Proteins associated with poly(A) and other regions of mRNA and hnRNA molecules as investigated by crosslinking

The proteins associated with poly(A) and other regions of mRNA and hnRNA molecules in mouse L cells were investigated with the aid of ultraviolet light-induced crosslinking of proteins to RNA. The poly(A)s of polyribosomal and free cytoplasmic mRNAs are associated with a protein, p78A. In contrast, the poly(A) of hnRNA is associated with a smaller protein, p60A, that differs from p78A in its partial peptide map. p78A occurs free in the cytoplasm, but p60A does not. There is a second 78 kd protein, p78X, associated with mRNA sequences other than poly(A). p78X differs from p78A in its partial peptide map. The total proteins crosslinked to polyribosomal and free cytoplasmic mRNAs are similar. However, the total proteins crosslinked to hnRNA are quite different from those crosslinked to mRNA. We suggest that newly synthesized mRNA molecules emerging from the nucleus into the cytoplasm shed the proteins with which they were associated in the nucleus and become associated with a new set of proteins derived from the cytosol. Furthermore, the cytoplasmic mRNA-associated proteins continue to exchange with free proteins.     

Metabolic heterogeneity of nuclear poly (A)-containing RNA in mouse liver.

The analysis by the approach to equilibrium labeling method has shown that the poly(A)+ fraction of liver hnRNA is not a uniform class of molecules, but is comprised of two distinct subclasses with half-lives of 5 and 60 min, while the poly(A)- hnRNA was metabolically homogeneous and turned over with a rather uniform half-life of 30 min. The results suggest that (a) poly(A) synthesis and addition is not limiting for the rate of hnRNA processing, and (b) there is a correlation between the kinetics of mRNA appearance in the cytoplasm and kinetic behavior of their possible nuclear precursors.     

Oligo(A) and double-stranded segments in polyadenylated and non-polyadenylated RNA from cytoplasm and nuclei of chick embryo

Chick embryonic RNA was fractionated by affinity chromatography on oligo(dT)-cellulose and poly(U)-Sepharose into three classes: poly(A)+RNA containing poly(A) segments of 100 and more residues, poly(A)-oligo(A)+RNA containing oligo(A) segments of about 25 residues, and poly(A)-oligo(A)-RNA which bound to neither of the beds used and which contained double-stranded segments of 300 and more base pairs. These three classes of RNA were found in cytoplasmic as well as in heterogeneous nuclear RNA. Double-stranded segments in hnRNA, unlike those in cytoplasmic RNA, were intermolecular in nature; this may explain the occurrence of "giant" molecules in hnRNA.     

Analysis of the message-sequence content of the pulse-labeled poly(A)+ heterogeneous nuclear RNA from HeLa cells by cDNA-excess hybridizations.

The message-sequence content of pulse-labeled poly(A)+ HeLa heterogenous nuclear RNA (hnRNA) has been examined by hybridizations to an excess of message cDNA. Control experiments show that the message cDNA accurately reflects the sequence distribution of the complex mixture of poly(A)+ messages present in the HeLa cytoplasm. Pulse-labeled poly(A)+ molecules in both the lamina-associated and shnRNA fractions contain message sequences, and approximately 65% of the poly(A)-adjacent hnRNA sequences are homologous to the 3' ends of mRNA. The majority of the pulse-labeled hnRNA molecules contain abundant message sequences. By use of these techniques it is also shown that some pulse-labeled polyadenylated message sequences are still synthesized in the presence of the adenosine analogue 5,6-dichloro-beta-D-ribofuranosylbenzimidazole under conditions where little or no new cytoplasmic mRNA is produced.     

5'-terminal structures of poly(A)+ cytoplasmic messenger RNA and of poly(A)+ and poly(A)- heterogeneous nuclear RNA of cells of the dipteran Drosophila melanogaster.

Structures at the 5′ terminus of poly (A)-containing cytoplasmic RNA and heterogeneous nuclear RNA containing and lacking poly(A) have been examined in RNA extracted from both normal and heat-shocked Drosophila cells. ³²P-labeled RNA was digested with ribonucleases T₂, T₁ and A and the products fractionated by a fingerprinting procedure which separates both unblocked 5′ phosphorylated termini and the blocked, methylated, “capped” termini, known to be present in the messenger RNA of most eukaryotes.Approximately 80% of the 5′-terminal structures recovered from digests of poly(A)-containing Drosophila mRNA are cap structures of the general form m⁷G^5′ppp^5′X(m)pY(m)pZp. With respect to the extent of ribose methylation and the base distribution, the 5′-terminal sequences of Drosophila capped mRNA appear to be intermediate between those of unicellular eukaryotes and those of mammals. Drosophila is the first organism known in which type 0 (no ribose methylations), type 1 (one ribose methylation), and type 2 (two ribose methylations) caps are all present. In contrast to mammalian cells, the caps of Drosophila never contain the doubly methylated nucleoside N⁶,2′-O-dimethyladenosine. Both purines and pyrimidines can be found as the penultimate nucleoside of Drosophila caps and there is a wide variety of X-Y base combinations. The relative frequencies of these different base combinations, and the extent of ribose methylation, vary with the duration of labeling. The large majority of poly(A)-containing cytoplasmic RNA molecules from heat-shocked Drosophila cells are also capped, but these caps are unusual in having almost exclusively purines as the penultimate X base.Greater than 75% of the 5′ termini of heterogeneous nuclear RNA (hnRNA) containing poly(A) and greater than 50% of the termini of hnRNA lacking poly (A) are also capped. Triphosphorylated nucleotides, common as the 5′ nucleotides of mammalian hnRNA, are rare in the poly(A)-containing hnRNA of Drosophila. The frequency of the various type 0 and type 1 cap sequences of cytoplasmic and nuclear poly (A)-containing RNA are almost identical. The caps of hnRNA lacking poly(A) are also quite similar to those of poly-adenylated hnRNA, but are somewhat lower in their content of penultimate pyrimidine nucleosides, suggesting that these two populations of molecules are not identical.     

Biosynthesis and utilization of extensively undermethylated poly(A)+ RNA in CHO cells during a cycloleucine treatment.

The role of RNA methylations in the control of mRNA maturation and incorporation into polysomes has been investigated through a study of the effects in vivo of cycloleucine, a specific inhibitor of S-adenosyl-methionine mediated methylation. During the cycloleucine treatment, the rate of biosynthesis of hnRNA and its subsequent polyadenylation were only slightly reduced as compared with untreated cells. However a significant lag-time in the cytoplasmic appearance of poly(A)+ undermethylated molecules was observed, in parallel with a transient shift in the average size of hnRNA towards higher molecular weight. Nevertheless, the total amount of pulse-labelled poly(A)+ mRNA transferred to cytoplasm after a long chase time (3 h.) was approximately the same for both cycloleucine-treated and control cells. Extensively undermethylated poly(A)+ cytoplasmic RNAs, possessing a 5' terminal cap were incorporated into polysomes in proportions very similar to control messenger molecules. These results suggest that a normal level of methylation is not stringently required for the production of the functional mRNA molecules although it appears to be of importance for the kinetics of the maturational process.     

Formation and spectral characterization of Cu(II)-poly(L-ornithine) complexes.

Cu(II)-Poly-(1-ornithine) complexes in aqueous solution have been studied using potentiometric titration and absorption and circular dichroism spectra. As in the case of Cu(II)-poly(L-arginine) complexes studied previously, two types of compounds have been detected, labeled complexes I and II. Complex I contains two amine nitrogens and two water molecules coordinated to the copper. Complex II, two amine and two amide nitrogens. Amide nitrogen coordination confers optical activity to the copper d-d transitions. Furthermore, amine and amide nitrogen coordination to the copper are characterized by charge transfer transitions at 250 and 320 nm respectively which were already identified in Cu(II)-poly(L-arginine) systems.     

Poly(A) shortening and degradation of the 3'' A+U-rich sequences of human c-myc mRNA in a cell-free system.

The early steps in the degradation of human c-myc mRNA were investigated, using a previously described cell-free mRNA decay system. The first detectable step was poly(A) shortening, which generated a pool of oligoadenylated mRNA molecules. In contrast, the poly(A) of a stable mRNA, gamma globin, was not excised, even after prolonged incubation. The second step, degradation of oligoadenylated c-myc mRNA, generated decay products whose 3' termini were located within the A+U-rich portion of the 3' untranslated region. These products disappeared soon after they were formed, consistent with rapid degradation of the 3' region. In contrast, the 5' region, corresponding approximately to c-myc exon 1, was stable in vitro. The data indicate a sequential degradation pathway in which 3' region cleavages occur only after most or all of the poly(A) is removed. To account for rapid deadenylation, we suggest that the c-myc poly(A)-poly(A)-binding protein complex is readily dissociated, generating a protein-depleted poly(A) tract that is no longer resistant to nucleases.     

Antisera to poly(A)-poly(U)-poly(I) contain antibody subpopulations specific for different aspects of the triple helix.

Rabbit antibodies to the triple-helical polynucleotide poly(A)-poly(U)-poly(I) were fractionated into three major antibody populations, each recognizing a different conformational feature of the triple-helical immunogen. Two distinct populations were purified from precipitates made with poly(A)-poly(U)-poly(U) and poly(A)-poly(I)-poly(I). The former reacted with double-stranded poly(A)-poly(U) or poly(I)-poly(C), and similar populations could be purified with either double-stranded form. The second population recognized the poly(A)-poly(I) region of the triple helix, and the third required all three strands for reactivity. These immunochemical studies suggest that the poly(A) and poly(U) have the same orientation in the triple-helicical poly(A)-poly(U)-poly(I) as in the double-helical poly(A)-poly(U), in which they have Watson-Crick base pairing.     

Complexity and characterization of polyadenylated RNA in the mouse brain

The complexity of nuclear RNA, poly(A)hnRNA, poly(A)mRNA, and total poly(A)RNA from mouse brain has been measured by saturation hybridization with nonrepeated DNA. These DNA populations were complementary, respectively, to 21, 13.5, 3.8, and 13.3% of the DNA. From the RNA Cot required to achieve half-sturation, it was estimated that about 2.5–3% of the mass of total nuclear RNA constituted most of the complexity. Similarly, complexity driver molecules constituted 6–7% of the mass of the poly(A)hnRNA. 75–80% of the poly(A)mRNA diversity is contained in an estimated 4–5% of the mass of this mRNA. Poly(A)hnRNA constituted about 20% of the mass of nuclear RNA and was comprised of molecules which sedimented in DMSO-sucrose gradients largely between 16S and 60S. The number average size of poly(A)hnRNA determined by sedimentation, electron microscopy, or poly(A) content was 4200–4800 nucleotides. Poly(A)mRNA constituted about 2% of the total polysomal RNA, and the number average size was 1100–1400 nucleotides. The complexity of whole cell poly(A)RNA, which contains both poly(A)hnRNA and poly(A)mRNA populations, was the same as poly(A)hnRNA. This implies that cytoplasmic polyadenylation does not occur to any apparent qualitative extent and that poly(A)mRNA is a subset of the poly(A)hnRNA population. The complexity of poly(A)hnRNA and poly(A)mRNA in kilobases was 5 × 10⁵ and 1.4 × 10⁵, respectively. DNA which hybridized with poly(A)mRNA renatures in the presence of excess total DNA at the same rate as nonrepetitive tracer DNA. Hence saturation values are due to hybridization with nonrepeated DNA and are therefore a direct measure of the sequence complexity of poly(A)mRNA. These results indicate that the nonrepeated sequence complexity of the poly(A)mRNA population is equal to about one fourth that observed for poly(A)hnRNA.     

Nuclear penetration and stimulation of nucleic acids synthesis by poly(A)-poly(U) in mammalian cells

The rapid penetration of poly(A)-poly(U) into cell nuclei is shown by radioautography, by recovery of acid-precipitable material from isolated nuclei and by sucrose gradient centrifugation of nuclear lysates. The majority of poly(A)-poly(U) remains intact in the nuclei for at least h. This penetration is increased 20-fold by pretreatment of the cells with DEAE Dextran. In cells treated with DEAE Dextran, DNA and RNA syntheses are stimulated by poly(A)-poly(U) from the time the polymer complex is added and for at least h.