A simple procedure for the isolation and purification of protamine messenger ribonucleic acid from trout testis.

Preparation of milligram quantities of purified poly(A)+ (polyadenylated) protamine mRNA from trout testis tissue was accomplished by a simple procedure using gentle conditions. This involves chromatography of the total nucleic acids isolated by dissociation of polyribosomes with 25 mM-EDTA to release messenger ribonucleoprotein particles and deproteinization of the total postmitochondrial supernatant with 0.5% sodium dodecyl sulphate in 0.25 M-NaCl by binding it to a DEAE-cellulose column. Total RNA was bound under these conditions, and low-molecular-weight RNA, lacking 18S and 28S RNA, could be eluted with 0.5 M-NaCl and chromatographed on oligo(dT)-cellulose columns to select for poly(A)+ RNA. Further purification of both the unbound poly(A)- RNA and the bound poly(A)+ mRNA on sucrose density gradients showed that both 18S and 28S rRNA were absent, being removed during the DEAE-cellulose chromatography step. Poly(A)- RNA sedimented in the 4S region whereas the bound poly(A)+ RNA fraction showed a main peak at 6S [poly(A+) protamine mRNA] and a shoulder in the 3-4S region. Analysis of the main peak and the shoulder on a second gradient showed that most of the main peak sedimented at 6S, whereas the shoulder sedimented slower than 4S. The identity of the poly(A)+ protamine mRNA was established by the following criteria: (1) purified protamine mRNA migrated as a set of four bands on urea/polyacrylamide-gel electrophoresis; (2) analysis of the polypeptides synthesized in the wheat-germ extract by starch-gel electrophoresis showed a single band of radioactivity which co-migrated exactly with the carrier trout testis protamine standard; and (3) chromatography of the polypeptide products on CM-cellulose (CM-52) showed the presence of three or four radioactively labelled protamine components that were co-eluted with the unlabelled trout testis protamine components added as carrier. The availability of large quantities of purified protamine mRNA should now permit a more thorough analysis of its physical and chemical properties.     

Heterogeneity of biologically active deadenylated protamine mRNA components isolated from rainbow trout testes.

Poly(A)+ protamine mRNA's were isolated from rainbow trout testes and deadenylated by treatment with calf thymus RNase H. Four subcomponents of deadenylated PmRNA (PmRNA1-4) were purified by electrophoresis on a 6% polyacrylamide gel in 8 M urea. Translation of each PmRNA subcomponent in the wheat germ S-30 cell-free system showed that all subcomponents are biologically active but each codes for two or more protamine polypeptides suggesting molecular heterogeneity. However, the deadenylated mRNA's can be categorized into two groups based on the spectrum of protamines whose synthesis they stimulate.     

Translation of partially purified poly(A)+ protamine messenger RNA components in wheat germ and rabbit reticulocyte cell-free systems. Evidence for translational control mechanisms.

The coding properties of individual poly(A)+ protamine mRNA subcomponents have been explored by analysis of their translation products in two different cell-free protein synthesis systems, the rabbit reticulocyte lysate and the wheat germ S-30, both of which can translate total protamine mRNA. The products synthesized in the reticulocyte lysate in the presence of total poly(A)+ PmRNA consisted mainly of protamine components CII and CIII with component CI only a minor product. However, in the wheat germ S-30, the same mRNA preparation supported the synthesis of all three protamine components, in approximately equal amounts. In addition a new polypeptide, a putative fourth protamine component, labelled CO, was also synthesized. The translation products of subcomponents of poly(A)+ PmRNA separated as individual bands on polyacrylamide gels were similarly analyzed and it was shown that each of the isolated poly(A)+ PmRNA species could stimulate the incorporation of [3H]arginine into protamines in both translational systems. Although each mRNA band stimulated the synthesis of one particular protamine polypeptide predominantly in a given cell-free system, the same RNA preparation was found to direct preferentially the synthesis of a different protamine component in the second cell-free system. The products synthesized in the rabbit reticulocyte lysate in the presence of the individual mRNA species still showed component CI present as a minor product.     

Protamine messenger RNA: partial purification and characterization of a heterogeneous family of polyadenylated messenger components.

Poly(A)+ protamine mRNA (pmRNA) components were isolated after separation on denaturing preparative polyacrylamide gels. The four size classes of protamine mRNA described previously were found to contain poly(A) tracts of different lengths. The pmRNA1 was found to be associated with (A)110, pmRNA2 with (A)90, pmRNA3 with (A)85, and pmRNA4 with (A)69. Following deadenylation with RNase H after duplex formation with oligo-dT, the isolated mRNAs were found to be still heterogeneous, although highly enriched in certain of the deadenylated components. DNA complementary to the isolated mRNAs (cDNA) was synthesized in vitro. Following depurination, the oligopyrimidine maps indicated that C7T4, corresponding to an Arg-Arg-Gly-Gly sequence in protamine and originally thought to be characteristic of all mRNA components, is present in only one or possibly tow of the components. Cross-hybridizations between the cDNAs and the four poly(A)+ pmRNAs indicated that a basic polynucleotide unit of substantial length is common to all four mRNAs and that the existing nucleotide sequence variations probably originate from one or both of the non-coding portions of the mRNA molecules.     

Purification and properties of biologically active rainbow trout testis protamine mRNA.

At least two classes of protamine mRNA are present in both trout testis polysomal RNA and RNA from the postribosomal supernatant fraction of trout testis hormogenate both of which direct the synthesis of protamine in a Krebs II ascites S-30. One contains poly(A) tracts and the other is devoid of poly(A). Sucrose gradient analyses showed that the poly(A) containing protamine mRNA (poly(A) (+)) sedimented IN THE 6 S region with a shoulder in the 4 S region while the protamine mRNA devoid of poly(A) (poly(A) (-)) appeared to sediment at about 4 S and could not be resolved from tRNA. Analysis of the poly(A) (+) protamine mRNA by boundary sedimentation in an analytical ultracentrifuge showed a sedimentation coefficient of 5.7 S, a value which gives rise to an estimate of 165 to 170 nucleotides per molecule. The poly(A) (+) protamine mRNA migrated as a single species in formamide-containing polyacrylamide gels and its mobility in relation to markers of tRNA (4 S) and 5 S RNA was consistent with its sedimentation velocity of 6 S. The RNA present in the major band on an aqueous polyacrylamide gel was extracted and shown to code for protamine in a wheat germ cell-free system.     

Complex population of nonpolyadenylated messenger RNA in mouse brain

The complexity of nonadenylated mRNA [poly(A)-mRNA] has been determined by hybridization with single-copy DNA (scDNA) and cDNA. Our results show that poly(A)- and poly(A)+ mRNA are essentially nonoverlapping (nonhomologous) sequence populations of similar complexity. The sum of the complexities of poly(A)+ mRNA and poly(A)- mRNA is equal to that of total polysomal RNA or total mRNA, or the equivalent of approximately 1.7 x 10(5) different sequences 1.5 kb in length. Poly(A)- mRNA, isolated from polysomal RNA by benzoylated cellulose chromatography, hybridized with 3.6% of the scDNA, corresponding to a complexity of 7.8 x 10(4) different 1.5 kb sequences. The equivalent of only one adenosine tract of approximately 20 nucleotides per 100 poly(A)- mRNA molecules 1.5 kb in size was observed by hybridization with poly(U). cDNA was transcribed from poly(A)- mRNA using random oligonucleotides as primers. Only 1-2% of the single-copy fraction of this cDNA was hybridized using poly(A)+ mRNA as a driver. These results show that poly(A)- mRNA shares few sequences with poly(A)+ mRNA and thus constitutes a separate, complex class of messenger RNA. These measurements preclude the presence of a complex class of bimorphic mRNAs [that is, species present in both poly(A)+ and poly(A)- forms] in brain polysomes.     

The majority of calf muscle cell messenger RNAs contain poly(A)

Previous studies from our laboratory have investigated messenger RNA metabolism in calf muscle cells in tissue culture. The analysis of mRNA was based on its poly(A) content. We have now examined directly the proportion of mRNA which contains poly(A) in these cells. After separation of poly(A)+ -and poly(A) - -RNA on oligo(dT) -cellulos, the two fractions were translated in a reconstituted, heterologous cell-free protein-synthesizing system and the products were compared with those from the translation of total RNA. The great majority of mRNA form either prefusion or postfusion cultures was poly(A)- containing; quantitative determinations show that about 70-90% of the actin mRNA is poly(A)-containing. In order to determine if a large fraction of the calf muscle mRNA can be translated by a heterologous cell-free system, [3H]-POLY(A)+ -RNA was added to reticulocyte lysates and the formation of initiation complexes was followed. These experiments suggest that the bulk of calf muscle cell mRNA would be utilized in such a system and justify the use of cell-free systems to examine the poly(A) content of total mRNA. Thus, differential polyadenylation does not seem to be an important aspect of mRNA metabolism in cultured muscle cells. The previous study of mRNA in these cells, based on poly(A) content, is apparently a valid measure of overall mRNA metabolism.     

Histone messenger RNAs of the mouse testis

A 6-12S RNA fraction has been isolated following sucrose gradient fractionation of mouse testis RNA, and further resolved into poly A+ and poly A- RNA fractions by oligo-(dt)-cellulose chromatography. Polyacrylamide gel electrophoresis of products formed in a reticulocyte lysate-dependent cell-free translation system has enabled identification of histone variants, H1t, H2S, H2A . X, an H4-like protein and a low Mr protein (presumably TP and/or protamine). Cell-free synthesis of a number of these histone variants appears to be directed by poly A+ mRNAs.     

Translational properties of a non-polyadenylated oligo(U)-containing RNA fraction from wheat embryos

A non-polyadenylated oligo(U)-containing RNA (poly(A)- . oligo(U)+ RNA) fraction was isolated from wheat embryo cytoplasm and its properties were compared with those of polyadenylated RNA (poly(A)+ RNA) from the same source. Both RNA preparations were highly heterogeneous and effectively stimulated [14C]leucine incorporation in a wheat germ cell-free translation system. Electrophoretic patterns of the translation products appearing in the non-polyadenylated RNA- and polyadenylated RNA-supplemented translation assays, respectively, differed from each other. The non-polyadenylated RNA-specific translation products included, in particular, a series of high molecular weight polypeptides. It is concluded that a specific class of non-polyadenylated oligo(U)-containing mRNA species (other than histone mRNAs) occurs in the wheat embryo cells.     

Further evidence that the majority of primary nuclear RNA transcripts in mammalian cells do not contribute to mRNA

Nuclear RNA from Chinese hamster ovary cells was effectively separated into polyadenylic acid [poly(A)]-containing [poly (A)+] and non-poly(A)-containing [poly(A)-] fractions so that -90% of the poly(A) was present in the (A)+ fraction. Only 25% of the 5'-terminal caps of the large nuclear molecules were present in the (A)+ class, but about 70% of the specific mRNA sequences (assayed with cDNA clones) were in the (A)+ class. It appears that many long capped heterogeneous nuclear RNA molecules are of a different sequence category from those molecules that are successfully processed into mRNA.     

Biochemical changes in progressive muscular dystrophy. XIV. Skeletal muscle myosin mRNA translatability in dystrophic mice

Variations in the content and translatability of the poly(A)+ RNA and mRNA molecules coding for myosin (M) were studied in the hind leg muscles of genetically dystrophic mice. The poly(A)+ RNA content of total skeletal muscle failed to increase normally during progression of the disease. M mRNA, isolated from dystrophic normally during progression of the disease. M mRNA, isolated from dystrophic murine muscle poly(A)+ RNA, was mostly found to be associated with the 26S RNA species. The translation of M mRNA in an in vitro heterologous wheat germ system was lower at 8 and 16 weeks in the dystrophic group as compared with the controls. Analysis of the translation products via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and densitometric autoradiographic tracing demonstrated the gradual disappearance of a protein band corresponding to M, the major component of skeletal muscle. cDNA was synthesized, using M mRNA that was isolated and purified from normal and dystrophic mouse muscle as a template. Total radioactivity was measured in some cDNA fractions produced from normal and dystrophic mouse muscle, while other fractions were utilized for separation and sizing of cDNA by disc gel electrophoresis. The cDNA from normal muscle was hybridized with M mRNA from normal and 16-week-old dystrophic mouse muscles. The cDNA probe, hybridization experiments, and studies involving the content and synthesis of M mRNA suggest that murine muscular dystrophy elicited a shorter species of mRNA or shorter sequences of the same species of mRNA coding for M. Not all poly(A)+ mRNA sequences coding for M, found in control mice, were present in their dystrophic counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyadenylated, noncapped RNA from the archaebacterium Methanococcus vannielii.

Polyadenylated [poly(A)+] RNA molecules have been isolated from Methanococcus vannielii by oligodeoxythymidylate-cellulose affinity chromatography at 4 degrees C. Approximately 16% of the label in RNA isolated from cultures allowed to incorporate [3H]uridine for 3 min at 37 degrees C was poly(A)+ RNA. In contrast, less than 1% of the radioactivity in RNA labeled over a period of several generations was contained in poly(A)+ RNA molecules. Electrophoretic separation of poly(A)+ RNA molecules showed a heterogeneous population with mobilities indicative of sizes ranging from 900 to 3,000 bases in length. The population of poly(A)+ RNA molecules was found to have a half-life in vivo of approximately 12 min. Polyadenylate [poly(A)] tracts were isolated by digestion with RNase A and RNase T1 after 3' end labeling of the poly(A)+ RNA with RNA ligase. These radioactively labeled poly(A) oligonucleotides were shown by electrophoresis through DNA sequencing gels to average 10 bases in length, with major components of 5, 9, 10, 11, and 12 bases. The lengths of these poly(A) sequences are in agreement with estimates obtained from RNase A and RNase T1 digestions of [3H]adenine-labeled poly(A)+ RNA molecules. Poly(A)+ RNA molecules from M. vannielii were labeled at their 5' termini with T4 polynucleotide kinase after dephosphorylation with calf intestine alkaline phosphatase. Pretreatment of the RNA molecules with tobacco acid pyrophosphatase did not increase the amount of phosphate incorporated into poly(A)+ RNA molecules by polynucleotide kinase, indicating that the poly(A)+ RNA molecules did not have modified bases (caps) at their 5' termini. The relatively short poly(A) tracts, the lack of 5' cap structures, and the instability of the poly(A)+ RNA molecules isolated from M. vannielii indicate that these archaebacterial poly(A)+ RNAs more closely resemble eubacterial mRNAs than eucaryotic mRNAs.