Assays of Adenylate Uridylate-Rich Element-Mediated mRNA Decay in Cells

Ysla, Riza M.; Wilson, Gerald M.; Brewer, Gary

doi:10.1016/s0076-6879(08)02403-8

Cited by 23 publications

(21 citation statements)

References 48 publications

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“…The next day, the medium was replaced with medium containing 2 g/ml doxycycline. At various times thereafter, the cells were washed with icecold phosphate-buffered saline (PBS) and lysed by adding TRIzol (Invitrogen), and total RNA was isolated, analyzed by Northern blotting (see below), quantified with a phosphorimager, and plotted using the statistical software package Prism (GraphPad, San Diego, CA) (30).…”

Section: Methodsmentioning

confidence: 99%

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LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner

Mattijssen

Maraia

2016

Molecular and Cellular Biology

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bLARP4 is a protein with unknown function that independently binds to poly(A) RNA, RACK1, and the poly(A)-binding protein (PABPC1). Here, we report on its regulation. We found a conserved AU-rich element (ARE) in the human LARP4 mRNA 3= untranslated region (UTR). This ARE, but not its antisense version or a point-mutated version, significantly decreased the stability of ␤-globin reporter mRNA. We found that overexpression of tristetraprolin (TTP), but not its RNA binding mutant or the other ARE-binding proteins tested, decreased cellular LARP4 levels. RNA coimmunoprecipitation showed that TTP specifically associated with LARP4 mRNA in vivo. Consistent with this, mouse LARP4 accumulated to higher levels in TTP gene knockout (KO) cells than in control cells. Stimulation of WT cells with tumor necrosis factor alpha (TNF-␣), which rapidly induces TTP, robustly decreased LARP4 with a coincident time course but had no such effect on LARP4B or La protein or on LARP4 in the TTP KO cells. The TNF-␣-induced TTP pulse was followed by a transient decrease in LARP4 mRNA that was quickly followed by a subsequent transient decrease in LARP4 protein. Involvement of LARP4 as a target of TNF-␣-TTP regulation provides a clue as to how its functional activity may be used in a physiologic pathway. L a-related protein 4 (LARP4), a member of the La family of proteins (1, 2), is a multimodular protein involved in mRNA metabolism. Unlike La protein, the most ancient member of the family, which binds most avidly to 3=-terminal oligo(U) tracts found on nascent transcripts and processing intermediates of small nuclear RNAs, LARP4 is predominantly cytoplasmic and binds preferentially to poly(A) RNA (2). LARP4 interacts with the MLLE domain of the cytoplasmic poly(A)-binding protein (PABPC1) via a well-characterized PAM2 motif near its N terminus (2). LARP4 independently interacts with the receptor for activated C kinase (RACK1), which associates with mRNAs and the 40S ribosome (2). Small interfering RNA (siRNA)-mediated knockdown of LARP4 decreased general protein synthesis (2). Modest overexpression of LARP4 conferred stability on some mRNAs more than others (2).LARP4 underwent a gene duplication event in an ancient vertebrate that gave rise to LARP4B, which has been independently maintained thereafter (3). The two genes are located on different human chromosomes and produce different proteins that each bind to PABP and RACK1 but likely exhibit different sequencespecific RNA binding (1, 2, 4). LARP4B promotes mRNA accumulation and translation by binding 3= untranslated region (UTR) AU-rich sequences of a set of mRNAs (4, 5). The data suggest that LARP4 and -4B modulate mRNA stability and translation but that they may target different sets of mRNAs.AU-rich elements (AREs) are a specific subset of AU-rich sequences found in mammalian cis-acting tracts of 50 to 150 nucleotides (nt) that in humans reside within the 3= UTRs of 5 to 8% of mRNAs (6) and that consist of AUUUA pentamers, either overlapping or near other AU-rich sequences (7). ARE-c...

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Section: Methodsmentioning

confidence: 99%

“…To test if the ARE predicted in LARP4 might have functional potential, we used a popular ␤-globin mRNA reporter assay system (30,36). The predicted 317-nt ARE (shown in Fig.…”

Section: Figmentioning

confidence: 99%

LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner

Mattijssen

Maraia

2016

Molecular and Cellular Biology

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“…For each time point, RNA was extracted as described above and cDNA was synthesized using primers specific for pgdA. The quantitative PCR (qPCR) data were used to calculate the pgdA mRNA half-life as described previously (24). Prism (GraphPad, San Diego, CA) was used with the equation…”

Section: Methodsmentioning

confidence: 99%

Aconitase-Mediated Posttranscriptional Regulation of Helicobacter pylori Peptidoglycan Deacetylase

Austin

Maier

2013

Journal of Bacteriology

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Some bacterial aconitases are bifunctional proteins that function in the citric acid cycle and act as posttranscriptional regulators in response to iron levels and oxidative stress. We explore the role of aconitase (AcnB) in Helicobacter pylori as a posttranscriptional regulator of the cell wall-modifying enzyme peptidoglycan deacetylase, PgdA. Under oxidative stress, PgdA is highly expressed and confers resistance to lysozyme in wild-type cells. PgdA protein expression as well as transcript abundance is significantly decreased in an acnB mutant. In the wild type, pgdA mRNA half-life was 13 min, whereas the half-life for the acnB strain was 7 min. Based on electrophoretic mobility shift assays and RNA footprinting, the H. pylori apo-AcnB binds to the 3=-untranslated region of the pgdA RNA transcript. Some of the protected bases (from footprinting) were localized in proposed stem-loop structures. AcnB-pgdA transcript binding was abolished by the addition of iron. The acnB strain is more susceptible to lysozyme-mediated killing and was attenuated in its ability to colonize mice. The results support a model whereby apo-AcnB directly interacts with the pgdA transcript to enhance stability and increase deacetylase enzyme expression, which impacts in vivo survival.

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“…Clontech pTRE-TIGHT) (Ysla et al 2008). Tet-Off HEK293 cells were plated at a concentration of 150,000 cells/mL in Tet-free media supplemented with 100 ug/mL G418 followed by overnight incubation.…”

Section: Lackey 23mentioning

confidence: 99%

Allele-specific SHAPE-MaP assessment of the effects of somatic variation and protein binding on mRNA structure

Lackey

Coria

Woods

et al. 2018

RNA

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The precise impact of inherited and somatic mutations on messenger RNA (mRNA) structure remains poorly understood. Recent technological advances that leverage next generation sequencing to obtain experimental structure data, such as SHAPE-MaP (Selective 2' Hydroxyl Acylation by Primer Extension and Mutational Profiling), can reveal structural effects of mutations especially when this data is rigorously incorporated in RNA structural modeling. Here we analyze the ability of SHAPE-MaP to detect the relatively subtle structural changes caused by single nucleotide mutations. We find that allele-specific sorting greatly improved the detection ability of SHAPE-MaP. Thus, we used SHAPE-MaP with a novel combination of clone-free robotic mutagenesis and allele-specific sorting to perform a rapid, comprehensive survey of non-coding somatic and inherited riboSNitches in two cancer-associated mRNAs, TPT1 and LCP1. Combined with rigorous thermodynamic modeling of the Boltzmann suboptimal ensemble we identified a subset of somatic and Lackey 2 inherited mutations that change TPT1 and LCP1 RNA structure, with approximately 14% of all variants identified acting as riboSNitches. To confirm that these in vitro structures were biologically relevant, we tested how dependent TPT1 and LCP1 mRNA structures are on their environments. We performed SHAPE-MaP on TPT1 and LCP1 mRNAs in the presence or absence of cellular proteins and found that both TPT1 and LCP1 have similar overall folds in all conditions. RiboSNitches identified within these mRNAs in vitro are likely to exist under biological conditions. Overall, these data reveal a remarkably robust mRNA structural landscape where differences in environmental conditions and most sequence variants do not significantly alter RNA structural ensembles. Finally, predicting riboSNitches in mRNAs from sequence alone remains particularly challenging; these data will provide the community with a benchmark for further algorithmic development.

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Assays of Adenylate Uridylate-Rich Element-Mediated mRNA Decay in Cells

Cited by 23 publications

References 48 publications

LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner

LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner

Aconitase-Mediated Posttranscriptional Regulation of Helicobacter pylori Peptidoglycan Deacetylase

Allele-specific SHAPE-MaP assessment of the effects of somatic variation and protein binding on mRNA structure

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