Silencing Xrn1 by dox-inducible shRNA
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ABSTRACT:
PROVIDER: PRJNA471893 | ENA |
REPOSITORIES: ENA
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Project description:Xrn1 is a cytoplasmic 5’-3’ exoribonuclease responsible for degradation of multiple types of RNA. Data obtained on yeast model described Xrn1 function in degradation of messenger (m)RNA, Rapid transfer (t)RNA Decay (RTD) of hypomodified and unstable tRNA transcripts as well as maturation of 25S ribosomal (r)RNA and small nucleolar (sno)RNAs. Our group discovered previously that deletion of Xrn1 gene results in stabilization of a group of long non-coding RNAs which are often transcribed antisense to protein-coding genes and may regulate their expression on transcriptional level. However much less is known about human (h)Xrn1 function. Data available from human cell lines confirm involvement of hXrn1 in degradation of mRNA, initiator tRNA methionine and some miRNA. The high sequence similarity of Xrn1 between yeast and man suggests that also its function should be well conserved for hXrn1. Therefore, in this experiment using inducible shRNA we want to define hXrn1 targets by analyzing RNAs that are stabilized after Xrn1 silencing with focus on both coding and non-coding transcripts.
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Project description:Unsupervised hierarchical clustering revealed a strong similarity in gene modulation resulting from either compound treatment or BRAF ablation mediated by RNA interference relative to DMSO-treated control samples . Experiment Overall Design: We have generated melanoma A375 cells stably expressing a shRNA construct for doxycycline-inducible knockdown of B-Raf (2 mg/ml Dox, 48h). Small molecule treatment was at 1000nM R341787, 24h.
2009-10-10 | E-GEOD-13487 | biostudies-arrayexpress
Project description:Despite the remarkable achievement of immune checkpoint blockade (ICB) therapy, the response rate is relatively low and only a subset of patients can benefit from the treatment. We hypothesize that targeting RNA decay machinery may lead to accumulation of aberrantRNA, triggering interferon (IFN) signaling and sensitizing tumor cells to immunotherapy. With this in mind, we identified an RNA exoribonuclease, XRN1 as a potential target. Silencing of XRN1 suppressed tumor growth in syngeneic immunocompetent mice and potentiated immunotherapy, while silencing of XRN1 alone did not affect tumor growth in immune deficient mice. Mechanistically, XRN1 depletion activated interferon signaling and viral defense pathway; both pathways play determinant roles in regulating immune evasion. In murine tumors engrafted on immmunocompetent mice, XRN1 depletion significantly enhanced immune cell infiltration in solid tumors especially in combinatory with PD-1 blockade. We identified aberrant-RNA sensing signaling proteins (RIG-I/MAVS and PKR) in mediating the expression of IFN genes, as depletion of each of them blunted the elevation of anti-viral/IFN signaling in Xrn1 silenced cells. Analysis of pan-cancer CRISPR screening data indicated that IFN signaling triggered by Xrn1 silencing is a common phenomenon, suggesting that the effect of Xrn1 silencing may be extend to multiple types of cancers.
2023-02-21 | GSE202587 | GEO
Project description:Despite the remarkable achievement of immune checkpoint blockade (ICB) therapy, the response rate is relatively low and only a subset of patients can benefit from the treatment. We hypothesize that targeting RNA decay machinery may lead to accumulation of aberrantRNA, triggering interferon (IFN) signaling and sensitizing tumor cells to immunotherapy. With this in mind, we identified an RNA exoribonuclease, XRN1 as a potential target. Silencing of XRN1 suppressed tumor growth in syngeneic immunocompetent mice and potentiated immunotherapy, while silencing of XRN1 alone did not affect tumor growth in immune deficient mice. Mechanistically, XRN1 depletion activated interferon signaling and viral defense pathway; both pathways play determinant roles in regulating immune evasion. We identified aberrant-RNA sensing signaling proteins (RIG-I/MAVS and PKR) in mediating the expression of IFN genes, as depletion of each of them blunted the elevation of anti-viral/IFN signaling in Xrn1 silenced cells. Analysis of pan-cancer CRISPR screening data indicated that IFN signaling triggered by Xrn1 silencing is a common phenomenon, suggesting that the effect of Xrn1 silencing may be extend to multiple types of cancers.
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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.
2011-11-08 | E-MEXP-27 | biostudies-arrayexpress
Project description:To study the role of the exonuclease Xrn1 in translational control, we performed ribosome profiling and RNA-seq in Xrn1-depleted cells. By using an auxin-inducible degron, we were able to study immediate effects of Xrn1 depletion in translational control. Therefore, we could overcome experimental limitations associated to stable deletion mutants.
2019-02-08 | GSE109734 | GEO
Project description:To study the role of NRAS mutations in cell proliferation and self-renewal in acute myeloid leukemia (AML), the human AML cell line, THP1, was modified to replace its naturally occurring heterozygous NRAS-G12D mutation with a doxycycline(dox)-inducible heterozygous NRAS-G12V mutation. The endogenous copies of the NRAS-G12D allele were deleted using CRISPR/Cas9 after a dox-inducible, CRISPR resistant, NRAS-G12V transgene was introduced into the THP1 cell line. The resulting cell line was named B11. RNA-seq data confirmed that endogenous NRAS G12D was successfully replaced by dox-inducible exogenous NRAS G12V in the B11 cell line. As expected, depletion of dox induced G1 cell cycle arrest. Interestingly, the B11 cells experienced ten-times higher expression of NRAS induced G2/S-phase cell cycle arrest. Forty-nine genes were identified as signaling responsible genes associated with high expression of NRAS.
2022-07-25 | GSE115911 | GEO
Project description:To study the role of the exonuclease Xrn1 in gene expression dynamics under osmotic stress conditions, we performed RNA-seq in Xrn1-depleted cells. By using an auxin-inducible degron, we were able to study immediate effects of Xrn1 depletion in gene expression dynamics. Therefore, we could overcome experimental limitations associated to stable deletion mutants.
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Project description:Histone profile for wild type MEF and secondary MEF with Dox-inducible vectors for Klf4, Sox2 and Oct4 (KSO).
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Project description:Wnt signaling plays a pivotal role in colorectal cancer. Intrinsic activation of Wnt by mutational events, such as mutations in the tumor suppressor gene APC, represents the most frequent initiating event in this disease background. Long truncated versions of APC retain partial functionality, which leads to a sub-maximal, “just right” activation state of Wnt signaling supposed to be beneficial for disease initiation. In order to study the transcriptomic alterations of an over-stimulated Wnt signaling pathway, conditional shRNA-mediated silencing of APC was performed in chromosomal instable HT-29 CRC cells which express a 1555 amino acid variant of APC protein able to bind and partially inactivate β-catenin. To achieve this, cells were stably transduced with lentiviral particles encoding for a doxycyline-inducible shRNA directed against APC, or, as a control, a non-silencing shRNA (pTRIPZ inducible shRNA vectors, RHS4740-EG324, Horizon Dharmacon, CO, USA). 72 hours after APC silencing, total RNA was isolated and quality controlled for subsequent RNA-Seq Analysis (DKFZ Heidelberg, Genomic and Proteomic Core Facility) on a HiSeq 2000 instrument (Illumina). Overall, we observed bona-fide Wnt target genes, such as NKD1, AXIN2, PTK7, ASCL2, and SMOC2, and additional putative direct or indirect targets of Wnt signaling up-regulated upon shRNA-mediated APC silencing.
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