carcinoma cell line.
2010-07-14 | E-GEOD-22918 | biostudies-arrayexpress
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