Poly(uridylic acid) sequences in messenger ribonucleic acid of HeLa cells

El, Korwek; Nakazato, N; Edmonds, Mary; Venkatesan, Sundararajan

doi:10.1021/bi00666a015

Cited by 39 publications

(24 citation statements)

References 20 publications

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“…We find that oligo(U)-rich sequences of 15-50 nucleotides are basepaired with hnRNA-derived poly(A) in a pancreatic RNaseresistant form that can be purified by oligo(dT)-cellulose chromatography. The size of these oligo(U) tracts is similar to that of U-rich sequences previously identified in total (deproteinized) HeLa hnRNA under conditions where a base-paired configuration with poly(A) would not have been preserved (12,19). Our results also suggest, but do not conclusively demonstrate, that proteins are associated with these oligo(U)-poly(A) duplex regions in hnRNP.…”

Section: Isolation Of Uridylate-rich Sequences Bound To Poly(a)supporting

confidence: 69%

Heterogeneous nuclear RNA secondary structure: oligo (U) sequences base-paired with poly (A) and their possible role as binding sites for heterogeneous nuclear RNA-specific proteins.

Kish¹,

Pederson²

1977

Proc. Natl. Acad. Sci. U.S.A.

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HeLa cell heterogeneous nuclear RNA derived from high-molecular-weight nuclear ribonucleoprotein (RNP) particles contains oligo(U) sequences of 15-50 nucleotides base-paired with poly(A). These duplexes are resistant to pancreatic RNase at 0.5 M NaCI in native RNP, remain so after chemical deproteinization of the RNP digests, and then copurify with poly(A)on oligo(dT)cellulose chromatography. (1,2). We have shown that poly(A)-rich sequences in HeLa cell hnRNA and polyribosomal mRNA are complexed with specific proteins (3, 4), and we have also found that a protein of similar molecular weight (74,000) is bound to poly(A) sequences in hnRNA of the cellular slime mold, Dietyostelium discoideum (5). However, our results (3-5) indicated that the poly(A) tracts were not protein-covered in their entirety. We therefore predicted that these proteins must be bound to nonpoly(A) sequences covalently or hydrogen-bonded to poly(A). In this paper-we show that oligo(U) sequences 15-50 nucleotides in length are base-paired with poly(A) in HeLa hnRNA. Our controls demonstrate that these oligo(U)-poly(A) duplexes are not generated during their isolation, but are an aspect of hnRNA structure in its native RNP form. Additional results raise the possibility that these A-U duplex regions are associated with proteins in hnRNA-RNP particles, and that these proteins staAbbreviations: RNP, ribonucleoprotein; hnRNA, heterogeneous nuclear RNA; TEN, 10 mM Tris-HCI, pH 7.5/10 mM Na2EDTA/0.5 M NaCl. * This is the sixth paper in a series entitled "Ribonucleoprotein organization of eukaryotic RNA." The preceding paper in this series is ref. bilize the oligo(U)-poly(A) duplexes at physiological ionic strength.MATERIALS AND METHODS The methods for growth and isotopic labeling of HeLa cells, isolation of hnRNP particles, RNase digestion of hnRNP, oligo(dT)-cellulose chromatography, base compositional analysis, and formamide/polyacrylamide gel electrophoresis have all been described in detail (6). Specific experimental conditions are given in the table and figure legends. RESULTS Isolation of Uridylate-Rich Sequences Bound to Poly(A).The point of departure for the present investigation was our observation that "poly(A)"-protein complexes isolated from HeLa cell hnRNP or mRNP particles contained a substantial fraction of UMP residues (18-24 mole %) (3, 4). The resistance of this UMP-rich material to pancreatic RNase could be explained either by complementary base-pairing with poly(A) or, alternatively, by a covalent linkage to poly(A) in combination with protection of the UMP-rich sequences against pancteatic RNase by proteins. Our present results support the first of these alternative explanations and also suggest that these base-paired A-U sequences are likely to be binding sites for proteins.hnRNP particles were isolated from HeLa cells pulse-labeled In the absence of proteins, there are two possible sources of pancreatic RNase-resistant [3H]uridine-labeled hnRNA: double-stranded sequences containing all four nucleotides and oligo(U) se...

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Section: Isolation Of Uridylate-rich Sequences Bound To Poly(a)supporting

confidence: 69%

Heterogeneous nuclear RNA secondary structure: oligo (U) sequences base-paired with poly (A) and their possible role as binding sites for heterogeneous nuclear RNA-specific proteins.

Kish¹,

Pederson²

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…The importance of the 5'-terminal cap structure in translation has been extensively reviewed by Shatkin (1 976), and reduced cap content was proposed to account for the inefficient translation of postpolysomal mRNA from mouse kidney cells (Ouellette et al, 1982). However, in the preceding paper, we have shown that oligo(U+) RNA from HeLa cells is not inherently less capped than total mRNA (Kulkosky et al, 1985). Reduced 5'-cap content was not the reason for the lowered translational efficiency of postpolysomal RNA from developing sea urchin embryos (Rudensey & Infante, 1979).…”

Section: Discussionmentioning

confidence: 77%

Sequence content of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

Wood¹,

Wallace²,

Edmonds³

1985

Biochemistry

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Oligo(uridylic acid)-containing [oligo(U+)] RNA was isolated from poly(adenylic acid)-containing [poly(A+)] mRNA from HeLa cells by using either formaldehyde pretreatment or poly(A) removal, both of which resulted in increased accessibility of oligo(U)-rich sequences to a poly(A)-agarose affinity column. In this report, we compared the sequence content of oligo(U+) RNA with that of molecules lacking oligo(U) [oligo(U-) RNA] by their relative hybridization to cDNA reverse-transcribed from poly(A+) mRNA and by comparison of their in vitro translation products synthesized in a rabbit reticulocyte lysate. Formaldehyde-modified poly(A+) RNA, treated to remove the formol adjuncts, was inactive as a template for in vitro protein synthesis; consequently, only depolyadenylated RNA, which retains its translatability, could be used in the translation studies. The hybridization kinetic experiments revealed that oligo(U+) RNA contained most of the sequence information present in oligo(U-) RNA but at a reduced level (ca. 25%), the majority of the oligo(U+) RNA sequences being poorly represented in the cDNA. This result was supported by one- and two-dimensional gel analysis of their in vitro translation products which showed that oligo(U+) RNA, although less effective as a template for translation than oligo(U-) RNA, coded for proteins, the most abundant of which were encoded by rare messages not highly represented in oligo(U-) RNA or the total poly(A+) RNA. Although some minor products were synthesized by both oligo(U+) and oligo(U-) RNA, at least 33 proteins were unique to or highly enriched in the pattern of products directed by oligo(U+) RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Approximately 8 x 107 cells in late logarithmic phase were labeled with 50 mCi of 32P04 (1 Ci = 37 GBq) at a concentration of4 x 106 cells per ml for 4 hr as described (1). Cell fractionation was by the method of Penman (2), except that the DNase step was omitted and the supernatant from the detergent wash ofthe nuclei was discarded.…”

Section: Methodsmentioning

confidence: 99%

Polyadenylylated nuclear RNA contains branches

Wallace

Edmonds

1983

Proc. Natl. Acad. Sci. U.S.A.

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121

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A highly charged component can be isolated from a total RNase T2 digest of nuclear polyadenylylated RNA from HeLa cells that is separable from caps by (dihydroxyboryl)aminoethyl-cellulose chromatography. Chemical and enzymatic analyses show that the component contains a 2'-5' phosphodiester bond that creates a branch at the 2'-hydroxyl group of one nucleotide already linked to an adjoining nucleotide through the usual 3'-5' phosphodiester bond.p_5'y3'_p N3-P-5'X2'/ This structure was confirmed by analysis of a similar component isolated from nuclease P1 digests of the same nuclear polyadenylylated RNA. Branches occur in roughly 10% of nuclear polyadenylylated RNAs, including those >1OS in size, but are absent from cytoplasmic polyadenylylated RNA. Possible implications for branches as intermediates in mRNA processing are discussed.The processing ofnuclear transcripts into mature mRNA in eukaryotic cells involves a variety of covalent modifications that include polyadenylylation, capping, methylation, and splicing of RNAs. The transient existence of the intermediates in these cellular processes and the low concentrations of individual mRNA precursors has greatly restricted their isolation in quantities sufficient for chemical characterization.The polyadenylylated heterogeneous nuclear RNA (hnRNA) of animal cells represents a precursor population containing many different mRNA sequences. It is sufficiently long lived to provide a source of labeled RNAs that undergo similar covalent modifications and can be examined for processing intermediates free of artifacts associated with the use of inhibitors.During an investigation of the "cap" structures in RNase digests of polyadenylylated nuclear RNA, we detected another highly negatively charged component. Chemical and enzymatic analyses indicate that it is derived from a RNA molecule containing a 2'-5' phosphodiester bond at the 2'-OH group of a nucleotide already joined by the normal 3'-5' phosphodiester bond to an adjacent nucleotide. The absence of such structures from polyadenylylated cytoplasmic mRNA raises the possibility that branched RNA may arise during RNA processing in the cell nucleus. We describe here the isolation and characterization of the structure at which RNA branching occurs. METHODSApproximately 8 x 107 cells in late logarithmic phase were labeled with 50 mCi of 32P04 (1 Ci = 37 GBq) at a concentration of4 x 106 cells per ml for 4 hr as described (1). Cell fractionation was by the method of Penman (2), except that the DNase step was omitted and the supernatant from the detergent wash ofthe nuclei was discarded. The cytoplasmic fraction was freed of mitochondria by centrifugation for 10 min at 10,000 x g. Nuclear and cytoplasmic RNA was extracted by the hot phenol/ NaDodSO4 method (3).Poly(A)+RNA was obtained by three cycles of binding to oligo(dT)-cellulose (P-L Biochemicals, type 7) as described (4). The 60°C heating step was included for each binding to minimize contamination with nonpolyadenylylated RNA. The efficiency of the third bind...

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Poly(uridylic acid) sequences in messenger ribonucleic acid of HeLa cells

Cited by 39 publications

References 20 publications

Heterogeneous nuclear RNA secondary structure: oligo (U) sequences base-paired with poly (A) and their possible role as binding sites for heterogeneous nuclear RNA-specific proteins.

Heterogeneous nuclear RNA secondary structure: oligo (U) sequences base-paired with poly (A) and their possible role as binding sites for heterogeneous nuclear RNA-specific proteins.

Sequence content of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

Polyadenylylated nuclear RNA contains branches

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