Unknown,Transcriptomics,Genomics,Proteomics

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RNAi knock down of known nonsense-mediated mRNA decay (NMD) factors in Drosophila cells

ABSTRACT: All eukaryotes studied to date have the capacity to detect and degrade mRNAs harboring premature translation termination codons (PTCs) in a process called nonsense-mediated mRNA decay (NMD) (reviewed in Wagner E & Lykke-Andersen J, 2002). This surveillance system allows the cell to prevent the expression of potentially harmful truncated proteins. trans-acting factors required for NMD in Drosophila are the proteins UPF1, UPF2, UPF3, SMG-1, SMG-5 and SMG-6. To identify mRNAs naturally regulated by NMD, we analyzed expression profiles in Drosophila cells RNAi-depleted of known NMD factors using high-density oligonucleotide arrays.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Jan Rehwinkel

PROVIDER: E-MEXP-202 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Nonsense-mediated mRNA decay factors act in concert to regulate common mRNA targets.

Rehwinkel Jan J Letunic Ivica I Raes Jeroen J Bork Peer P Izaurralde Elisa E

RNA (New York, N.Y.) 20051001 10

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades mRNAs containing nonsense codons, and regulates the expression of naturally occurring transcripts. While NMD is not essential in yeast or nematodes, UPF1, a key NMD effector, is essential in mice. Here we show that NMD components are required for cell proliferation in Drosophila. This raises the question of whether NMD effectors diverged functionally during evolution. To address this question, we examined expression profi ...[more]

PMID: 16199763

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Project description:We used a modification of GINI analysis to identify genes containing premature translation termination codons (PTC) generated by nonsense or frameshift mutations in prostate cancer cell lines. The analysis was performed in two steps. In the first step nonsense mediated mRNA decay (NMD) was inhibited in prostate cancer cell lines using incubation with medium containing caffeine for 4 hours. Gene expression analysis of caffeine treated or untreated cells after this step detects mRNA accumulation that takes place for genes containing PTC and as well as for genes that show induction of transciption due to stress caused by NMD inhibition. In the second step either both transcription and NMD or transcription only are blocked by incubating cell in a medium containing either both actinomycin D and caffeine or actinomacin D only for 4 hours. Gene expression analysis after this second step detects mRNA degradation for genes containing PTC as well as for genes that show induction of transciption due to stress caused by NMD inhibition. The efficiency of mRNA degradation for genes containing PTC during this step depends on whether NMD is inhibited or not. The efficiency of mRNA degradation for stress response genes does not depend on whether NMD is inhibited or not. Our modified GINI protocol allows efficient separation of genes containing PTC from the genes that show mRNA increases due to stress response to NMD inhibition. Experiment Overall Design: The LNCaP and 22Rv1 cells were seeded into four 60 mm tissue culture plates each. Caffeine (10 mM) was added to three plates and one plate was incubated without caffeine as a control. Following four hours? incubation, the medium was removed from one plate (with caffeine) and from the control plate and total RNA was prepared using TRIZOL reagent, according to manufacturer?s instructions, and used for an Affymetrix U133Plus2.0 oligonucleotide array hybridization. The caffeine-containing medium of the two remaining plates was removed, the cells were washed twice with phosphate-buffered saline and the medium with actinomycin D (2 mkg/ml) together with caffeine (10 mM) was added to one plate. Actinomycin D alone was added to the other plate. Following another four hours? incubation, the total RNA from both plates was prepared and used for an Affymetrix U133Plus2.0 oligonucleotide array analysis.

Project description:SMARD1 is an infantile autosomal recessive motor neuron (MN) disease, caused by mutations in the Immunoglobulin mu binding protein 2 (IGHMBP2). We investigated the potential of a spinal cord neural stem cell population isolated on the basis of aldehyde dehydrogenase activity (ALDH) to modify disease progression of nmd mice, an animal model of SMARD1. ALDHhiSSClo stem cells are self-renewing and multipotent and when intratechally transplanted in nmd mice generate motor neurons properly localized in the spinal cord ventral horns. Transplanted nmd animals presented delayed disease progression, sparing of motor neurons and ventral root axons and increased life-span. To further investigate the molecular events responsible for these differences, microarray and Real time RT-PCR analysis of wild-type, mutated and transplanted nmd spinal cord were undertaken. We demonstrated a down-regulation of genes involved in excitatory amino acid toxicity and oxidative stress handling, as well as an up-regulation of genes related to the chromatin organization in nmd compared to wild-type mice, suggesting that they may play a role in SMARD1 pathogenesis. Spinal cord of nmd transplanted mice expressed high transcript levels for genes related to neurogenesis such as Doublecortin (DCX), LIS1 and drebrin. The presence of DCX-expressing cells in adult nmd spinal cord suggests that both exogenous and endogenous neurogenesis may contribute to the observed nmd phenotype amelioration. Experiment Overall Design: We intratechally transplanted a spinal cord neural stem cell population isolated on the basis of aldehyde dehydrogenase activity (ALDH) in nmd mice, an animal model of SMARD1. Experiment Overall Design: We analyzed the lumbar spinal cord tract of transplanted nmd mice (n=3), untransplanted nmd mice (n=3) and wild-type mice (n=3) (all mice are of male gender). The latter groups underwent surgical procedure with vehicle. The surgical procedure and cell transplantation were done at P1 and the animals were sacrificed at 6 weeks of age.

Dataset Information

RNAi knock down of known nonsense-mediated mRNA decay (NMD) factors in Drosophila cells

Publications

Nonsense-mediated mRNA decay factors act in concert to regulate common mRNA targets.

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