Histone messenger RNA from HeLa cells: evidence for modified 5' termini

Stein, Janet L.; Stein, Gary S.; McGuire, Patricia

doi:10.1021/bi00629a026

Cited by 11 publications

(6 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The cell cycle-dependent histone genes do not contain nonprotein-coding intervening sequences (Stein et al, 1984;Sierra et al, 1982;Sittman et al, 1981;Seiler-Tuyns and Birnsteil, 1981;Heintz et al, 19811, the mRNAs are not polyadenylated at 3' termini Wedes, 1979), and newly transcribed histone mRNAs leave the nucleus within several minutes after transcription (Perry and Kelley, 1968;Schochetman and Perry, 1972). The only processing of histone gene transcripts that has been documented is 5' capping (Stein et al, 1977;Moss et al, 1977) and a limited trimming of 3' sequences (Birchmier et al, 1984;Krieg and Melton, 1984;Stunnenburg and Birnsteil, 1982). Although a very low level of H4 histone mRNA was observed in the nuclear matrix fraction following long exposures of autoradiograms from S1 nuclease analysis, this amounted to less than 0.05% of the cellular tran-Faiferman, I., and Pogo, A.O.…”

Section: Representation Of Histone M R N H In Nuclear Fractionsmentioning

confidence: 99%

See 1 more Smart Citation

Localization of human histone gene transcripts predominantly in the nonmatrix nuclear fraction

Bandyopadhyay¹,

Stein²,

Stein³

1986

Journal Cellular Physiology

View full text Add to dashboard Cite

Using S1 nuclease protection assays, we have examined the representation of cell cycle-dependent H4 histone RNAs in the nuclear matrix and nonmatrix nuclear fractions of human cells. Cytoplasmic and nuclear fractions were prepared from exponentially growing HeLa S3 cells by double detergent (sodium deoxycholate and NP40) lysis. The nuclear matrix and nonmatrix nuclear fractions were then prepared by digestion of nuclei with RNase-free DNase I and subsequent high-salt [0.4 M (NH4)2SO4] extraction. Subcellular fractions were characterized by 1) DNA, RNA, and protein composition; 2) electrophoretic analysis of the proteins in each fraction; 3) the representation of 45S ribosomal RNA precursors and processed 18S and 28S ribosomal RNAs; and 4) the presence of mitochondrial RNAs. In contrast to ribosomal and messenger RNA precursors, which are largely associated with the nuclear matrix, the human H4 histone RNAs in the nucleus were found predominantly in the nonmatrix nuclear fraction. The presence of H4 histone RNA in the nonmatrix nuclear fraction appeared to be coupled to DNA replication, since inhibition of DNA synthesis by hydroxyurea resulted in a loss of histone RNA from the nucleus. Our results suggest either that the association of histone RNAs with the nuclear matrix is very transient or that posttranscriptional modifications of the rapidly processed histone gene transcripts do not involve the nuclear matrix.

show abstract

Section: Representation Of Histone M R N H In Nuclear Fractionsmentioning

confidence: 99%

“…Transcripts are not polyadenylated and undergo only a limited amount of processing: 5' capping (Stein et al, 1977;Moss et al, 1977) and elimination of 3' sequences (Birchmier et al, 1984;Krieg and Melton, 1984). Functional mRNAs reach the polysomes within several minutes after transcription (Perry and Kelley, 1968;Schochetman and Perry, 1972).…”

mentioning

confidence: 99%

Localization of human histone gene transcripts predominantly in the nonmatrix nuclear fraction

Bandyopadhyay¹,

Stein²,

Stein³

1986

Journal Cellular Physiology

View full text Add to dashboard Cite

show abstract

“…The Pl-digested caps were fractionated in the presence of unlabeled markers by two-dimensional thin-layer chromatography (22) on cellulose thin-layer plates followed by autoradiography. This chromatography system is capable of separating all the common methylated and unmethylated nucleotides found in cap structures, as well as the core cap structures m7G5'ppp5'Gm and m7G5'ppp5'Am, which we have previously shown to be the only core cap structures found in a preparation containing H3, H2A, H2B, and H4 histone mRNAs (26). Radioactive material associated with spots A, B, and C of H4-1 and H4-2 histone mRNAs (Fig.…”

Section: Resultsmentioning

confidence: 99%

Multiple forms of H4 histone mRNA in human cells.

Lichtler¹,

Detke²,

Phillips³

et al. 1980

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

View full text Add to dashboard Cite

Two seies of H4 histone mANA were isolated from the polysomes of S phase HeLa S3 cells. Electrophoresis under denaturing and nondenaturing conditions indicates that the two H4 mRNAs differ in size. Both mRAs translate H4 histones in vitro, lack poly(A) at their 31 termini, and are capped at their 5' termini. Polyacrylamide gel electrophoresis and tryptic peptide analysis suggest. that the Polypeptides synthesized by the two mRNAs are indistinguishable. Histones play an important role in structural and transcriptional properties of the eukaryotic genome (reviewed in refs. 1-10). Hence, over the past several years a considerable amount of attention has been focused on histone genes and on the regulation of histone gene expression. Histone genes are intriguing from a biological standpoint for a number of reasons. Of particular importance are the presence of five classes of histone polypeptides and the transcription of histone mRNAs from reiterated genes. The fact that there are multiple copies of histone genes raises important considerations regarding the identity and functional significance of the histone sequences. Have all copies of the genes coding for individual histones been conserved? Are all copies of the histone genes expressed simultaneously? While fractionating histone mRNAs with the objective of isolating mRNAs for individual histones, we isolated from the polysomes of S phase HeLa cells two mRNAs that code for the arginine-rich H4 histone. We have therefore been addressing the questions of whether the two mRNAs are distinctively different species and whether there are variations in the proteins coded by the H4 histone mRNAs. MATERIALS AND METHODS Preparative Polyacrylamide Gel Electrophoresis of HeLaCell Histone mRNA. 32P-Labeled polysomal RNA was isolated from 1 liter of S phase HeLa cells synchronized by double thymidine block (11). Cells were lysed in 20 mM Tris-HCI, pH 7.5/25 mM NaCl/5 mM MgCl2/0.1 mM spermidine/1% deoxycholate/1% Triton X-100 and centrifuged at 8000 X g for 10 min. The supernatant was centrifuged through a cushion of 2 M sucrose in lysis buffer without detergent at 100,000 X g for 2 hr, and the polysomal RNA pellet was extracted with phenol/chloroform/isoamyl alcohol (12). 7-11S RNA was isolated on a 5-30% sucrose gradient as described (12). Unlabeled 7-1lS RNA was isolated by using similar procedures from 30 liters of S phase HeLa cells, mixed with 32P-labeled RNA, and electrophoresed on a 6% polyacrylamide slab gel polymerized in 36 mM Na2HPO4/30 mM Tris-HCl/2 mM EDTA/0.2% NaDodSO4 (at pH 7.8) as described (12, 13). Bands were located by autoradiography and excised and the RNA was eluted electrophoretically (13).Cell-Free Protein Synthesis. Cell-free translation of the excised bands was carried out according to Weber et al. (14) with 20 ,Ci of [3H]lysine and 1-5 .ug of RNA per 50-Al reaction mixture. Aliquots of the translation system were mixed with unlabeled HeLa cell marker histones, dialyzed against 0.9 M acetic acid/2.5 M urea/0.4% 2-mercaptoethanol, and electropho...

show abstract

“…At least one H 4 histone mRNA species that is observed in WI-38 cells is not represented in the polysomal RNA of HeLa cells, suggesting a possible difference in expression of H 4 histone mRNA species in different human cell types. 0 We have begun to assign H4 histone mRNA species to individual H 4 histone genes using a modification of the Berk and Sharp procedure.1° As shown in FIGURE 8, when H 4 histone mRNA species collectively were annealed with cloned genomic H4 histone sequences, each H4 histone gene formed an S1-nuclease-resistant hybrid with only one H4 mRNA. These results provide convincing evidence that the various H4 histone mRNA species do not represent post-transcriptional processing.…”

Section: Structure and Organization Of Human Histone Genesmentioning

confidence: 99%