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A specialized mechanism of translation mediated by FXR1a-associated microRNP in cellular quiescence

ABSTRACT: MicroRNAs predominantly decrease gene expression; however, specific mRNAs are translationally upregulated in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes by an FXR1a-associated microRNP (microRNA-protein complex) that lacks the microRNP repressor, GW182. We conducted global proteomic analysis in THP1 cells depleted of FXR1 to globally identify activation targets of more than one microRNA, since FXR1 is required for microRNAmediated translation activation in THP1 G0 cells by FXR1-microRNPs.Since proteomic data changes could also be due to changes at the RNA level, total RNA levels in FXR1knockdown compared to control shRNA cells were examined in parallel by microarray analysis using Affymetrix Human GeneChip 2.0 ST. We used microarrays to get total RNA levels from FXR1 knockdown and control shRNA cells, to normalize the mass spectrometry data in order to provide normalized protein data or translation efficiency.

ORGANISM(S): Homo sapiens

PROVIDER: GSE77512 | GEO | 2016/02/03

SECONDARY ACCESSION(S): PRJNA310658

REPOSITORIES: GEO

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A specialized mechanism of translation mediated by FXR1a-associated microRNP in cellular quiescence

Project description:MicroRNAs predominantly decrease gene expression; however, specific mRNAs are translationally upregulated in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes by an FXR1a-associated microRNP (microRNA-protein complex) that lacks the microRNP repressor, GW182. We conducted global proteomic analysis in THP1 cells depleted of FXR1 to globally identify activation targets of more than one microRNA, since FXR1 is required for microRNAmediated translation activation in THP1 G0 cells by FXR1-microRNPs.Since proteomic data changes could also be due to changes at the RNA level, total RNA levels in FXR1knockdown compared to control shRNA cells were examined in parallel by microarray analysis using Affymetrix Human GeneChip 2.0 ST. We used microarrays to get total RNA levels from FXR1 knockdown and control shRNA cells, to normalize the mass spectrometry data in order to provide normalized protein data or translation efficiency. FXR1 knockdown and shRNA control cells were induced with 1ug/ml of doxycycline continually to cause knockdown of FXR1; the cells were grown in serum for 3 days to induce the shRNAs, followed by 2 days of serum-starvation to induce G0. After harvesting, the cells were lysed in proteinase K buffer (Truesdell et al., 2012) and total RNA was isolated using Trizol (Invitrogen) and hybridized on Affymetrix Human GeneChip 2.0 ST microarray.

2016-02-03 | E-GEOD-77512 | biostudies-arrayexpress

Transcriptome and translatome analysis of THP1 cells with doxycycline induced FXR1 Knockdown

Project description:FXR1 depletion in THP1 cells causes changes in polysome associated mRNAs. This leads to a distinct translatome in the cell.

2022-08-25 | GSE211933 | GEO

Transcriptome and translatome analysis of constitutively overexpression of FXR1 and control THP1 cells

Project description:FXR1 overexpression in THP1 cells causes changes in polysome associated mRNAs. This leads to a distinct translatome in the cell that contributes to chemo- and immune- survival

2022-08-25 | GSE211932 | GEO

shRNA RNA-seq from HepG2 (ENCSR009PPI)

Project description:shRNA knockdown against FXR1 in HepG2 cells followed by RNA-seq. (FXR1-BGHLV12) For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODE_Data_Use_Policy_for_External_Users_03-07-14.pdf

2016-05-26 | GSE80837 | GEO

shRNA RNA-seq from K562 (ENCSR780YFF)

Project description:shRNA knockdown against FXR1 in K562 cells followed by RNA-seq. (FXR1-LV08) For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODE_Data_Use_Policy_for_External_Users_03-07-14.pdf

2016-05-26 | GSE80925 | GEO

MicroRNA Directly Enhances Mitochondrial Translation during Muscle Differentiation.

Project description:MicroRNAs are well known to mediate translational repression and mRNA degradation in the cytoplasm. Various microRNAs have also been detected in membrane-compartmentalized organelles, but the functional significance has remained elusive. Here we report that miR-1, a microRNA specifically induced during myogenesis, efficiently enters the mitochondria where it unexpectedly stimulates, rather than represses, the translation of specific mitochondrial genome-encoded transcripts. We show that this positive effect requires specific miR:mRNA base-pairing and Ago2, but not its functional partner GW182, which is excluded from the mitochondria. We provide evidence for the direct action of Ago2 in mitochondrial translation by Ago2 CrossLinking ImmunoPrecipitation coupled with sequencing (CLIP-seq), functional rescue with mitochondria-targeted Ago2, and selective inhibition of the microRNA machinery in the cytoplasm. These findings unveil a positive function of microRNA in mitochondrial translation and suggest a highly coordinated myogenic program via miR-1 mediated translational stimulation in the mitochondria and repression in the cytoplasm. Examination of miRNA's regulation function in mitochondria in C2C12 myoblasts cells and myotubes cells with CLIP-seq (Ago2).

2014-08-01 | E-GEOD-57596 | biostudies-arrayexpress

Arginine methylation-mediated FXR1 binding with RNAs in cancer cells

Project description:FXR1 is an RNA binding protein and it is post traslationally modified by a methyltrasferase to facilitate its RNA binding activity FXR1 is methylated by PRMT5, an arginine methyltransferase, to promote its stability and facilitate its association with mRNAs. Both genetic and small molecule PRMT5 inhibition failed to methylate recombinant as well as the endogenous FXR1, resulting in protein instability and downregulation of FXR1 target mRNA levels in HNSCC cells. To study the differentially regulated gene under the loss of PRMT5, we knocked- down the PRMT5 using shRNA and confirmed the transcript and protein levels using qRT PCR and immunoblot respectively.

2024-04-08 | GSE256352 | GEO

Interactome of Nematostella GW182 in HEK293 cells

Project description:Here, we dissect the function of GW182 protein in the cnidarian Nematostella, separated by 600 million years from other Metazoa. Using cultured human cells, we show that Nematostella GW182 recruits the CCR4-NOT deadenylation complexes via its tryptophan-containing motifs, thereby inhibiting translation and promoting mRNA decay. Further, similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with Argonaute proteins, and functions downstream to repress mRNA. Thus, our work suggests that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria. Despite this remarkable mechanistic conservation Argonaute and GW182 differentially co-evolved in bilaterians and cnidarians seemingly leading to distinct interaction surfaces.

2016-09-27 | PXD004668 | Pride

RNAi knock down of GW182 in Drososphila cells compared to cells with knock down of AGO1, a key effector of miRNA-mediated gene silencing

Project description:RNA silencing pathways are conserved gene regulation mechanisms that lead to degradation, translational repression or transcriptional silencing of target-transcripts selected based on complementarity with small RNA molecules. Using firefly luciferase reporters, we previously were able show that the RNA binding protein GW182 plays a role in microRNA-mediated gene silencing (Rehwinkel, J. et al., RNA J. 2005). In this study, we provide further evidence for the role GW182 in the miRNA pathway by determining targets regulated by this protein at the genomic level. We examined expression profiles in Drosophila cells depleted of GW182 and compared these profiles to data obtained from cells depleted of AGO1, a key effector of miRNA-mediated gene silencing.

2006-10-31 | E-MEXP-487 | biostudies-arrayexpress

Arginine methylation-mediated FXR1-G4 RNA binding in cancer cells

Project description:The RBP, FXR1, is overexpressed in many epithelial tumors containing a canonical RGG box domain. FXR1 controls post-transcriptional gene regulation through changes in mRNA turnover and translation of target genes. Here we show that arginine methyltransferase PRMT5- mediated specific arginine methylation of FXR1 increases its stability in cancer cells. FXR1-dependent gene signatures show decreased expression and instability of G4-rich sequences containing mRNAs such as CDKN1A, PLK2, TCN2, and TRAF4. Furthermore, structural features of FXR1 and G4-RNA interactions provide novel insights into the critical arginine amino acids of FXR1 essential for G4-RNA-binding and turnover activity. In addition, analysis of the eCLIP data provides various RNA targets and the G-rich sequence motifs of FXR1 in head and neck cancer cell line. Thus, our results indicate that PRMT5-mediated methylation of FXR1 binding with G4-RNAs favors mRNA stability and turnover, contributing to cancer cell growth and proliferation.

2024-04-08 | GSE252916 | GEO

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