Demonstration of Globin Messenger Sequences in Giant Nuclear Precursors of Messenger RNA of Avian Erythroblasts

Abstract

Highly purified globin mRNA from ducks was copied with RNA-directed DNA polymerase from avian myeloblastosis virus into anti-messenger DNA. With excess RNA, more than 90% of this DNA annealed back to its template with a Cot/2 value of 7.5 X 10-4 mol-sec* liter-'; the melting temperature of the hybrid was 860. Giant nuclear RNA fractions with sedimentation coefficients of more than 50 S formed hybrids of almost equal stability at Cot/2 values of 0.05-0.42 mol sec liter-1, indicating amRNA content of 0.3-1.5%. 12S RNA from the same polyribosomes and nuclear giant RNA from HeLa cells did not cross-hybridize. Although a large part of the giant RNA broke down in 99% dimethylsulfoxide gradients, RNA fractions sedimenting faster than 28S rRNA still were found to consist of up to 0.03% globin mRNA sequences. Thus, the mRNA sequences are contained in the covalent structure of giant nuclear precursors, which are termed precursor-mRNA.Since the first observation of giant messenger-like RNA with a molecular weight of more than 3.5 X 106 (miRNA) (1)(2)(3)(4)(5) in the nuclei of animal cells, the function of this class of RNA has been questioned. Indirect evidence first led us to postulate that this RNA was a precursor to mRNA (3,4,6), in analogy to the nucleolar precursor of ribosomal RNA (rRNA) (1,7,8). High Cot hybridization-competition experiments demonstrated base-sequence homology between polyribosomal mRNA and giant nascent mIRNA from HeLa cells (9). Viralspecific RNA in SV40-transformed (10) and polyoma-infected (11) cells are transcribed as giant RNA molecules several times the size of the viral genome.We report here direct evidence that giant nascent mlRNA does indeed contain the same nucleotide sequence as functional mRNA. Globin mRNA from ducks was transcribed by RNA-directed DNA polymerase (12,13) from oncornaviruses into anti-messenger DNA (amDNA). This DNA could be annealed with giant nascent nuclear RNA, forming a hybrid almost as stable as that with the 9S mRNA itself. METHODS9S and 1RS Polyribosomal ImRNA Was Prepared from immature duck erythrocytes as described (14), followed by hot phenol extraction, DNase treatment (5 ug/ml, 30 min, 00), and elimination of DNase by Macaloid adsorption. The 9S preparations thus obtained directed the synthesis of all duck Abbreviations: pre-mRNA, precursor to messenger RNA [replaces the largely synonymous mlRNA (6), HnRNA (5), and D-RNA (1, 2)]; rRNA, ribosomal RNA; pre-rRNA, precursor to rRNA; amDNA, DNA strand copied from mRNA by the RNAdirected DNA polymerase (anti-messenger DNA); Hb, hemoglobin; AMV, avian myeloblastosis virus; Tm, melting tempera- Al for up to 2 days. Hybrid formation was assayed by rinsing the incubation mixture into 200 ul of 30 mM Na-acetate (pH 4.5), 0.15 M NaCl, 1 mM ZnSO4, and 5 ug of denatured salmon-sperm DNA, containing 2 units/ml of partially purified DNase Si (18). Incubation was for 90 min at 370, followed by trichloroacetic acid precipitation and assay for radioactivity. The (Fig. 1). With relatively low concentrations (40-50 MM...

Size Distribution of Rat Liver Nuclear RNA Containing mRNA Sequences

“…The experiments published at present have been performed only with specific messenger sequences. Using cDNA prepared from globin mRNA [12, hnRNA molecules of various sizes appear to contain globin messenger sequences. Whereas the existence of a 15-S globin mRNA nuclear precursor in mouse Friend-virus transformed cells was recently established [17], ovalbumin messenger sequences in the chicken oviduct can be identified only in molecules of mRNA size [18].…”

The Secondary Structure and Poly(A) Content of Globin Messenger RNA as a Pure RNA and in Polyribosome-Derived Ribonucleoprotein Complexes

“…This is based on the analysis of the mRNA by high-resolution gel electrophoresis [12], as well as on the specific activity of the mRNA in translation and the kinetics of back-hybridization [39] of the specific anti-messenger DNA (obtained with RNA-dependant DNA polymerase). A similar estimation was recently reported for rabbit globin mRNA preparations [29].…”

Intergenic transcription and transinduction of the human β-globin locus

“…Transcription has been detected both in a microlocus fragment containing HS1-HS4 linked together and in HS2 as part of a chimeric construct, raising the possibility that the ␤-globin LCR is transcribed (Collis et al 1990;Tuan et al 1992). In addition, the existence of large transcripts from the avian and murine ␤-globin clusters, up to seven times longer than the globin mRNAs, was described over two decades ago (Imaizumi et al 1973;Bastos and Aviv 1977). Although the human ␤-globin cluster represents one of the best characterized gene loci in eukaryotes, the extent of transcription throughout the locus has not been directly investigated.…”