Addition of poly(A) to nuclear RNA occurs soon after RNA synthesis

A kinetic analysis of the appearance of [3H]uridine label in RNA sequences that neighbor poly(A), as well as the incorporation of [3H]adenosine label into both the RNA chain and the poly(A) of poly(A)-containing molecules, shows that poly(A) is added within a minute or so after RNA chain synthesis in Chinese hamster ovary cells and HeLa cells. Previous conclusions by several groups (5-7) that poly(A) might be added as long as 20-30 min after RNA synthesis appear to be in error, and the present conclusion seems much more in line with several different types of recent studies with specific mRNAs that suggest prompt poly(A) addition (13-16).     

The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells.

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.     

Primer specificity of ribosome-associated poly(A) polymerase from Ehrlich ascites tumour cells

The reaction product of the ribosomal poly(A) polymerase [ATP(UTP):RNA nucleotidyltransferase] is analyzed. Two systems are used in vitro: (a) isolated polyribosomes with endogenous enzyme and RNA primer and (b) purified enzyme with total polyribosomal RNA as primer. In the polyribosome system about 50% of the [3H]AMP label is in poly(A)-containing mRNA. This RNA displays a heterogeneous size ditribution in the range of 8--30 S with a maximum at about 14 S. Upon denaturation the maximum is shifted towards the 10-S zone. The poly(A) polymerase catalyzes the addition of 12--18 adenylate residues to pre-existing mRNA poly(A) sequences of 40--160 residues. The [3H]AMP incorporated into poly(A)-lacking RNA is mainly in a fraction with an electrophoretic mobility corresponding to 4-S RNA. In the purified enzyme system, specificity towards poly(A)-containing mRNA is lost to a considerable extent. Only 10% of the [3H]AMP label is retained by oligo(dT)-cellulose. The bulk of the product is in 18-S rRNA and heterogeneous small molecular weight RNA. We conclude that the ribosome-associated poly(A) polymerase is most likely the enzyme responsible for the cytoplasmic polyadenylation of poly(A)-containing mRNA in vivo.     

Subcellular distribution and properties of poly(A)-containing RNA from cultured plant cells.

Cultured sycamore cells rapidly incorporate [3H]uridine or [32P]orthophosphate into rRNA precursors and polydisperse RNA. Mature rRNA accumulates only after a lag period of approximately 40 min. Fractionation of pulse-labelled cells and analysis of the RNA shows that after 30 min the rRNA precursors, together with some polydisperse RNA, are confined to the nucleus. In consequence radioactive polydisperse RNA can be isolated from polyribosomes in the complete absence of labelled rRNA. Approximately 40% of this RNA is retained by an oligo(dT)-cellulose column and by this criterion is judged to contain poly(A) sequences. A smaller proportion of nuclear polydisperse RNA also contains poly(A). The tendency for poly(A)-containing RNA to aggregate complicates molecular weight determinations. Denaturation of poly(A)-containing RNA in 8 M urea prior to gel electrophoresis produces a broad peak of RNA with an average Mr = 10(6). Analysis of the nucleotide composition of total cell poly(A)-containing RNA shows that it contains 41% AMP. Roughly 6% of this RNA is resistant to digestion by ribonuclease A and T1. AMP is the only nucleotide detectable in these fragments. From their mobility during electrophoresis in 8 M urea at 60 degrees C with 5.8-S, 5-S and tRNA as molecular weight markers it is concluded that the poly(A) regions contain an average of 160 nucleotides.     

Effect of amines on fibrinogen synthesis

L-Epinephrine, serotonin, and isoproterenol stimulate the incorporation of [14C]leucine into thrombin-induced clottable protein; this stimulation was abolished by actinomycin D. The incorporation of 32P into total RNA of rat liver, the site of fibrinogen synthesis, was stimulated by epinephrine and was highest at 2 h after 32P administration. [14C]Orotic acid incorporation into polysomal RNA of liver was also increased significantly by epinephrine and serotonin. The immunoprecipitation of newly synthesized protein by monospecific antibody raised against pure rat fibrinogen clearly demonstrates that L-epinephrine increased fibrinogen formation in vivo under the experimental condition. Translation of poly (A)-containing RNA from total polysomal RNA clearly indicates that L-epinephrine increased mRNA specific for fibrinogen.     

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.     

Early effects of dimethylnitrosamine on the initiation of protein synthesis in mouse liver.

1. Dimethylnitrosamine (37.5 mg/kg body wt.) was administered to mice by a single intraperitoneal injection, and the early effects on protein synthesis and related functions were studied in a liver S-30 system. 2. The incorporation of [14C]leucine into protein decreased rapidly after dimethylnitrosamine administration. The effect was associated with a decreased ability of the system to utilize methionyl-tRNAfMet and formyl-methionyl-tRNAfMet for 80 S ribosomal initiation-complex formation (primary initiation), and a loss of poly(A)-containing RNA from the postmicrosomal fraction. All the three effects developed simultaneously, and were clearly demonstrable within 15 min. 3. Initiation-complex formation in the polyribosomal fraction (re-initiation) was decreased to the same extent as the primary initiation, indicating that the initiation defect was not a result of the decrease in free mRNA. 4. The inhibition of initiation was only manifest at the joining of the 40 S pre-initiation complex to 60 S ribosomal subunits. It was not a result of methionyl-tRNAfMet deacylation. The functions between the formation of the methionyl-tRNAfMet-containing 80 S ribosomal complex and the first translocation on the ribosome were not involved, since the incorporation of formylmethionine into N-terminal polypeptides decreased to the same extent as the 80 S initiation-complex formation. 5. Inhibitors of protein synthesis (cycloheximide and pactamycin) decreased poly(A)-containing RNA in the postmicrosomal fraction in a similar way to dimethylnitrosamine.     

Enrichment of the poly (A) sequence and lack of enhancement of total RNA synthesis in cultured Xenopus hepatocytes by estradiol-17 beta

The effect of estradiol-17 beta on RNA synthesis and the amounts of total RNA and polyadenylic acid were determined in primary cultures of Xenopus laevis liver parenchymal cells. Results showed that estradiol did not alter the RNA content significantly; control cells contained 11.9 +/- 0.34 micrograms and estradiol-treated cells 12.4 +/- 0.17 micrograms per 10(6) cells on day 2 of estradiol treatment, and 22.0 +/- 0.61 micrograms and 24.0 +/- 1.09 micrograms on day 5. Hybridization with [3H]poly(U) revealed that estradiol increased the poly(A) content about 1.2-fold more than in the controls on day 2 and 1.6-fold on day 5 of estradiol treatment. The actual rate of RNA synthesis was estimated from analyses of the kinetics of [3H]adenosine incorporation into the ATP pool and into RNA. The initial rate of incorporation of ATP into RNA on day 5 of estradiol treatment was 29.38 pmol/min/10(6) cells and the rate of the controls of 29.35. Subsequent accumulation kinetics of [3H]adenosine into RNA showed no difference between estradiol and the control cells. Thus, estradiol did not alter the rate of total RNA synthesis and the total RNA content significantly, but it did increase the poly(A) content.     

RNA synthesis in whole cells and protoplasts of centaurea: a comparison

Protoplasts enzymically isolated from suspension cultures of Centaurea cyanus L. incorporate radioactive precursors into RNA with kinetics similar to that of whole cells. There are differences, however, in several other aspects of RNA metabolism. The proportion of total RNA that contains poly(A) sequences (25 to 30%) is similar in both freshly isolated protoplasts and whole cells after a 20-minute pulse with [³H]adenosine. After a 4-hour pulse, however, poly(A)-containing RNA makes up 30% of the total RNA in protoplasts whereas it drops to 8% in whole cells. There appears to be a faulty processing of ribosomal precursor into the mature ribosomal species, as the precursor seems to accumulate to higher levels relative to the mature 18S and 25S rRNAs in protoplasts as compared to whole cells. Additional differences are seen in the size distributions of poly(A)-containing RNA, although the length of the poly(A) segment is similar in both protoplasts and whole cells. Within 24 hours protoplasts appear to have resumed a pattern of RNA synthesis similar to that of whole cells.     

Control of synthesis of functional mRNA coding for phenylalanine ammonia-lyase from Rhodosporidium toruloides

The regulation of functional mRNA coding for phenylalanine ammonia-lyase (PAL) from Rhodosporidium toruloides was investigated. Polyadenylic acid [poly(A)]-containing RNA was an efficient template for in vitro translation in rabbit reticulocyte lysate. Non-poly(A)-containing RNA did not stimulate in vitro protein synthesis. Several lines of experimental evidence indicate that mRNA from R. toruloides directs PAL synthesis in reticulocyte lysate: (i) the major radioactive product in immunoprecipitates when lysates, incubated with yeast poly(A)-containing RNA, were reacted with PAL-antiserum had the same molecular weight as native PAL (75,000); (ii) this major radioactive product competes with authentic PAL for binding to PAL-antiserum; and (iii) partial proteolytic peptide maps of the in vitro translation product were very similar to those of native PAL. The levels of functional mRNA coding for PAL, when R. toruloides was grown in different physiological conditions, were determined by quantitation of PAL synthesized in vitro when RNA was added to reticulocyte lysate. Functional PAL mRNA was six times higher in yeast grown on phenylalanine compared with glucose-phenylalanine minimal medium. No functional PAL mRNA was detected in yeast grown on glucose-ammonia minimal medium in the presence or absence of phenylalanine. These observed changes in functional PAL mRNA were similar to levels of PAL catalytic and antigenic activity. The kinetics of functional PAL mRNA synthesis and degradation were studied. Maximum levels of functional PAL mRNA were observed within 60 min of transfer to PAL-inducing growth conditions. Poly(A)-containing RNA and functional PAL mRNA were rapidly degraded when cells were transferred from phenylalanine to glucose-ammonia minimal medium, with half-lives of 25 and 10 min, respectively. Thus, it is suggested that the alterations in the amount of PAL in cells of R. toruloides grown in different physiological conditions primarily result from alteration in the amount of functional mRNA coding for the enzyme.     

Extensive homology between poly(A)-containing mRNA and purified nominal poly(A)-lacking mRNA in mouse kidney

To investigate poly(A)-lacking mRNA in mouse kidney, we studied a fraction of renal mRNA that does not bind to oligo(dT)-cellulose but can be purified by benzoylated cellulose chromatography. Nominal poly(A)-lacking mRNA and poly(A)-containing mRNA have complete nucleotide sequence homology, suggesting that kidney does not contain mRNAs that are not represented in the polyadenylated RNA fraction. Translation products directed by nominal poly(A)-lacking mRNA and poly(A)-containing mRNA are qualitatively and quantitatively similar in one-dimensional polyacrylamide gels. [3H]cDNA transcribed from poly(A)-containing mRNA hybridizes with its template and with nominal poly(A)-lacking mRNA to the same extent (95%) and with the same kinetics; reaction of [3H]cDNA to nominal poly(A)-lacking mRNA with the two mRNA populations gives the same result. The extensive homology these two mRNA populations share is important to the interpretation of mRNA lifetime and to the analysis of authentic poly(A)-lacking mRNAs.     

Polyadenylic acid-containing RNA and its polyadenylic acid sequences newly synthesized in isolated embryonic cells of Xenopus laevis.

Newly synthesized polyriboadenylic acid [poly(A)]-containing RNA and its poly(A) sequences were isolated and characterized in Xenopus embryonic cells. Upon sedimentation analysis, the poly(A)-containing RNA labeled for 30 min showed a very heterogeneous size distribution ranging from 9 to >40 S. After 5 hr of labeling, the profile became much less heterogeneous and the main component was distributed in the 9–28 S region. The average molecular weight of 6.5–7.0 × 10⁵ daltons was calculated for the 5-hr labeled RNA. This poly(A)-containing RNA, comprising about 10% of the total labeled RNA, was metabolically stable and accumulated linearly for 5 hr. Gel electrophoresis of the RNA revealed the presence of little or no free poly(A) sequences. Most of the poly(A) sequences, which were isolated from 30-min labeled poly(A)-containing RNA migrated as a single discrete component approximately 150 nucleotides long. In contrast, they were slightly smaller (130 nucleotides long) and more heterogeneous, when obtained from the poly(A)-containing RNA labeled for 5 hr. From these results, it may be likely that the embryonic poly(A)-containing RNA is similar in size to the steady-state population of the poly(A)-containing RNA reported to occur in vitellogenic oocytes and cultured kidney cells of the same species.     

Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa.

Ribonucleic acid (RNA) extracted from Neurospora crassa has been fractionated by oligodeoxythymidylic acid [oligo(dT)]-cellulose chromatography into polyadenylated messenger RNA [poly(A) mRNA] and unbound RNA. The poly(A) mRNA, which comprises approximately 1.7% of the total cellular RNA, was further characterized by Sepharose 4B chromatography and polyacrylamide gel electrophoresis. Both techniques showed that the poly(A) mRNA was heterodisperse in size, with an average molecular weight similar to that of 17S ribosomal RNA (rRNA). The poly(A) segments isolated from the poly(A) mRNA were relatively short, with three major size classes of 30, 55, and 70 nucleotides. Gel electrophoresis of the non-poly(A) RNA indicated that it contained primarily rRNA and 4S RNA. The optimal conditions were determined for the translation of Neurospora mRNA in a cell-free wheat germ protein-synthesizing system. Poly(A) mRNA stimulated the incorporation of [14C]leucine into polypeptides ranging in size from 10,000 to 100,000 daltons. The RNA that did not bind to oligo(dT)-cellulose also stimulated the incorporation of [14C]leucine, indicating that this fraction contains a significant concentration of mRNA which has either no poly(A) or very short poly(A) segments. In addition, the translation of both poly(A) mRNA and unbound mRNA was inhibited by 7-methylguanosine-5'-monophosphate (m7G5'p). This is preliminary evidence for the existence of a 5'-RNA "cap" on Neurospora mRNA.     

Biochemical studies of mammalian oogenesis: kinetics of accumulation of total and poly(A)-containing RNA during growth of the mouse oocyte

Kinetics of accumulation of total and poly(A)-containing RNA have been measured during growth of the mouse oocyte. Total RNA from oocytes isolated at discrete stages of growth was determined by two independent microassays. The full-grown oocyte contained about 0.60 ng of RNA. Kinetics of accumulation of total RNA with respect to oocyte volume were biphasic. Small, growing oocytes (about 30 pl) contained about 0.20 ng of RNA/oocyte. The amount of RNA increased in a quasi-linear fashion until oocyte volume was about 160 pl, at which point there was about 0.57 ng of RNA/oocyte. Thus oocytes about 65% of their final volume had accumulated about 95% of the total amount of RNA present in the fully-grown oocyte. The relative amount of poly (A)-containing RNA in oocytes of various size was determined by in situ hybridization of [3H] poly (U) to ovarian sections from juvenile mice of known age, followed by autoradiography. The kinetics of accumulation of poly (A)-containing RNA were similar to those of total RNA; oocytes about 70% of their final volume had accumulated about 95% of the amount of poly (A)-containing RNA present in the fully-grown oocyte. The poly(A)-containing RNA resided predominantly in the cytoplasm and no obvious cytoplasmic localization was observed. Kinetics of accumulation of total RNA, which is mainly ribosomal, and poly (A)-containing RNA were consistent with levels of RNA polymerases I and II measured by others during oocyte growth (Moore and Lintern-Moore, '78). The number of ribosomes that could be made from the amount of rRNA present at various stages of growth was compared to the actual number of ribosomes calculated from a published morphometric study (Garcia et al., '79). Kinetic differences in accumulation between the theoretical and actual number of ribosomes suggested oocyte ribosomes are recruited into cytoplasmic lattice structures. These structures accumulate during oocyte growth and have been postulated to be a ribosomal storage form. In addition, the results from this study are compared to results derived from lower species.     

Comparison of cytoplasmic and nuclear poly(A)-containing RNA sequences in Xenopus liver cells.

Poly(A)-containing RNAs from cytoplasm and nuclei of adult Xenopus liver cells are compared. After denaturation of the RNA by dimethysulfoxide the average molecule of nuclear poly(A)-containing RNA has a sedimentation value of 28 S whereas the cytoplasmic poly(A)-containing RNA sediments slightly ahead of 18 S. To compare the complexity of cytoplasmic and nuclear poly(A)-containing RNA, complementary DNA (cDNA) transcribed on either cytoplasmic or nuclear RNA is hybridized to the RNA used as a template. The hybridization kinetics suggest a higher complexity of the nuclear RNA compared to the cytoplasmic fraction. Direct evidence of a higher complexity of nuclear poly(A)-containing RNA is shown by the fact that 30% of the nuclear cDNA fails to hybridize with cytoplasmic poly(A)-containing RNA. An attempt to isolate a specific probe for this nucleus-restricted poly(A)-containing RNA reveals that more than 10(4) different nuclear RNA sequences adjacent to the poly(A) do not get into the cytoplasm. We conclude that a poly(A) on a nuclear RNA does not ensure the transport of the adjacent sequence to the cytoplasm.     

Photoaffinity labeling of the ribosomal peptidyl transferase site with synthetic puromycin analogues.

A photoaffinity labeling puromycin analogue, Nepsilon-(2-nitro-4-azidophenyl)-L-lysinyl puromycin aminonucleoside (NAP-Lys-Pan), was synthesized and used for investigation of the peptidyl transferase center of 70S riobsomes. Visible light irradiation of NAP-Lys-Pan led to covalent linkage of the analogue with Escherichia coli ribosomes. In a subsequent step, poly(uridylic acid) was employed to direct Ac[14C]Phe-tRNA to the P sites of the photolabeled ribosomes. Transpeptidation of Ac[14C]phenylalanine to the bound NAP-Lys-Pan resulted in selective incorporation of radioactive label into the peptidyl transferase A site. Dissociation of the ribosomes into subunits, and digestion of the RNA components, indicated that the radioactive label was incorporated into a protein fraction of the 50S subunit.