Size of the poly(A) region in mouse globin messenger RNA

A 9S RNA fraction from mouse reticulocytes, containing the active - and -globin mRNAs, has been isolated by hybridization of the polyadenylate regions in the mRNAs to oligo(dT)-cellulose. The adenylate-rich sequence isolated by limited RNase digestion of the globin mRNAs migrates between 4S and 5S RNA standards when co-electrophoresed on 12% polyacrylamide gels. Poly(A) standards, 28 and 84 nucleotides in length, showed anomolous migration relative to the 4S and 5S RNAs. The average size of the adenylate-rich sequence, estimated by its migration relative to the poly(A) standards, is about 50 nucleotides. The polyadenylate stretch in mouse globin mRNA is therefore much shorter than those found in other mRNAs.     

Isolation and characterization of trout testis protamine mRNAs lacking poly (A)

Poly(A)+ protamine mRNA was isolated from trout testis cells in a very pure form, and artificial poly(A)- protamine mRNA molecules were derived from it by enzymatic deadenylation with RNAase H from calf thymus after hybridization with oligo(dT). The deadenylated protamine mRNA was found to be active in a wheat germ cell-free system and yielded a labeled product which co-migrated with authentic protamine. These deadenylated mRNA molecules were subsequently used as markers on denaturing polyacrylamide gels to identify and allow the purification of the poly(A)- protamine components known to exist in vivo in the total cellular poly(A)- RNA. RNA species of molecular weights similar to the enzymatically deadenylated subcomponents of protamine mRNA were observed in the natural poly(A)-RNA population of the testis cells. These naturally occurring poly(A)- protamine mRNAs were isolated by preparative gel electrophoresis and further characterized by 3H-poly(U) hybridization assay, by hybridization to complementary DNA made against highly purified poly(A)+ protamine mRNA, and by their ability to direct protamine synthesis in a cell-free system.     

Biosynthesis of poly(A)-RNA and differentiation in chick erythrocytes

The rates of synthesis of poly(A)-containing RNA species in the nuclei of erythrocytes from 4- to 9-day-old chick embryos were determined by poly(T)-cellulose chromatography and were found to vary according to the developmental stages of the chick embryos. The rate appeared to increase 1 day prior to the onset of hemoglobin differentiation. The enzymatic activities of ATP polymerization in the nucleus of these erythrocytes were also examined. The enzymatic activity was resolved into two fractions on O-(diethylaminoethyl) cellulose. The ratio of the two enzymatic activities remained relatively constant in erythrocytes from 4- to 19-day-old embryos. However, a threefold increase in the total poly(A) polymerase activities was observed 1 day prior to the onset of hemoglobin differentiation. These results indicate that hemoglobin differentiation in these erythrocytes is associated with an increase in the rate of synthesis of poly(A)-containing RNA and in the activities of poly(A) polymerases.     

U2 snRNP: not just for poly(A) mRNAs

Polyadenylated mRNAs and replication-dependent histone mRNAs, which are not polyadenylated, share factors involved in 3' end formation. Friend et al. (2007) now report that the U2 snRNP, required for pre-mRNA splicing, is also required for histone mRNA 3' end formation.     

Polypeptide composition of the globin poly(A)-protein complex from rabbit reticulocytes

Polypeptides associated with the rabbit reticulocyte poly(A)-protein complex, isolated by oligo (dT)-cellulose chromatography, were analyzed by SDS-polyacrylamide gel electrophoresis. The complex derived from EDTA released total polysomal mRNP and 20S globin mRNP by RNase treatment contained four polypeptides of molecular weight 58000, 67000, 71000 and 79000. A 79000 molecular weight polypeptide, which was also a major component of the reticulocyte low molecular weight cytoplasmic fraction, was shown to form tenacious associations with poly(A) in vitro.     

The poly(A) tail of mRNAs: bodyguard in eukaryotes,scavenger in bacteria

In eukaryotes, poly(A) tails usually act as stabilizers of intact mRNAs, whereas in E. coli they serve to accelerate the destruction of fragments. The mechanisms underlying these contrasting effects of the same RNA modification are discussed.     

Novel alternative splicing of mRNAs encoding poly(A) polymerases in Arabidopsis

The Arabidopsis thaliana genome possesses four genes whose predicted products are similar to eukaryotic poly(A) polymerases from yeasts and animals. These genes are all expressed, as indicated by RT/PCR and Northern blot analysis. The four Arabidopsis PAPs share a conserved N-terminal catalytic core with other eukaryotic enzymes, but differ substantially in their C-termini. Moreover, one of the four Arabidopsis enzymes is significantly shorter than the other three, and is more divergent even within the conserved core of the protein. Nonetheless, the protein encoded by this gene, when produced in and purified from E. coli, possesses nonspecific poly(A) polymerase activity. Genes encoding these Arabidopsis PAPs give rise to a number of alternatively spliced mRNAs. While the specific nature of the alternative splicing varied amongst these three genes, mRNAs from the three "larger" genes could be alternatively spliced in the vicinity of the 5th and 6th introns of each gene. Interestingly, the patterns of alternative splicing vary in different tissues. The ubiquity of alternative splicing in this gene family, as well as the differences in specific mechanisms of alternative processing in the different genes, suggests an important function for alternatively spliced PAP mRNAs in Arabidopsis.     

The putative 15 S precursor of globin mRNA contains a poly(A) sequence

[³H]Uridine or [³H]adenosine pulse-labelled nuclear RNA was isolated from chicken immature red blood cells and separated on denaturing formamide sucrose gradients. RNA of each gradient fraction was hybridized with unlabelled globin DNA complementary to mRNA (cDNA) and subsequently digested by RNAase A and RNAase T₁. The experiments revealed two RNA species with globin coding sequences sedimenting at 9 S and approx. 15 S, the latter probably representing a precursor of 9 S globin mRNA.A poly(A) sequence was demonstrated in this RNA by two different approaches. Nuclear RNA pulse-labelled with [³H]uridine was fractionated by chromatography on poly(U)-Sepharose. Part of the 15 S precursor was found in the poly(A)-containing RNA. In the second approach 15 S RNA pulse-labelled with [³H]adenosine was hybridized with globin cDNA, incubated with RNAase A and RNAase T₁ and subjected to chromatography on hydroxyapatite. The hybrids were isolated and after separation of the strands degraded with DNAase I, RNAase A and RNAase T₁. By this procedure poly(A) sequences of approximately 100 nucleotides could be isolated from the 15 S RNA with globin coding sequences. The poly(A) sequence was completely degraded by RNAase T₂.     

The putative 15 S precursor of globin mRNA contains a poly (A) sequence

[3H] Uridine or [3H] adenosine pulse-labelled nuclear RNA was isolated from chicken immature red blood cells and separated on denaturing formamide sucrose gradients. RNA of each gradient fraction was hybridized with unlabelled globin DNA complementary to mRNA (cDNA) and subsequently digested by RNAase A and RNAase T1. The experiments revealed two RNA species with globin coding sequences sedimenting 9 S and approx. 15 S, the latter probably representing a precursor of 9 S globin mRNA. A poly (A) sequence was demonstrated in this RNA by two different approaches. Nuclear RNA pulse-labelled with [3H] uridine was fractionated by chromatography on poly (U)-Sepharose. Part of the 15 S precursor was found in the poly(A)-containing RNA. In the second approach 15 S RNA pulse-labelled with [3H]adenosine was hybridized with globin cDNA, incubated with RNAase A and RNAase T1 and subjected to chromatography on hydroxyapatite. The hybrids were isolated and after separation of the strands degraded with DNAase I, RNAase A and RNAase T1. By this procedure poly(A) sequences of approximately 100 nucleotides could be isolated from the 15 S RNA with globin coding sequences. The poly(A) sequence was completely degraded by RNAase T2.     

Localization and sequence analysis of poly(A) sites generating multiple dihydrofolate reductase mRNAs

The murine dihydrofolate reductase (DHFR) gene gives rise to multiple polyadenylated mRNAs displaying heterogeneity in the length of the 3' untranslated region. These species are present in the cytoplasm at levels that vary over 2 orders of magnitude, suggesting that certain poly(A) sites are preferred over others. Previous observations have shown that three out of the four major sites of polyadenylation do not display consensus hexanucleotide (AATAAA, ATTAAA) signals. We have further analyzed the sequences involved in directing multiple polyadenylation events on the DHFR gene by focusing our attention on the 4.1- and 5.6-kilobase mRNAs, the lowest abundance DHFR species observed on RNA blot analysis. Identification and sequence analysis of the poly(A) addition sites corresponding to these species revealed appropriately positioned consensus hexanucleotide signals; additional nearby poly(A) sites were also detected which apparently do not use consensus hexanucleotides to direct poly(A) addition to DHFR mRNAs of relatively lower abundance. We have also identified polyadenylation sites downstream of the 4.1- and 5.6-kilobase sites which display consensus hexanucleotide signals and correspond to messenger species too rare for detection by routine RNA blot analysis. Our data bring to 11 the number of known functional poly(A) addition sites associated with the DHFR gene.     

Molecular cloning and sequencing of mRNAs coding for minor adult globin polypeptides of Xenopus laevis.

Globin mRNA was isolated from immature red blood cells of an adult Xenopus laevis female. mRNA/cDNA hybrids were integrated in the Pst I cleavage site of pBR 322 by G/C tailing, and cloned in Escherichia coli strain HB 101. By restriction site analysis as well as hybridization behaviour we identified two clones coding for minor adult alpha and beta globin chains. Nucleotide sequence analysis and derived amino acid sequences are presented.     

Ribonucleotide sequences non-adjacent to poly(A) participate in the poly(A)-protein complex in 15S duck globin mRNP particles.

The study of the interaction between mRNA and proteins in the polyribosomal 15 S duck globin messenger ribonucleoprotein complex showed that proteins protect specific mRNA sequences against digestion by the nonspecific micrococcal nuclease (Nucleic Acids Research 6 (8) 2787, 1979). Here we report the isolation of the poly(A)-protein RNP complex from nuclease digested 15 S mRNP by two different methods: sucrose gradient sedimentation and oligo(dT)-cellulose chromatography. We show by fingerprint analysis, that aprt from the periodically fragmented poly(A) segment, mRNA sequences adjacent and non-adjacent to the poly(A) segment are protected by the poly(A) binding proteins against nuclease digestion. The duck globin poly(A)-protein RNP complex, with a sedimentation coefficient between 7 S and 10 S, shows a characteristic protein composition, with a major 73,000 MW polypeptide and some minor components. The results are discussed in view of a dynamic ribonucleoprotein structure.     

Polypeptides synthesized in vitro by poly(A)+ and poly(A)- mRNAs of rat ascites hepatoma AH 7974 cells.

Polysomal RNA of rat ascites hepatoma AH 7974 cells was fractionated into poly(A)+ and poly(A)- mRNAs and these RNAs were translated into polypeptides in a protein-synthesizing system derived from wheat germ. The analysis of polypeptides synthesized in vitro by two-dimensional gel electrophoresis revealed that there are three classes of polypeptides. The first group can be synthesized equally by both poly(A)+ and poly(A)- mRNAs; the second and third groups are synthesized predominantly by poly(A)+ and poly(A)- mRNAs, respectively. These results suggest that the three classes of polypeptides can be characterized by the presence or absence of poly(A) tails of the corresponding mRNAs.     

Nucleotide sequence complexities, molecular weights, and poly(A) content of the vesicular stomatitis virus mRNA species.

Poly(A)-containing vesicular stomatitis virus mRNA species synthesized in vesicular stomatitis virus-infected cells have been separated into four bands by electrophoresis on formamide-polyacrylamide gels. Two-dimensional fingerprints of ribonuclease T-1 and ribonuclease A digests of the RNA from each band show that they contain unique oligonucleotide sequences as well as 60 to 125 nucleotides of poly(A). The fingerprints were used to determine the nucleotide sequence complexities of RNA from three of the bands. Two contain nucleotide sequences which account completely for their molecular weights (0.70 times 10-6 and 0.55 times 10-6) determined by gel electrophoresis and sedimentation rate, and, therefore, these are radiochemically pure RNA species. The most rapidly migrating band must contain two ro three different RNA species since it has a molecular weight of 0.28 times 10-6, determined by physical methods, and a nucleotide sequence complexity two to three times that expected for a pure RNA species of this size. These data are in complete accord with translational studies (accompanying paper) which show that each of the two pure RNA species codes for a distinct viral protein, whereas the third codes for two viral proteins. From the molecular weight and sequence complexity determinations on mRNA from the bands, we conclude that most of the vesicular stomatitis virus genome is transcribed into discrete mRNA species.