Non-polysomal poly(A)-containing messenger ribonucleoproteins of cryptobiotic gastrulae of Artemia salina

Non-polysomal poly(A)-containing messenger ribonucleoprotein (mRNP) of Artemia salina has been isolated by thermal chromatography on oligo(dT)-cellulose in moderate (250 mM) and low (50 mM NaCl and 5 mM MgCl2) ionic strength. The purified particles sedimented between 5 S and 30 S and banded at a density of 1.38-1.40 g/cm3 and 1.26-1.27 g/cm3 in CsCl and sucrose isopycnic centrifugation, respectively. The translatability of the mRNP in a cell-free system depended on the conditions of isolation. The protein composition of the free mRNP is independent of the conditions used in oligo(dT)-cellulose chromatography. The proteins have Mr of 87,000, 76,000, 65,000, 50,000, 45,000, 38,000 and 23,500. A specific set of proteins is associated wtih different ribonucleoproteins, although some proteins are present on multiple particles. The main 17 +/- 2-S particle is composed of proteins with Mr of 87,000, 76,000, 45,000 and 38,000. Approximately the same proteins were present on free mRNP and mRNP isolated from non-polysomal mRNP-ribosome complexes. Poly(A)-binding proteins have Mr of 38,000 and 23,500. The 38,000-Mr protein comprised at least 60% of the total mRNP protein. Poly(A)-binding proteins with Mr of 38,000 and 76,000 are also present in a free state in the cytoplasm. A relation between the main poly(A)-binding mRNP protein and the helix-destabilizing protein HD40 [Marvil, D. K., Nowak, L., and Szer, W. (1980) J. Biol. Chem. 255, 6466-6472] is discussed.     

The primer specificity of cytoplasmic poly(A) polymerase from cryptobiotic gastrulae of Artemia salina

Poly(A) polymerase has been purified to near homogeneity from the cytoplasm of Artemia salina as described previously (Roggen, E and Slegers, H. (1985) Eur. J. Biochem. 147, 225–232). Affinity chromatography on poly(A)-Sepharose 4B separates the enzyme preparation into two fractions. In standard assay conditions poly(A) polymerase fraction I (poly(A)-Sepharose 4B unbound) and fraction II (poly(A)-Sepharose 4B bound) have specific activities of 2.4 and 8.0 μmol AMP/h per mg enzyme, respectively. Poly(A) polymerase fraction II shows a high primer specificity towards the 17 S poly(A)-containing mRNP. Depending on the reaction conditions used, poly(A) sequences of 140 ± 15 AMP residues/μg enzyme are synthesized on the latter primer. In contrast, poly(A) polymerase fraction I only elongates oligo(A) primers efficiently. An endogenous RNA is detected in poly(A) polymerase II preparations. This RNA has a length of 83 ± 2 nucleotides and is a component of a 60 kDa particle. After removal of the latter the specificity of poly(A) polymerase fraction II for the 17 S poly(A)-containing mRNP is abolished and the characteristics of the enzyme resemble those of poly(A) polymerase I.     

Template activity for histones of poly(A)-minus RNA fraction from different developmental stages of Artemia salina embryos

Template activity for histones of RNA fractions derived from Artemia salina embryos at different developmental stages were measured in a Krebs ascites cell-free system. Appreciable amounts of acid-soluble polypeptides comigrating with Hela cell histone markers on acrylamide gel electrophoresis were detected only when RNA fractions from nauplii were used. Tryptic peptide analysis by high voltage electrophoresis of the translational products had a pattern qualitatively similar to that of in vivo labeled histone markers from Hela.     

Isolation and characterization of cytoplasmic poly(A) polymerase from cryptobiotic gastrulae of Artemia salina

Poly(A) polymerase has been purified to near homogeneity from the cytoplasm of Artemia salina cryptobiotic gastrulae by ion-exchange chromatography on DEAE-cellulose, DEAE-Sepharose CL-6B and phosphocellulose P11, gel filtration on CL-Sepharose 6B, affinity chromatography on poly(A)-Sepharose 4B and ATP-agarose. The enzyme is fully dependent on exogeneous oligo(riboadenylic acid) and is free of any nuclease or other enzyme activities. In standard assay conditions the enzyme preparation has a specific activity of 5.6 mumol AMP . h-1 . (mg protein)-1. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals the presence of only two proteins with Mr 94 000 and 70 000. The Mr-70 000 protein has been identified as poly(A) polymerase. The enzyme is exclusively activated by Mn2+. Addition of Ca2+, Mg2+, Zn2+, NH4+, K+ or Na+ inhibits the enzymatic reaction. The activity is specific for ATP and competitive inhibition is observed in the presence of other ribonucleoside 5'-triphosphates. AMP incorporation is time-dependent and is increased non-linearly with protein and primer concentration.     

Characterization of protein components of poly(A)-containing messenger ribonucleoproteins from cryptobiotic gastrulae of Artemia salina

Conditions for the isolation of intact poly(A)⁺mRNP from cryptobiotic gastrulae ofA. salina are described. In the presence of Mg²⁺ ions nucleolytic cleavage occurs in vitro in the vicinity of the 3-poly(A) segment of mRNP during the isolation procedure. The resulting two parts of poly(A)⁺mRNP complex are separated by thermal elution from oligo(dT)-cellulose affinity column. Analysis by SDS-gel electrophoresis of protein components associated with intact poly(A)⁺mRNP has revealed the existence of 20–30 S RNP complex containing five major proteins with M_r 68,000, 53,000, 50,000, 45,000 and 38,000, respectively, but completely lacking the poly(A)-specific M_r 76,000 protein.     

Evidence of a store of informational RNA in encysted embryos of Artemia salina

RNA prepared from dormant cysts and developmental stages of the brine shrimp Artemia salina stimulated the incorporation of ¹⁴C-leucine into polypeptide by a cell-free Escherichia coli system. Preparations from cysts were about as active as those from hatching embryos or nauplii. When analysed by density gradient centrifugation the activity of cyst RNA showed a heterodisperse distribution, not quantitatively related to the absorbance profile. These results and evidence from similar experiments with crude ribosome preparations indicated that the contribution of 18S and 28S ribosomal RNA to the template-like activity was fairly limited. The experiments suggest that RNA with latent messenger activity is present in Artemia cysts during the resting stage.     

Initiation factor eIF2 associated with non-polysomal poly(A)-containing messenger ribonucleoproteins of cryptobiotic gastrulae of Artemia salina

Non-polysomal poly(A)-containing mRNP of A. salina cryptobiotic embryos is separated in mRNP active in protein synthesis and in repressed mRNP by sucrose gradient centrifugation. In the translationally active fraction the presence of eukaryotic initiation factor 2 (eIF2) is demonstrated by electroblotting of sodium dodecylsulphate/polyacrylamide gels on nitrocellulose and anti-eIF2 antibody detection. mRNP proteins with Mr of 40 000 and 42 000 are identified as the alpha and beta subunits of eIF2. The repressed mRNP is devoid of eIF2 and is associated with an inhibitor ribonucleoprotein composed of a small 85 +/- 2-nucleotide-long RNA and a protein with Mr of 64 000. The latter ribonucleoprotein is a potent inhibitor of the translationally active mRNP.     

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.     

Study of the double-stranded fragments in the poly(A)-containing cytoplasmic RNA of the rat liver

The double-stranded sequences in the poly(A)-containing cytoplasmic RNA (c-dsRNA) hybridize mainly with the repetitive DNA. 70-80% of double-stranded regions in the cytoplasmic poly (A)-RNA are identical to double-stranded regions of heterogeneous nuclear RNA in normal as well as in cortisone-treated rats. The thermal stability of cytoplasmic double-stranded regions is higher in the presence of polyadenylate sequences than in their absence. It is suggested that the double-stranded sequences in the poly (A) +RNA interact with poly (A) stretches and form higher order structures. The thermal stability of c-dsRNA isolated from cortisone-treated rats is higher than that from control rats.     

'Cap' structures in maize poly(A)-containing RNA.

Poly(A)-containing RNA was isolated from maize embryos by chromatography on columns of oligo(dT)-cellulose and exhaustively digested with ribonucleases T2, T1, and A. Fractionation of the digests by two-dimensional electrophoresis revealed the presence of three 7-methylguanosine-terminated 'cap structures' of the type m7GpppNp.     

Differential accumulation and localization of maternal poly(A)-containing RNA during early development of the ascidian,Styela

The regional distribution of poly(A)⁺ RNA was examined in sections of Styela oocytes and fertilized eggs by in situ hybridization with [³H]poly(U). The nucleus and cytoplasm of previtellogenic oocytes contain equivalent densities of [³H]poly(U) binding sites. The concentration of these sites is reduced in the cytoplasm, but not the nucleus, during vitellogenesis. Consequently, the germinal vesicle (GV) plasm of mature oocytes is characterized by an eightfold elevation in [³H]poly(U) binding activity relative to the surrounding cytoplasm. The distinctive cytoplasmic regions of the mature oocyte do not exhibit differential concentrations of [³H]poly(U) binding sites. Following fertilization which triggers GV breakdown, meiosis, and ooplasmic segregation, the high density of [³H]poly(U) binding sites characteristic of the GV plasm is conserved in the basophilic cytoplasm during its extensive migration and eventual accumulation in the animal hemisphere of the egg. The insensitivity of the [³H]poly(U) binding sites of the basophilic cytoplasm to actinomycin D suggests that they are of maternal origin. It is concluded that maternal poly(A)⁺ RNA is subject to differential accumulation in the GV plasm and its derivative ooplasm during the early development of Styela.     

Characterization and messenger activity of poly(A)-containing RNA from Chlorella.

Poly(A)-containing RNA was isolated by cellulose column chromatography from total RNA extracted from Chlorella fusca var. vacuolata 211/8p. RNA retained by the column was identified as poly(A)-containing RNA because it contained ribonuclease-resistant tracts, 25 to 55 nucleotides in length, from which not less than 80% of base was found to be adenine after acid hydrolysis. The base composition of poly(A)-containing RNA differed from that of RNA (largely ribosomal) which did not adsorb to cellulose, having a higher adenine content and a lower guanine content. Poly(A)-containing RNA was polydisperse including molecules with mobilities from 10S to 40S with a mean of about 20S. In an in vitro system derived from wheat-germ, protein synthesis was stimulated by adding poly(A)-containing RNA from Chlorella. Optimum conditions were established in this system with respect to the amount of poly(A)-containing RNA added and the concentration of KCl and Mg-2+. It is proposed that, in Chlorella, poly(A)-containing RNA includes cytoplasmic mRNA as has been shown for some other eucaryotic organisms.     

Changes in polyribosomes and poly(A)-containing polyribosomal RNA in the uterus of the ovariectomized rat in response to oestradiol-17 beta treatment.

Polyribosome formation and the characteristics of polyribosomal poly(A)-containing RNA from uteri of ovariectomized rats responding to a single dose of oestradiol-17 beta was investigated. The mean proportion of polyribosomes in the atrophic uterus was 65%. In response to 10 micrograms of oestradiol-17 beta/100 g body mass, the amount of polyribosomes increased to 88% 24 h after stimulation. Thereafter the proportion of polyribosomes decreased to a value of 48% at 72h. The pattern of amino acid incorporation in oocytes from Xenopus laevis injected with these polyribosomes was similar to the changes in polyribosome formation and degradation. The polyribosomal poly(A)-containing RNA from the controls consisted of a heterogeneous population of RNA with sedimentation values between 5S and 25S. The hormone stimulation resulted in an increase in both the amount and the size (13S to 35S) of the RNA.     

Characterization of a poly(A)+RNA-containing structural component directly associated with cytoplasmic membranes in dormant Artemia cysts

Poly(A)+RNA-containing material was extracted from the purified cytoplasmic membranes of dormant Artemia cysts by treatment with mild detergents. Sedimentation analysis of the extracts showed a predominant poly(A)-containing fraction at 40 S, associated with about 6% of the extracted proteins. Only limited amounts of poly(A)-containing material were found in the heavier fractions. Poly(A)+RNA extracted from the 40-S fraction sedimented around 14 S. The poly(A)-containing 40-S structures could be purified by treatment with non-ionic or zwitterionic detergents followed by resedimentation in sucrose gradients in the presence or absence of detergent. When the 40-S fraction was analyzed by isopycnic centrifugation in Cs2SO4 gradients, the main part of the poly(A)-containing material banded at a density of 1.27 g/ml. Electron-microscopic examination of this fraction revealed circular or slightly bullet-shaped profiles measuring 17-26 nm. When the 40-S fraction had been submitted to mild RNAase treatment prior to density gradient centrifugation, the material was displaced towards lower density and became less distinct. Purified 40-S particles showed a complex protein pattern not very similar to that of polyribosomal poly(A)+RNA-containing particles from developing embryos, but with components in common with unfractionated membranes. The particles also contained some lipids. The experiments indicate that a major part of the membrane-bound, latent poly(A)+RNA in dormant Artemia cysts occurs in the form of relatively uniform, detergent- and Cs2SO4-resistant structures, independent of ribosomes, but intimately associated with membrane components.