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RNA-seq of ER-targeted and cytosolic mRNAs with control or CNOT1-targeting siRNA treatment in HEK293 cells

ABSTRACT: Determination of mRNA localisation in HEK293 cells with and without the depletion of CNOT1

ORGANISM(S): Homo sapiens

PROVIDER: GSE183148 | GEO | 2021/09/07

REPOSITORIES: GEO

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Project description:Gene expression is balanced by transcription and mRNA degradation. Shortening of polyadenosine (poly(A)) tails (deadenylation) is the initial step in the decay of most mRNAs. However, the mechanism by which deadenylation controls mRNA expression is not fully understood. This experiment aimed to determine changes in transcriptional activity in livers of control and Cnot1 tamoxifen-inducible liver-specific knockout mice. Control and Cnot1 tamoxifen-inducible liver-specific knockout mice placed on tamoxifen-containing food at 6 weeks of age for 2 weeks. Lysates from livers were subjected to ChIP assay with Histone H3 (tri methyl K4) (H3K4me3) antibody (ab8580; abcam) using SimpleChIP Enzyatic Chromatin IP Kit (#9003; Cell Signaling Technology). Libraries for DNA sequence were prepared from DNA isolated by ChIP assays with a KAPA Hyper Prep Kit (Illumina). 109 base-pair pair-end read DNA-seq was performed with Hiseq PE Rapid Cluster Kit v2-HS and Hiseq Rapid SBS Kit v2-HS (200 Cycle) on Illumina Hiseq2500.

2020-03-30 | E-MTAB-5887 | biostudies-arrayexpress

RNA-seq of livers in control and Cnot1 tamoxifen-inducible liver-specific knockout mice

Project description:Gene expression is balanced by transcription and mRNA degradation. Shortening of polyadenosine (poly(A)) tails (deadenylation) is the initial step in the decay of most mRNAs. However, the mechanism by which deadenylation controls mRNA expression is not fully understood. This experiment aimed to determine changes in gene expression and mRNA stability in livers of control and Cnot1 tamoxifen-inducible liver-specific knockout mice. For comprehensive mRNA half-life profiling, we injected control and Cnot1 tamoxifen-inducible liver-specific knockout mice, which were placed on a tamoxifen-containing diet at 6 weeks of age for 2 weeks, intraperitoneally with 2 mg actinomycin D per g body weight for 4 and 8 hr. 1 g of total RNA was used for RNA-seq library preparation with Illumina TruSeq Stranded mRNA LT Sample Prep Kit. 109 base-pair pair-end read RNA-seq was performed with Hiseq PE Rapid Cluster Kit v2-HS and Hiseq Rapid SBS Kit v2-HS (200 Cycle) on Illumina Hiseq2500.

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Project description:The Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin is involved in the spreading of H3K9me3 repressive marks across genes and transgenes such as ZNF encoding genes, ribosomal DNAs, retroelements, including LINE-1 elements, or the integrated HIV provirus. The deposit of these repressive marks leads to heterochromatin formation and inhibits gene expression. The precise mechanisms of HUSH-mediated silencing are still poorly understood, but HUSH presents structural homology with the RNA-induced transcriptional silencing (RITS) complex found in fission yeast. During transcription elongation by RNA polymerase II, RITS recruits a TRAMP-like RNA degradation complex composed of CNOT1 partners, MTR4 and the exosome, to ultimately repress gene expression via H3K9me3 deposit. Here, we show that TASOR depletion or HIV-2 Vpx expression, which induces TASOR degradation, increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. Furthermore, using a yeast 2-hybrid screen, we identified new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 interact in vivo and synergistically repress HIV expression from its LTR. Similar to the RITS complex, we show that TASOR interacts and cooperates with MTR4 and the exosome. We also highlight an interaction between TASOR and RNA Polymerase II, predominantly under its elongating state, and between TASOR and some HUSH-targeted nascent transcripts. Finally, we show that TASOR overexpression facilitates the association of the aforementioned RNA degradation proteins with RNA polymerase II and is detected at transcriptional centers. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral gene expression.

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