Cloning of ARE-Containing Genes by AU-Motif-Directed Display

Domı́nguez, Orlando; Ashhab, Yaqoub; Sabater, Lidia; Belloso, Eva; Caro, Pepi; Pujol-Borrell, Ricardo

doi:10.1006/geno.1998.5548

Cited by 16 publications

(11 citation statements)

References 18 publications

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“…Our differential display protocol was adapted from a previously reported procedure, which was designed to specifically amplify target genes that contain the AUUUA motif (7). The differential display PCR products would correspond to the 3Ј-UTR of the target genes, from the AUUUA motif to the poly(A) tail (7). RNA samples from sham-operated and I/R mouse kidneys (taken at 6 h postreperfusion) were subjected to differential display analysis.…”

Section: Resultsmentioning

confidence: 99%

“…The experimental design was adapted from a previously published method (7). Four sets of PCRs were carried out for each reverse transcription (RT) reaction, with primers that differ in the anchoring base residue (common AT-rich sequences are underlined) (RT1, 5Ј-GGGGGGGTATT TATTTANTTTTTTTTTTTTTTT(A/C/G)-3Ј; PCR1, 5Ј-GGGGGGGTATTTA TTTAA-3Ј, 5Ј-GGGGGGGTATTTATTTAC-3Ј, 5Ј-GGGGGGGTATTTATTT AG-3Ј, and 5Ј-GGGGGGGTATTTATTTAT-3Ј; RT2, 5Ј-GGTGGGTGGTAT TTATTTANTTTTTTTTTTTTTTT(A/C/G)-3Ј; and PCR2, 5Ј-GGTGGGTGG TATTTATTTAA-3Ј, 5Ј-GGTGGGTGGTATTTATTTAC-3Ј, 5Ј-GGTGGGTG GTATTTATTTAG-3Ј, and 5Ј-GGTGGGTGGTATTTATTTAT-3Ј).…”

Section: Methodsmentioning

confidence: 99%

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IRIP, a New Ischemia/Reperfusion-Inducible Protein That Participates in the Regulation of Transporter Activity

Jiang

Prokopenko

Wong

et al. 2005

Molecular and Cellular Biology

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We report the identification and characterization of a new ischemia/reperfusion-inducible protein (IRIP), which belongs to the SUA5/YrdC/YciO protein family. IRIP cDNA was isolated in a differential display analysis of an ischemia/reperfusion-treated kidney RNA sample. Mouse IRIP mRNA was expressed in all tissues tested, the highest level being in the testis, secretory, and endocrine organs. Besides ischemia/reperfusion, endotoxemia also activated the expression of IRIP in the liver, lung, and spleen. The transporter regulator RS1 was identified as an IRIP-interacting protein in yeast two-hybrid screening. The interaction between IRIP and RS1 was further confirmed in coimmunoprecipitation assays. A possible role of IRIP in regulating transporter activity was subsequently investigated. IRIP overexpression inhibited endogenous 1-methyl-4-phenylpyridinium (MPP ؉ ) uptake activity in HeLa cells. The activities of exogenous organic cation transporters (OCT2 and OCT3), organic anion transporter (OAT1), and monoamine transporters were also inhibited by IRIP. Conversely, inhibition of IRIP expression by small interfering RNA or antisense RNA increased MPP ؉ uptake. We measured transport kinetics of OCT2-mediated uptake and demonstrated that IRIP overexpression significantly decreased V max but did not affect K m . On the basis of these results, we propose that IRIP regulates the activity of a variety of transporters under normal and pathological conditions.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

IRIP, a New Ischemia/Reperfusion-Inducible Protein That Participates in the Regulation of Transporter Activity

Jiang

Prokopenko

Wong

et al. 2005

Molecular and Cellular Biology

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“…Previous attempts of arbitrary PCR/RNA fingerprinting protocols that target gene families, including zinc fingers, 3Ј-ARE-containing UTR regions, and MADS-box coding sequences (Asson-Batres et al 1994;Fischer et al 1995;Johnson et al 1996;Gonsky et al 1997;Dominguez et al 1998), were tried but had limitations that our ARE-cDNA procedure avoids. For example, in a study that targeted G-protein coupled receptors (Lopez-Nieto and Nigam 1996;Consalez et al 1999), a statistically designed primer set targeted at the protein-coding regions of mammalian G-protein coupled receptors needed 496 pair of primer sets to span 77.7%.…”

Section: Discussionmentioning

confidence: 99%

“…The detection rate of the two subsets of ARE-mRNAs, those containing two and three ARE pentamers, using the computational approach exceeds 80% with only a definitive set of the primers that equals 16 primers. Procedures by others targeted the short regions between the 3Ј ends and the AREs (AssonBatres et al 1994;Gonsky et al 1997;Dominguez et al 1998) that use 3Ј primers to the polyA tail and 5Ј primers to ARE regions, resulting in largely AT-rich regions that are characteristically homologous, redundant, and in which size distribution is restricted. Also, none of these approaches by others address the computational targeting of large or full-length protein-coding regions as described in this paper.…”

Section: Discussionmentioning

confidence: 99%

An Integrated Computational and Laboratory Approach for Selective Amplification of mRNAs Containing the Adenylate Uridylate-Rich Element Consensus Sequence

Khabar

Dhalla²,

Bakheet³

et al. 2002

Genome Res.

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Messenger RNAs that have the stability determinants, adenylate uridylate-rich elements (AREs), in their 3Ј untranslated region (UTR) code for key products that regulate early and transient biological responses. We used a computational laboratory approach for amplification of large, including full-length, protein-coding regions for ARE genes. Statistical analysis of the initiation regions in the 5Ј UTR of ARE-mRNAs was performed. Accordingly, several 5Ј primers and a single universal 3Ј primer that targeted the initiation consensuses and ARE regions, respectively, were designed. Using optimized conditions, the primers were able to enrich and amplify large protein-coding regions for the ARE gene family. The selective amplification of ARE cDNAs was verified using specific polymerase chain reactions (PCRs) to known ARE mRNA molecules and monitoring the abundance of the non-ARE ␤-actin signal. A mini-library from the amplified ARE products was constructed for further confirmation of ARE selection. Distinct ARE amplified cDNA pools were selectively generated by distinct 5Ј primers. The biological utility of the method was shown with differential display. The up-regulation of several ARE-mRNAs, including the full-length coding region of the small inducible cytokine A4 (SCYA4) gene, was shown in endotoxin-stimulated monocytic cells. The integrated computational and laboratory approach should lead to enhanced capability for discovery and expression analysis of early and transient response genes.

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“…Several laboratories have designed primers to improve sensitivity or enrich for moderate-to low-abundance mRNAs by targeting specific sequences including the polyadenylation signal AATAAA or ATTTA, a motif in short-lived mRNAs (150), and AU sites (31). AU-rich elements are regulatory sequences found in the 3Ј UTR of labile mRNAs encoding cytokines, proto-oncoproteins and transcription factors.…”

Section: Modificationsmentioning

confidence: 99%

Applications of Differential-Display Reverse Transcription-PCR to Molecular Pathogenesis and Medical Mycology

Sturtevant

2000

Clinical Microbiology Reviews

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Cloning of ARE-Containing Genes by AU-Motif-Directed Display

Cited by 16 publications

References 18 publications

IRIP, a New Ischemia/Reperfusion-Inducible Protein That Participates in the Regulation of Transporter Activity

IRIP, a New Ischemia/Reperfusion-Inducible Protein That Participates in the Regulation of Transporter Activity

An Integrated Computational and Laboratory Approach for Selective Amplification of mRNAs Containing the Adenylate Uridylate-Rich Element Consensus Sequence

Applications of Differential-Display Reverse Transcription-PCR to Molecular Pathogenesis and Medical Mycology

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