RNase H/oligonucleotide-directed mRNA purification (ROMP) of apoll mRNA

MacDonald, Clinton C.; Williams, David L.

doi:10.1093/nar/21.3.765

Cited by 5 publications

(4 citation statements)

References 5 publications

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“…From the results of this analysis, a protocol was developed (Fig. 2), using a modification of a previous strategy (21), to isolate similar segments from the coding regions of two specific GA mRNAs. Subsequent sequencing analysis confirmed the previously hypothesized relationship between 4.5-kb and 5.0-kb porcine GA mRNAs (25).…”

Section: Discussionmentioning

confidence: 99%

Complexity and species variation of the kidney-type glutaminase gene

Porter

Ibrahim

Taylor

et al. 2002

Physiological Genomics

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Increased expression of rat kidney-type glutaminase (KGA) during metabolic acidosis results from selective mRNA stabilization. This process is mediated by an 8-base AU-sequence that functions as a pH-response element (pHRE). LLC-PK1-FBPase+ cells, a pH-responsive porcine kidney cell line, express four distinct GA mRNAs. RNase H mapping indicated that three of the GA mRNAs are generated by use of alternative polyadenylation sites and are homologs of the rat KGA mRNA, while the fourth contains a different COOH-terminal coding and 3'-untranslated sequence. PCR cloning and sequencing established that the latter GA mRNA is the homolog of the human GAC mRNA. A rat GAC cDNA was also cloned from a rat kidney library. The 3'-untranslated regions of the GAC mRNAs, but not the porcine or human KGA mRNAs, contain identifiable pHREs. The human KGA gene spans 82 kb and is composed of 19 exons. The unique sequence from the hGAC cDNA is contained in a single exon. Thus in humans, alternative splicing of the initial transcript could produce two GA mRNAs, only one of which may be increased during acidosis.

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Section: Discussionmentioning

confidence: 99%

Complexity and species variation of the kidney-type glutaminase gene

Porter

Ibrahim

Taylor

et al. 2002

Physiological Genomics

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“…Total RNA was prepared from frozen tissues using TRIzol (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer's directions. RNA integrity was estimated by examination of mobilities of 18S and 28S rRNA after ethidium bromide staining of formaldehyde gels 32…”

Section: Methodsmentioning

confidence: 99%

The mRNA Encoding τCstF‐64 Is Expressed Ubiquitously in Mouse Tissues

Huber

Monarez

Dass

et al. 2005

Annals of the New York Academy of Sciences

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Polyadenylation is a process of endonucleolytic cleavage of the mRNA, followed by addition of up to 250 adenosine residues to the 3' end of the mRNA. Polyadenylation is essential for eukaryotic mRNA expression, and CstF-64 is a subunit of the CstF polyadenylation factor that is required for accurate polyadenylation. We discovered that there are two forms of the CstF-64 protein in mammalian male germ cells, one of which (CstF-64) is expressed in all tissues, the other of which (tauCstF-64) is expressed only in male germ cells and in brain (albeit at significantly lower levels in the brain). Therefore, we were surprised to find that, using reverse transcription-PCR, cDNA cloning, and RNA blot analyses, tauCstF-64 mRNA was expressed at higher levels in brain than in testis. Also, tauCstF-64 mRNA was expressed at lower but detectable levels in all tissues tested, including epididymis, heart, kidney, liver, lung, muscle, ovary, spleen, thymus, and uterus. These results suggest the hypothesis that tauCstF-64 mRNA is regulated at the translational or post-translational level.

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“…Then the mixture was subjected to UV irradiation, and photo cross-linked 64K protein-RNA complexes were isolated by immunoprecipitation with a 64K protein-specific monoclonal antibody (27). The protein-RNA complex was hybridized to an oligodeoxyribonucleotide complementary to 12 to 15 nt of the substrate RNA, and the resulting RNA-DNA hybrid was hydrolyzed with RNase H (10). Finally, the cleaved RNA was analyzed by denaturing gel electrophoresis to determine which of the resulting fragments contained the cross-linked 64K protein.…”

Section: Methodsmentioning

confidence: 99%

The 64-kilodalton subunit of the CstF polyadenylation factor binds to pre-mRNAs downstream of the cleavage site and influences cleavage site location.

MacDonald¹,

Wilusz²,

Shenk³

1994

Mol. Cell. Biol.

211

197

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The CstF polyadenylation factor is a multisubunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. Using an RNase H-mediated mapping technique, we show that the 64-kDa subunit of CstF can be photo cross-linked to pre-mRNAs at U-rich regions located downstream of the cleavage site of the simian virus 40 late and adenovirus L3 pre-mRNAs. This positional specificity of cross-linking is a consequence of CstF interaction with the polyadenylation complex, since the 64-kDa protein by itself is cross-linked at multiple positions on a pre-mRNA template. During polyadenylation, four consecutive U residues can substitute for the native downstream U-rich sequence on the simian virus 40 pre-mRNA, mediating efficient 64-kDa protein cross-linking at the downstream position. Furthermore, the position of the U stretch not only enables the 64-kDa polypeptide to be cross-linked to the pre-mRNA but also influences the site of cleavage. A search of the GenBank database revealed that a substantial portion of mammalian polyadenylation sites carried four or more consecutive U residues positioned so that they should function as sites for interaction with the 64-kDa protein downstream of the cleavage site. Our results indicate that the polyadenylation machinery physically spans the cleavage site, directing cleavage factors to a position located between the upstream AAUAAA motif, where the cleavage and polyadenylation specificity factor is thought to interact, and the downstream U-rich binding site for the 64-kDa subunit of CstF.Polyadenylation of precursor mRNAs involves a series of distinct steps: recognition of pre-mRNA substrates which contain the hexanucleotide sequence AAUAAA (19) by polyadenylation factors; endonucleolytic cleavage of the premRNA at a distinct site five to 40 nucleotides (nt) 3' of the hexanucleotide; and addition of approximately 200 adenosine residues at the 3' end (11,22,29). In vivo, these processes are tightly coupled, but the steps can be readily uncoupled in vitro. Previous studies have identified U-rich or GU-rich regions, located as far as 50 nt downstream of the cleavage site, which influence polyadenylation both in vivo and in vitro (6, 13-15, 20, 21, 23, 24, 34). To date, the mechanism by which these downstream sequence elements influence polyadenylation has not been elucidated.A 64-kDa (64K) polypeptide can be efficiently photo crosslinked to AAUAAA-containing RNA substrates that are undergoing polyadenylation in nuclear extracts of HeLa cells (31,16). The 64K polypeptide is a subunit of cleavage and polyadenylation stimulation factor (CstF [27,28]; also termed CF1 [7,8]). A role for the factor in poly(A) addition was confirmed by the ability of antibodies specific for the 64K polypeptide to deplete CstF and block in vitro polyadenylation (27). The 64K polypeptide will specifically cross-link to AAUAAA-containing mRNAs as part of the CstF complex only in the presence of a second multisubunit factor (33) termed the cleavage and polyadenylation specificity factor (CPSF; formerly...

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RNase H/oligonucleotide-directed mRNA purification (ROMP) of apoll mRNA

Cited by 5 publications

References 5 publications

Complexity and species variation of the kidney-type glutaminase gene

Complexity and species variation of the kidney-type glutaminase gene

The mRNA Encoding τCstF‐64 Is Expressed Ubiquitously in Mouse Tissues

The 64-kilodalton subunit of the CstF polyadenylation factor binds to pre-mRNAs downstream of the cleavage site and influences cleavage site location.

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