Secondary structure in heterogeneous nuclear RNA: Involvement of regions from repeated DNA sites

Jelinek, Warren R.; Molloy, George R.; Fernández-Muñoz, Rafael; Salditt, M.; Darnell, James

doi:10.1016/0022-2836(74)90597-x

Cited by 96 publications

(33 citation statements)

References 18 publications

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“…After 25 min, [ 3Hluridine (25 mCi/ILmol ; 200 ,uc/ml) was added and samples (2 x 10' cells/sample) were taken at indicated times and total nuclear RNA extracted. The RNA was treated for 30 min at 0°C with 0.2 M NaOH in ETS (0.01 M EDTA, 0.01 M Tris, and 0.12% SDS) as described (23) to reduce the RNA chains to an average length of 500 t 250 nucleotides (confirmed by gel analysis, data not shown; see Fig. 2 in reference 23) .…”

Section: Resultsmentioning

confidence: 99%

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Addition of poly(A) to nuclear RNA occurs soon after RNA synthesis.

Salditt-Georgieff¹,

Harpold²,

Sawicki³

et al. 1980

The Journal of Cell Biology

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A kinetic analysis of the appearance of [3H]uridine label in RNA sequences that neighbor poly(A), as well as the incorporation of [3H]adenosine label into both the RNA chain and the poly(A) of poly(A)-containing molecules, shows that poly(A) is added within a minute or so after RNA chain synthesis in Chinese hamster ovary cells and HeLa cells. Previous conclusions by several groups (5-7) that poly(A) might be added as long as 20-30 min after RNA synthesis appear to be in error, and the present conclusion seems much more in line with several different types of recent studies with specific mRNAs that suggest prompt poly(A) addition (13-16).

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Section: Resultsmentioning

confidence: 99%

“…after 8 min of exposure to actinomycin to measure what was thought to be synthesis of new poly(A) (23) . 15% as much [3H]adenosine was incorporated in 2 min into poly(A) as in control cells.…”

Section: Effects Of Actinomycin On Poly(a) Synthesismentioning

confidence: 99%

Addition of poly(A) to nuclear RNA occurs soon after RNA synthesis.

Salditt-Georgieff¹,

Harpold²,

Sawicki³

et al. 1980

The Journal of Cell Biology

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“…Growth and labeling of cells were carried out as described by Robertson et al (29) by an extensively modified version of the procedures described earlier (14,17) for preparation of nuclei and nuclear RNA. HeLa cells (type S3) and mouse L cells were from the collection of I. Tamm, The Rockefeller University, whereas KB cells (American Type Culture Collection) were the generous gift of S. Altman, Yale University.…”

Section: Methodsmentioning

confidence: 99%

“…Roughly 5% of its total mass consists of highly repetitive, double-stranded regions approximately 300 base pairs in length. These structured regions can be analyzed by nuclease treatment (11,14,17,21,23, 28), electron microscopy (9), hybridiza- …”

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confidence: 99%

Structure and distribution of Alu family sequences or their analogs within heterogeneous nuclear RNA of HeLa, KB, and L cells.

Robertson¹,

Dickson²

1984

Mol. Cell. Biol.

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We studied the distribution of repetitive sequence elements capable offorming double-stranded regions in nuclear RNA of HeLa, KB, and L cells. In human RNA populations, we called these regions duplex Alu family RNA (dAfRNA) because they represent transcripts of the highly reiterated family of DNA regions known as "Alu family DNA" (Rubin et al., Nature (London) 284:372-374, 1980). Although the dAfRNA populations of both human cell lines (HeLa and KB) have low sequence complexity, they represent 5% of the total heterogeneous nuclear RNA and have identical fingerprints; mouse L-cell dAf-like RNA (which has a similar complexity) represents only 2% of the total heterogeneous nuclear RNA and has an entirely different fingerprint. We utilized Escherichia coli RNase III as a highly specific reagent for the recognition of RNA:RNA duplex structure. This enzyme cleaves within the six characteristic RNase Tl-resistant oligonucleotides of HeLa-and KB-cell dAfRNA (Robertson et al., J. Mol. Biol. 115:571-589, 1977). In addition, the size of heterogeneous nuclear RNA from all three cell types is reduced from greater than 32S to about 15S after RNase III treatment. We conclude that this size shift is a result of cleavage within dAfRNA regions and that such regions are present in most or all of the large RNA transcripts of these cells.The heterogeneous nuclear RNA (hnRNA) of HeLa cells ranges in size up to at least 20,000 nucleotides. Roughly 5% of its total mass consists of highly repetitive, double-stranded regions approximately 300 base pairs in length. These structured regions can be analyzed by nuclease treatment (11,14,17,21,23, 28), electron microscopy (9), hybridiza- MATERIALS AND METHODSPreparation and characterization of 32P-labeled nuclear RNA species. Growth and labeling of cells were carried out as described by Robertson et al. (29) by an extensively modified version of the procedures described earlier (14,17) for preparation of nuclei and nuclear RNA. HeLa cells (type S3) and mouse L cells were from the collection of I. Tamm, The Rockefeller University, whereas KB cells (American Type Culture Collection) were the generous gift of S. Altman, Yale University. Cell densities were concentrated 10-fold to give a final titer of 5 x 106 cells per ml during the labeling periods (3 h in the presence of 1 to 2 mCi of 32p per ml). KB and HeLa cells were preincubated for 30 min with actinomycin D (0.05 ,ug/ml) before addition of 32p. In the case of L cells, however, this concentration of actinomycin D was insufficient to inhibit synthesis of rRNA prgcursors; a concentration of 0.12 ,ug of actinomycin D per ml was therefore used.Nuclear RNA was isolated as described previously (29) and subjected to preparative sucrose density gradient centrifugation in 15 to 30% sucrose gradients in NETS buffer (0.01 M Tris-hydrochloride [pH 7.5], 0.01 M NaCl, 0.01 M EDTA, and 0.2% sodium dodecyl sulfate). dAfRNA was prepared from pooled fractions of nuclear RNA sedimenting faster than 32S by a procedure involving (i) digestion by pancreatic DN...

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“…Thus, superimposed on the simple « ribonucleosomal » model, more complex structures exist. In addition to specific protein-protein or protein-RNA interactions, RNA base-pairing can occur within the same molecule (Jelinek and Darnell, 1972 ;Jelinek et al, 1974 ;Kish and Pederson, 1977) or with other RNA (Brunei et al, 1981 ;Setyono and Pederson, 1984). All these highly organized configurations of RNP might be expected to influence processing events.…”

Section: Introductionmentioning

confidence: 99%