Comparison of proteins bound to heterogeneous nuclear RNA and messenger RNA in HeLa cells.

Ribonucleoprotein particles containing either heterogeneous nuclear RNA or polyribosomal messenger RNA were isolated from growing HeLa cells in order to compare their respective protein components. The major obstacle to analysing the proteins bound to HeLa cell mRNA proved to be the cosedimentation of a large fraction of the mRNP² particles with ribosomal subunits following puromycin or EDTA disassembly of polyribosomes. This was circumvented by oligo(dT)-cellulose chromatography, in which essentially all of the ribosomal subunits passed through the column without retention, while approximately 80% of the pulse-labeled, poly(A)-containing mRNP became bound and could be eluted with formamide. Polyacrylamide gel electrophoresis of the non-bound fraction (ribosomal subunits) revealed polypeptides between 15,000 and 55,000 molecular weight, with no detectable components greater than 55,000. The oligo-(dT)-bound mRNP contained a much simpler protein complement, consisting of three major components having molecular weights of 120,000, 76,000 and 52,000.In the case of the nuclear ribonucleoprotein particles that contain heterogeneous nuclear RNA, oligo(dT)-cellulose chromatography revealed two classes of particles. The first contained 10 to 20% of the hnRNA, did not bind to oligo(dT)-cellulose in 0.25 m-NaCl, 10 mm-sodium phosphate buffer, pH 7.0 (4 °C), and contained primarily a single polypeptide component having an estimated molecular weight of 40,000 (“informofers”). A second population of hnRNP particles comprised approximately 80% of the hnRNA, displayed strong binding to oligo(dT)-cellulose at 0.25 m-NaCl, and contained a very complex population of proteins, having molecular weights between 40,000 and 180,000, the same as unfractionated hnRNP. The results indicate that, at the resolution of gel electrophoresis and at the sensitivity of Coomassie blue dye, the proteins bound to HeLa cell hnRNA are qualitatively distinct from those bound to polyribosomal mRNA and, in addition, that the hnRNP proteins are the more complex of the two. These results are discussed in relation to the possible nucleotide sequence elements in hnRNA and mRNA to which these specific proteins are bound.     

Intermolecular duplexes in heterogeneous nuclear RNA from HeLa cells

Rapidly sedimenting hnRNA complexes contain regions of stable intermolecular duplex. Disruption of such complexes, as judged by a reduction in sedimentation rate, requires conditions sufficient to denature the duplex regions. Rapidly sedimenting molecules reappear only when the complementary sequences reanneal — that is, the formation of such complexes is dependent upon time and the concentration of homologous RNA. These experiments lead us to the conclusion that rapidly sedimenting hnRNA complexes consist of two or more largely single-stranded RNA molecules held together by short duplex regions. Precisely such structures have been visualized in the electron microscope. Rapidly sedimenting fractions of native nuclear RNA from preparative sucrose gradients consist primarily of large, multi-molecular complexes interconnected by duplex regions averaging 300 base pairs in length. Exposure of the RNA to severely denaturing conditions eliminates such complexes. Reannealing of the RNA reconstitutes complexes which are indistinguishable from those observed in preparations before denaturation.     

Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells

A variety of methylated oligonucleotides were derived from mouse L cell messenger RNA and heterogeneous nuclear RNA by digestion with specific ribonucleases, and the cap-containing oligonucleotides separated from those containing internal m⁶A by chromatography on diborylaminoethyl-cellulose. Cap-containing sequences of the type m⁷GpppX^mpG, m⁷GpppX^mpY^(m)pG, m⁷GpppX^mpY^(m) pNpG and m⁷GpppX^mpY^(m)p(Np)_{> 1}G have distinctive non-random compositions of the 2′-O-methylated constituent X^m; yet sequences of a particular type and composition occur with a remarkably similar frequency in mRNA and hnRNA². For example, approximately 20% of the cap sequences in both hnRNA and mRNA are m⁷Gppp(m⁶)A^mG, whereas less than 1% are m⁷GpppU^mpG. The high degree of similarity in cap sequences is consistent with the previously postulated precursor-product relationship between hnRNA caps and mRNA caps.The composition of the Y position in capped hnRNA molecules was determined to be (29% G, 20% A, 51% Py), which differs considerably from the composition of Y^m in the cap II forms of mRNA (8% G^m, 11% A^m, 81% Py). Given the precursor-product relationship between hnRNA caps and mRNA caps, this result provides strong evidence that only a restricted subclass of mRNA molecules receive the secondary methylation at position Y.In both hnRNA and mRNA the internal m⁶A occurs in well-defined sequences of the type: $\text{-N}{}_1\text{-(}\text{G}\text{A}\text{)-m}{}^6\text{A-C-N}{}_2\text{-}$, the 5′ nearest-neighbor of m⁶A being G in about three-quarters of the molecules and A in about one-quarter of the molecules. The nucleotide N¹ is a purine about 90% of the time and the nucleotide N² is rarely a G. These same sequences are present in large (> 50 S), as well as small (14 S to 50 S) hnRNA. These results raise the possibility that the internal m⁶A, like caps, may be conserved during the processing of large hnRNA into mRNA. Two models based on this idea are discussed.     

N-6-methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA

In the biogenesis of adenovirus type 2 messenger RNAs, methylation occurs at the 5′ end (cap) and to internal adenosine residues to yield N⁶-methyl-adenosine (m⁶A) (Sommer et al., 1976; Moss &; Koczot, 1976; Wold et al., 1976). The kinetics of accumulation of ³H from methyl-labeled methionine and ¹⁴C from uridine into Ad-2²-specific RNA was measured late in Ad-2 infection. As reported previously (Nevins &; Darnell, 1978a), the rate of accumulation of [¹⁴C]uridine label in nuclear RNA was approximately four- to fivefold faster than in the cytoplasmic RNA, indicating a conservation of about 20% for the total RNA. The initial rates of [³H]methyl label in m⁶A in nuclear RNA and in the cytoplasmic RNA were approximately equal, suggesting a complete (or nearly complete) conservation of m⁶A.In accord with the accumulation kinetics, the ratio of ³H to ¹⁴C was higher in cytoplasmic RNA than in nuclear RNA that hybridized to equivalent regions of the Ad-2 DNA.A mathematical model was designed to evaluate the accumulation of methyl label in m⁶A, taking into consideration the three major parameters that affect the accumulation curves: equilibration of the S-adenosyl-methionine pool, the nuclear dwell time of sequences destined to be mRNA, and the cytoplasmic stability of mRNA. The half-time ($\text{t}\text{1}\text{2}$) for pool equilibration was determined experimentally to be 22 minutes and the nuclear dwell time and the mean life-time of cytoplasmic mRNA were estimated from ¹⁴C label to be about 30 and 70 minutes, respectively.The model gave an excellent fit to the data when the $\text{t}\text{1}\text{2}$ for pool equilibration time of 24 ± 2 minutes, a nuclear dwell time of 25 ± 10 minutes, and a mean cytoplasmic mRNA life-time of 75 ± 30 minutes were used to evaluate accumulation curves. Even when data from a restricted region of the genome, $\text{40.5–52.6}$, which encodes the main portion of at least five 3′ co-terminal mRNAs whose spliced junction with the tripartite leader sequence varies from $\text{38, 40, 43, 45}$, and $\text{48}$ was analyzed, it appeared that m⁶A was conserved.Finally, m⁶A was found to be added in a brief label (3.5 min) mainly to nuclear molecules that were longer than any cytoplasmic RNA. The conservation of m⁶A and its addition prior to splicing raise the possibility that internal methylations are involved, in the formation of mRNA.     

Ribonucleoprotein organization of polyadenylate sequences in HeLa cell heterogeneous nuclear RNA.

Ribonucleoprotein particles containing heterogeneous nuclear RNA (Pederson, 1974) were isolated from HeLa cells and digested with ribonucleases A and T₁ at high ionic strength. The nuclease-resistant material, comprising 9.4% of the initial acid-insoluble [³H]adenosine radioactivity, was further fractionated by poly(U)-Sepharose chromatography. The bound fraction eluted from the column with 50% formamide and banded in cesium sulfate gradients (without aldehyde fixation) at a buoyant density characteristic of ribonucleoprotein (1.45 g/cm³). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this material revealed two Coomassie blue-stained bands. The major polypeptide had a molecular weight of 74,000 a less prominent band had a molecular weight of 86,000. The RNA components contained 74.4 mol % AMP and 17.7 mol % UMP. Polyacrylamide gel electrophoresis of the RNA, labeled with [³H]adenosine, demonstrated the presence of molecules 150 to 200 nucleotides in length (poly(A)), as well as molecules 20 to 30 nucleotides long (oligo(A)). Both poly(A) and oligo(A) sequences have previously been identified in HeLa heterogeneous nuclear RNA. These data demonstrate that both the poly (A) and oligo(A) sequences in HeLa heterogeneous nuclear RNA exist in vivo tightly complexed with specific proteins.     

The absolute frequency of labeled N-6-methyladenosine in HeLa cell messenger RNA decreases with label time

When HeLa cells were labeled with either [³H]adenosine or H₃³²PO₄, the absolute frequency of m⁶A (N-6-methyladenosine) decreased 2 to 2.5-fold between a short and long label. The frequency of caps (m⁷GpppXmpYp) remained constant at a level which suggests that most if not all mRNAs are capped in either a short or a long label. Since the inhibition of heterogeneous nuclear RNA synthesis with either actinomycin D or 5,6-dichloro-I-β-d-ribofuranosyl-benzimidazole halted the labeling of both m⁶A and caps within 10 to 15 minutes and no direct cytoplasmic addition of m⁶A could be detected, m⁶A must be lost faster than caps from the cytoplasmic mRNA. This preferential loss could be due to one or more of the following: (1) a m⁶A demethylase; (2) excision of m⁶A containing sequences followed by splicing of the remaining parts of the mRNA; or (3) a minority of the mRNA with a high frequency of m⁶A leaving the cytoplasmic compartment rapidly, presumably by being degraded.     

Poly(adenylic acid)-containing and -deficient messenger RNA of mouse liver

RNA was isolated and fractionated into poly(A)-containing and -deficient classes by oligo(dT) chromatography. Approximately 99% of the poly(A) material bound to the oligo(dT); that which did not bind contained substantially shorter poly(A) chains. All RNA fractions retained an ability to initiate cell-free translation, with the poly(A)-deficient fraction containing half the total translational activity, i.e., mRNA. Two-dimensional polyacrylamide gel analysis of the cell-free translation products revealed three classes of mRNA: 1, mRNA preferentially containing poly(A), including the abundant liver mRNA species; 2, poly(A)-deficient mRNA, including many mid- and low-abundant mRNAs exhibiting less than 10% contamination in the poly(A)-containing fraction fraction; and 3, bimorphic species of mRNA proportioned between both the poly(A)-containing and -deficient fractions. Poly(A)-containing and bimorphic mRNA classes were further characterized by cDNA hybridizations. The capacity of various RNA fractions to prime cDNA synthesis was determined. Compared to total RNA, the poly(A)-containing RNA retained 70% of the priming capacity, while 20% was found in the poly(A)-deficient fraction. Poly(A)-containing, poly(A)-deficient, and total RNA fractions were hybridized to cDNAs synthesized from (+)poly(A)RNA. Poly(A)-containing RNA hybridized with an average R0t 1/2 approximately 20 times faster than total RNA. Poly(A)-deficient RNA hybridized with an average R0t 1/2 approximately 3-4 times slower than total RNA. These R0t 1/2 shifts indicated that in excess of three-quarters of the total hybridizable RNA was recovered in the poly(A)-containing fraction and that less than one-quarter was recovered in the poly(A)-deficient RNA fraction. Abundancy classes were less distinct in heterologous hybridizations. In all cases the extent of hybridization was similar, indicating that while the amount of various mRNA species varied among the RNA fractions, most hybridizing species of RNA were present in each RNA fraction. cDNA to the abundant class of mRNAs was purified and hybridized to both (+)- and (-)poly(A)RNA. Messenger RNA corresponding to the more abundant species was enriched in the poly(A)-containing fraction at least 2-fold over the less abundant species of mRNA, with less than 10% of the abundant mRNAs appearing inthe poly(A)-deficient fraction.