Identification of hn 18-brse c/s-element in the 5-untrhnslhted region of human folrte receptor-a mana which specificrllv binds 46-kda cytosolic (trans-factor) proteins

X. L. Sun, Asok Antony

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Folate receptors (PR) are inversely regulated by extracellular folate concentrations. Since FR regulation is primarily mediated at the translational level in cervical carcinoma (HeLa-IU,) cells which express FR-a mRNA, the potential for interaction of ciselements (FR-0 mRNA) and Irons-factors from these cells was determined. Using gelshift assays, two signals were identified which specifically derived from interaction of the 5′-untranslated region of FR-t.mRNA with cytosolic extracts from HeLa-IU, cells. RNase T l mapping revealed that both signals were from protein(s) interacting with two partially overlapping RNA sequences between nucleotides -133 to -116 and -158 to -116, upstream of the translation start site. Selective RNase H cleavage following hybridization with 18-mer antisense DNA complementary to the 18-base RNA fragment abolished both signals indicating that the 18-base RNA sequence is the cis-element. The interaction of this c/s-element and cytosolic protein was competed by poly(C), but not by poly(U), homopolymers. Cross-linking studies using ultra-violet light and Northwestern blot analysis confirmed that the 18-base cis-element specifically bound -46-kDa proteins. From the standpoint of assigning a function to the interaction of this cu-element and Svw-factor within the context of FR and folate metabolism, preliminary studies revealed that although these 46-kDa proteins were unaltered by extracellular folate concentrations, they were widely distributed in cells expressing little to no FR. Thus, the functional significance of this RNA-protein interaction warrants further study.

Original languageEnglish
JournalJournal of Investigative Medicine
Volume44
Issue number3
StatePublished - 1996

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Folic Acid
RNA
Proteins
HeLa Cells
Messenger RNA
Antisense DNA
Ribonuclease H
Poly C
Poly U
5' Untranslated Regions
Homopolymerization
Metabolism
Assays
Nucleotides
Cells
Iron
Carcinoma
Light

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

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title = "Identification of hn 18-brse c/s-element in the 5-untrhnslhted region of human folrte receptor-a mana which specificrllv binds 46-kda cytosolic (trans-factor) proteins",
abstract = "Folate receptors (PR) are inversely regulated by extracellular folate concentrations. Since FR regulation is primarily mediated at the translational level in cervical carcinoma (HeLa-IU,) cells which express FR-a mRNA, the potential for interaction of ciselements (FR-0 mRNA) and Irons-factors from these cells was determined. Using gelshift assays, two signals were identified which specifically derived from interaction of the 5′-untranslated region of FR-t.mRNA with cytosolic extracts from HeLa-IU, cells. RNase T l mapping revealed that both signals were from protein(s) interacting with two partially overlapping RNA sequences between nucleotides -133 to -116 and -158 to -116, upstream of the translation start site. Selective RNase H cleavage following hybridization with 18-mer antisense DNA complementary to the 18-base RNA fragment abolished both signals indicating that the 18-base RNA sequence is the cis-element. The interaction of this c/s-element and cytosolic protein was competed by poly(C), but not by poly(U), homopolymers. Cross-linking studies using ultra-violet light and Northwestern blot analysis confirmed that the 18-base cis-element specifically bound -46-kDa proteins. From the standpoint of assigning a function to the interaction of this cu-element and Svw-factor within the context of FR and folate metabolism, preliminary studies revealed that although these 46-kDa proteins were unaltered by extracellular folate concentrations, they were widely distributed in cells expressing little to no FR. Thus, the functional significance of this RNA-protein interaction warrants further study.",
author = "Sun, {X. L.} and Asok Antony",
year = "1996",
language = "English",
volume = "44",
journal = "Journal of Investigative Medicine",
issn = "1081-5589",
publisher = "Lippincott Williams and Wilkins",
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TY - JOUR

T1 - Identification of hn 18-brse c/s-element in the 5-untrhnslhted region of human folrte receptor-a mana which specificrllv binds 46-kda cytosolic (trans-factor) proteins

AU - Sun, X. L.

AU - Antony, Asok

PY - 1996

Y1 - 1996

N2 - Folate receptors (PR) are inversely regulated by extracellular folate concentrations. Since FR regulation is primarily mediated at the translational level in cervical carcinoma (HeLa-IU,) cells which express FR-a mRNA, the potential for interaction of ciselements (FR-0 mRNA) and Irons-factors from these cells was determined. Using gelshift assays, two signals were identified which specifically derived from interaction of the 5′-untranslated region of FR-t.mRNA with cytosolic extracts from HeLa-IU, cells. RNase T l mapping revealed that both signals were from protein(s) interacting with two partially overlapping RNA sequences between nucleotides -133 to -116 and -158 to -116, upstream of the translation start site. Selective RNase H cleavage following hybridization with 18-mer antisense DNA complementary to the 18-base RNA fragment abolished both signals indicating that the 18-base RNA sequence is the cis-element. The interaction of this c/s-element and cytosolic protein was competed by poly(C), but not by poly(U), homopolymers. Cross-linking studies using ultra-violet light and Northwestern blot analysis confirmed that the 18-base cis-element specifically bound -46-kDa proteins. From the standpoint of assigning a function to the interaction of this cu-element and Svw-factor within the context of FR and folate metabolism, preliminary studies revealed that although these 46-kDa proteins were unaltered by extracellular folate concentrations, they were widely distributed in cells expressing little to no FR. Thus, the functional significance of this RNA-protein interaction warrants further study.

AB - Folate receptors (PR) are inversely regulated by extracellular folate concentrations. Since FR regulation is primarily mediated at the translational level in cervical carcinoma (HeLa-IU,) cells which express FR-a mRNA, the potential for interaction of ciselements (FR-0 mRNA) and Irons-factors from these cells was determined. Using gelshift assays, two signals were identified which specifically derived from interaction of the 5′-untranslated region of FR-t.mRNA with cytosolic extracts from HeLa-IU, cells. RNase T l mapping revealed that both signals were from protein(s) interacting with two partially overlapping RNA sequences between nucleotides -133 to -116 and -158 to -116, upstream of the translation start site. Selective RNase H cleavage following hybridization with 18-mer antisense DNA complementary to the 18-base RNA fragment abolished both signals indicating that the 18-base RNA sequence is the cis-element. The interaction of this c/s-element and cytosolic protein was competed by poly(C), but not by poly(U), homopolymers. Cross-linking studies using ultra-violet light and Northwestern blot analysis confirmed that the 18-base cis-element specifically bound -46-kDa proteins. From the standpoint of assigning a function to the interaction of this cu-element and Svw-factor within the context of FR and folate metabolism, preliminary studies revealed that although these 46-kDa proteins were unaltered by extracellular folate concentrations, they were widely distributed in cells expressing little to no FR. Thus, the functional significance of this RNA-protein interaction warrants further study.

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