Size Distribution of Rat Liver Nuclear RNA Containing mRNA Sequences

Abstract

Total rat liver poly(A)-containing polysomal mRNA was size-fractionated on polyacrylamide gels in 98 % formamide. Complementary DNA (cDNA) was prepared from the 8 -14-S mRNA fraction and separated into sequences representing abundant and non-abundant mRNAs. The cDNA complementary to the abundant small mRNA of the rat liver cell (approximately 20 species) was hybridized to nuclear RNA of different lengths to determine the size distribution of nuclear RNA molecules which contain these messenger sequences. It was found that :1. All abundant 8 -14 -S poly(A)-containing mRNAs have larger nuclear precursor molecules ; 20% of the different messenger sequences are found in nuclear RNA of several times their cytoplasmic length.2. 70 % of the mass of the examined nuclear messenger sequences is in RNA molecules of a size similar to their polysomal mRNA; 30% are in larger than 18-S RNA and 2% are between 37 S and 44 S.3 . The majority of small messenger-containing RNA molecules in the RNA prepared from isolated nuclei are of true nuclear origin, since their frequency distribution differs significantly from that of the polysomal 8 -14-S mRNA.To understand the regulation of gene expression in eucaryotic cells it is crucial to know more about nuclear processes involved in the biogenesis of mRNA. Several features of the synthesis and structure of hnRNA indicate its role in mRNA metabolism (for review see [l-51). Indirect evidence first led to the postulate that hnRNA contained precursor molecules to mRNA [6,7]. Hybridization-competition experiments later demonstrated base-sequence homology between polyribosomal mRNA and hnRNA from HeLa cells [8]. Furthermore, it was found that viral-specific RNAs in and polyoma-infected [lo] cells are transcribed as giant RNA molecules. However, owing to the enormous sequence complexity, the large size and the high turnover of hnRNA, a detailed describtion of the nuclear pathway of messenger sequences has not yet Abbreviations. Hepes. 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; AMV, avian myeloblastosis virus; cDNA, DNA copy complementary to mRNA ; hnRNA, polydisperse high-molecularweight nuclear RNA; rot value, concentration of RNA (as mol phosphate/l) times time of hybridization (in s); rotllz value, rot value at which half the hybridizable cDNA is hybridized; vgt value, RNA derived from the amount of liver tissue (g), hybridized in IO-pI assay volume, times the hybridization time (h).Enzyme. S 1 nuclease, single-strand-specific endonuclease from Aspergillus oryzae (EC 3.1.4.21).been possible. The use of molecular hybridization with complementary DNA copies of specific mRNA molecules to identify messenger sequences in steadystate nuclear RNA has resulted in an increased amount of new information, but no general picture has emerged from these studies. For example, larger nuclear precursor RNA molecules for globin mRNA have been found, although with conflicting results as to their largest size [ l l -171. For the case of ovalbumin mRNA, no larger nuclear RNA containing the messenger se...

Intracellular Distribution of Rat-Liver Polyribosomes Synthesizing Coeruloplasmin

“…It should be noted that about 90% of the intracellular pool of cytoplasmic coeruloplasmin mRNA is engaged in translation and only a minor fraction is recovered from the non-polysomal compartment. Similar values were obtained by independent methods of calculation, assuming that each h e r cell contains 180000 molecules of polyadenylated polysomal mRNA with a weightaverage molecular weight of 560000 [20]. The estimated proportion of coeruloplasmin mRNA sequences in poly(A)-containing RNA from liver polysomes is 0.29 % (see Table 2) and the molecular weight of coeruloplasmin mRNA is 1 .I3 x lo6 [7,8].…”

“…In order to calculate the proportion of coeruloplasminsynthesizing ribosomes in the total population of translating ribosomes in the rat liver, it was assumed that: (a) each liver cell contains 180000 molecules of polysomal mRNA and each average mRNA molecule is translated simultaneously by nine ribosomes [20]; (b) each liver cell contains 327 molecules of coeruloplasmin mRNA, each one being translated by 18 ribosomes. Hence, the total number of translating ribosomes is 9 x 180000 = 1620000/cell while the number of polysomes engaged in coeruloplasmin synthesis is 18 x 327 = 5886.…”

“…If we suggest that the rates of elongation and termination are equal for different mRNAs, an expected proportion of the newly formed coeruloplasmin in the total product of rat liver protein synthesis could be calculated. A single round of translation of a population of polysomal mRNAs results in the release of 180000 different polypeptides with a weight-average molecular weight of 33000 [20]. If a concentration of translated coeruloplasmin mRNA is 327 molecules/cell (see above) and the molecular weight of procoeruloplasmin released from polysomes in vivo is 80000 [I41 the expected proportion of coeruloplasmin is 0.44% of the total yield of product per round of translation.…”

Polypyrimidine tracts isolated from inverted repeat sequences of rat DNA containing the repeat sequence d(CTC)

“…The steady-state nuclear RNA from rat liver nuclei was prepared according to [24]. The size of the RNA, determined by gel electrophoresis on 2% agarose under denaturing conditions (6 M urea, pH 3.5) [25], was between 28 and 35 S, i.e., in the range described [24].…”

Repression of the albumin gene in Novikoff hepatoma cells.

“…Polysomes were prepared from both rat liver and Novikoff hepatoma by a modification of the method described by Sippel et al (34). Fresh tissue was homogenized in 6 volumes of 140 mM sucrose and 2 mg of heparin per ml in polysomal buffer (25 mM Tris-hydrochloride [pH 7.5], 25 mM NaCl, 5 mM MgCl2), and then Triton X-100 and sodium deoxycholate were added successively to a final concentration of 0.5%.…”