Dawn E. Kelley

On the lability of poly(A) sequences during extraction of messenger RNA from polyribosomes

Extraction of mRNA from polyribosomes with sodium dodecyl sulfate and phenol at near neutral pH results in a considerable loss of the poly(A)-containing mRNA from the aqueous phase, and also in cleavage of poly(A) from the mRNA. The loss and cleavage are associated with some specific proteins in the polyribosomes, and can be avoided if these proteins are destroyed before extraction or if extractions are done with chloroform plus phenol rather than with phenol alone.

Messenger RNA-protein complexes and newly synthesized ribosomal subunits: Analysis of free particles and components of polyribosomes*

As a prerequisite for a study of the transport and functional forms of messenger RNA and ribosomes in cultured L cells, a method has been developed for preparing cytoplasmic particles rid of the nuclear contaminants routinely found with conventional procedures. This method, which uses a low concentration of nonionic detergent and avoids mechanical disruption, was used to obtain polyribosomes that were demonstrably free of co-sedimenting ribonucleoprotein. Polyribosomes from cells pulse-labeled with [ 3 H]uridine were analyzed by sucrose gradient sedimentation and equilibrium banding in CsCl before and after dissociation with EDTA. Newly synthesized messenger RNAs were released from the polyribosomes as a polydisperse array of ribonucleoprotein complexes (mRNP) with a relatively uniform RNA to protein ratio. The protein, termed m-protein, comprises about 60% of the weight of the complex. The mRNP released from polyribosomes was demonstrated to be similar in several respects to other RNP complexes in the cytoplasmic extracts which exist free of attached ribosomes. A more detailed examination of these latter structures indicated that roughly three-quarters of their protein could be reversibly removed by exposure to 0·55 m -LiCl-0·01 m -Mg 2+ , a condition which removes very little protein from the ribosomal subunits. Previously we have demonstrated that new ribosomal subunits entering the cytoplasm contain more protein than the subunits in the steady-state pool. The present experiments indicate that this extra protein, termed accessory protein, is released from the subunits when they are incorporated into polyribosomes.

Existence of methylated messenger RNA in mouse L cells

Abstract Messenger RNA of mouse L cells is methylated in both base and ribose moieties. On the average there are about 2.2 methyl groups per 1000 nucleotides in mRNA, a proportion which is about one-sixth that of mammalian ribosomal RNA. The variety of methylated bases in mRNA is more limited than in ribosomal RNA. A very low level of methylation is detected in heterogeneous nuclear RNA, suggesting that methylation, like polyadenylation, may constitute a post-transcriptional modification of messenger RNA precursor in eucaryotic cells.

Buoyant densities of cytoplasmic ribonucleoprotein particles of mammalian cells: distinctive character of ribosome subunits and the rapidly labeled components.

The buoyant densities of the cytoplasmic ribonucleoprotein particles of strain L cells were determined by banding in CsCl gradients. Stability of the particles at the high salt concentrations was achieved by means of fixation with formaldehyde. Under these conditions, the bulk of the material in monoribosome, 60 s and 40 s particles banded at characteristic densities, ρ = 1·55, 1·57 and 1·49 g/cm 3 , respectively. Preparations of 60 s and 40 s particles also yielded minor components of lower density which apparently possess a lower proportion of RNA to protein. The rapidly labeled 18 s RNA associated with the 40 s particles was found to exist in structures which band at ρ = 1·43, a density considerably lower than that characteristic of the bulk of the 40 s material. The results of pulse-chase and double-labeling experiments are consistent with the idea that the ρ = 1·43 components are lower-density precursors of the small ribosome subunits. No significant amount of labeled 40 s material banded at a density higher than the subunit itself. Such a higher density would be expected of complexes of messenger RNA and intact subunits, and therefore their presence is not indicated.

Comparison of methylated sequences in messenger rna and heterogeneous nuclear rna from mouse l cells

Abstract 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 m7GpppXmpG, m7GpppXmpY(m)pG, m7GpppXmpY(m) pNpG and m7GpppXmpY(m)p(Np)> 1G have distinctive non-random compositions of the 2′-O-methylated constituent Xm; yet sequences of a particular type and composition occur with a remarkably similar frequency in mRNA and hnRNA † . For example, approximately 20% of the cap sequences in both hnRNA and mRNA are m7Gppp(m6)AmG, whereas less than 1% are m7GpppUmpG. 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 Ym in the cap II forms of mRNA (8% Gm, 11% Am, 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: -N 1 -( G A )-m 6 A-C-N 2 - , 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.

Synthesis and turnover of nuclear and cytoplasmic polyadenylic acid in mouse L cells

Abstract A study was made of the kinetics of adenosine incorporation into the poly(A) segments of heterogeneous nuclear RNA, polyribosomal messenger RNA and total cytoplasmic RNA of mouse L cells. Special care was taken to ensure that the precursor pools used for incorporation remained at a constant specific activity over the entire course of an experiment. A comparison of the labeling of the poly(A) and the non-poly(A) portions of mRNA indicated that polyadenylation is a relatively late post-transcriptional step, occurring in the latter third of the interval between transcription of the mRNA precursor and appearance of the mRNA in cytoplasmic polyribosomes. The rates of labeling of messenger poly(A) and total cytoplasmic poly(A) reach a maximum after only a very brief lag and do not increase thereafter, in spite of that fact that the specific activity of nuclear poly(A) continues to increase for several hours. Such a result is not compatible with a simple precursor-product relation between all of the nuclear and all of the cytoplasmic poly(A), but rather, it suggests that there is a reasonably large intranuclear turnover of poly (A). The fact that nuclear poly(A) is not quantitatively converted to cytoplasmic poly(A) indicates that polyadenylation of a heterogeneous RNA molecule is not sufficient to ensure that it will be properly processed and transported to the cytoplasm.

CHD1 INTERACTS WITH SSRP1 AND DEPENDS ON BOTH ITS CHROMODOMAIN AND ITS ATPASE/HELICASE-LIKE DOMAIN FOR PROPER ASSOCIATION WITH CHROMATIN

Abstract. CHD1, an Mr∼200,000 protein that contains a chromodomain (C), an ATPase/helicase-like domain (H) and a DNA-binding domain (D), was previously shown to be associated with decompacted interphase chromatin in mammalian cells and with transcriptionally active puffs and interbands in Drosophila polytene chromosomes. We now show by transient transfection experiments with genes expressing wild-type and mutant forms of CHD1 that both the C and H domains are essential for its proper association with chromatin. We also present evidence for an in vivo interaction between CHD1 and a novel HMG box-containing protein, SSRP1, which involves an amino-terminal segment of CHD1 that does not include the chromodomain. Immunocytochemical analyses indicated that CHD1 and SSRP1 colocalize in both mammalian nuclei and Drosophila polytene chromosomes.

Immunoglobulin messenger RNAs in murine cell lines that have characteristics of immature B lymphocytes

Abstract The immunoglobulin-encoding messenger RNAs in a pre-B cell-like lymphoma line, 70Z/3, were characterized according to size, concentration in nuclear and cytoplasmic compartments and response to stimulation by the B cell mitogen, lipopolysaccharide. The μ heavy chain and κ light chain mRNAs produced by these cells were size-fractionated on methylmercuryhydroxide-agarose gels, transferred to diazotized cellulose paper and identified by hybridization with radioactive cloned probes containing C μ or C κ sequences. Unstimulated cells, which produce only intracellular μ chains, contain negligible amounts of κ mRNA and a few hundred molecules of μ mRNA per cell. The ratio of nuclear to cytoplasmic heavy chain mRNA sequences in these cells is about 50 times greater than in an immunoglobulin-secreting plasma cell. Stimulation of 70Z/ 3 cells by lipopolysaccharide, which causes the cells to express surface IgM, results in a greater than 10 fold increase in the concentration of κ mRNA, but does not markedly affect the level of μ mRNA. Four distinct μ mRNA components ranging in size from approximately 2.1 to 3.0 kb were observed in 70Z/3 cells as well as in a cloned derivative, 70Z/3–12. Two of these components, which were found in the membrane-bound polyribosomes, may code for the membrane and secretory forms of the μ heavy chain. The other two mRNAs, which were confined to the cytoplasmic fraction containing free polyribosomes and nonribosome-associated mRNP, may code for intracellular μ chains. The two membrane-bound μ mRNAs were observed in another immature B cell line, WEHI 231. In contrast, mature IgM-secreting plasma cells produce a single μ mRNA component of uniform size. The multiple species of μ mRNA may arise from different arrangements of the C μ genetic elements on allelic chromosomes and/or from alternative processing of C μ -containing transcripts.

Secondary structure maps of ribosomal RNA: II. Processing of mouse L-cell ribosomal RNA and variations in the processing pathway☆

Secondary structure mapping in the electron microscope was applied to ribosomal RNA and precusor ribosomal RNA molecules isolated from nucleoli and the cytoplasm of mouse L-cells. Highly reproducible loop patterns were observed in these molecules. The polarity of L-cell rRNA was determined by partial digestion with 3′-exonuclease. The 28 S region is located at the 5′-end of the 45 S rRNA precursor. Together with earlier experiments on labeling kinetics, these observations established a processing pathway for L-cell rRNA. The 45 S rRNA precursor is cleaved at the 3′-end of the 18 S RNA sequence to produce a 41 S molecule and a spacer-containing fragment (24 S RNA). The 41 S rRNA is cleaved forming mature 18 S rRNA and a 36 S molecule. The 36 S molecule is processed through a 32 S intermediate to the mature 28 S rRNA. This pathway is similar to that found in HeLa cells, except that in L-cells a 36 S molecule occurs in the major pathway and no 20 S precusor to 18 S RNA is found. The processing pathway and its intermediates in L-cells are analogous to those in Xenopus laevis, except for a considerable size difference in all rRNAs except 18 S rRNA. The arrangement of gene and transcribed spacer regions and of secondary structure loops, as well as the shape of the major loops were compared in L-cells, HeLa cell and Xenopus rRNA. The over-all arrangement of regions and loop patterns is very similar in the RNA from these three organisms. The shapes of loops in mature 28 S RNA are also highly conserved in evolution, but the shapes of loops in the transcribed spacer regions vary greatly. These observations suggest that the sequence complementarity that gives rise to this highly conserved secondary structure pattern may have some functional importance.

The coupling between enhancer activity and hypomethylation of kappa immunoglobulin genes is developmentally regulated.

Previous studies have indicated that immunoglobulin enhancers are essential for establishing transcriptional competence but not for maintaining the activity of constitutively transcribed genes. To understand the basis for this developmental shift away from dependence on enhancer function, we have investigated the relationship between transcriptional activity and methylation status of the immunoglobulin kappa light-chain genes (kappa genes) in mouse cell lines representing different stages of B-cell maturation. Using pre-B-cell lines in which the level of a critical kappa enhancer-binding factor, NF-kappa B, was controlled by the administration or withdrawal of lipopolysaccharide and plasmacytoma lines that either contain or lack this factor, we studied the properties of endogenous kappa genes and of transfected kappa genes which were stably integrated into the genomes of these cells. In the pre-B cells, the exogenous (originally unmethylated) kappa genes, as well as endogenous kappa genes, were fully methylated and persistently dependent on enhancer function, even after more than 30 generations in a transcriptionally active state. In plasmacytoma cells, the endogenous kappa genes were invariably hypomethylated, whereas exogenous kappa genes were hypomethylated only in cells that contain NF-kappa B and are thus permissive for kappa enhancer function. These results indicate that the linkage of hypomethylation to enhancer-dependent activation of kappa transcription occurs after the pre-B-cell stage of development. The change in methylation status, together with associated changes in chromatin structure, may suffice to eliminate or lessen the importance of the enhancer for the maintenance of the transcriptionally active state.