The spatial distribution of maternal mRNA is determined by a cortical cytoskeletal domain in Chaetopterus eggs

Messenger RNA molecules are localized in the cortical region of eggs and unevenly segregated to the embryonic cells during early development of the annelid Chaetopterus. The egg cortex is enriched in two organelles, ectoplasmic spherules and associated structures, which are similar in appearance to nuage. The physical basis of cortical mRNA localization was examined in stratified eggs and in eggs extracted with the nonionic detergent Nonidet P-40 (NP-40). The cortical organelles were displaced to the most centrifugal zone of stratified eggs. In situ hybridization with poly(U) or cloned DNA probes showed that a large proportion of the poly(A)+RNA, histone mRNA, and actin mRNA molecules was also displaced to the centrifugal zone. Extraction with NP-40 revealed a detergent-insoluble cytoskeletal domain (CD) in the egg cortex which contained the remnants of ectoplasmic spherules and nuage embedded in a fibrous network. Although most of the total protein and RNA was extracted by NP-40, a large proportion of the poly(A)+RNA, histone mRNA, and actin mRNA molecules was retained in the CD. In situ hybridization of stratified eggs extracted with NP-40 indicated that the CD, with its associated organelles and mRNA molecules, is displaced to the centrifugal zone as a unit. The results suggest that the tenacious association of mRNA molecules with the cortical CD may be responsible for maternal mRNA localization during early development.     

Localization of actin messenger RNA during early ascidian development

The spatial distribution of RNA sequences during early development of the ascidian, Styela plicata, was determined by in situ hybridization with poly(U) and cloned DNA probes. Styela eggs and embryos contain three colored cytoplasmic regions of specific morphogenetic fates, the ectoplasm, endoplasm, and myoplasm. These cytoplasmic regions participate in ooplasmic segregation after fertilization and are distributed to different cell lineages during early embryogenesis. n situ hybridization with poly(U) suggests that poly(A)+RNA is unevenly distributed in eggs and embryos, with about 45% in the ectoplasm, 50% in the endoplasm, and only 5% in the myoplasm. In situ hybridization with a histone DNA probe showed that histone RNA sequences were not localized in eggs or embryos and distributed between the three cytoplasmic regions according to their volumes. In situ hybridization with an actin DNA probe showed actin RNA was localized in the myoplasm and ectoplasm of eggs and embryos with about 45% present in the myoplasm, 40% in the ectoplasm, and only 15% in the endoplasm. These results suggest that a large proportion of the egg actin mRNA is localized in the myoplasm, participates in ooplasmic segregation after fertilization, and is differentially distributed to the mesodermal cell lineages during embryogenesis. Analysis of the translation products of egg mRNA suggests that the localized mRNA codes for a cytoplasmic actin isoform.     

Spatial distribution of messenger RNA in the cytoskeletal framework of ascidian eggs

Maternal poly(A)⁺RNA, histone mRNA, and actin mRNA exhibit unique spatial distributions in the different ooplasmic regions of ascidian eggs. These RNAs also appear to migrate with their respective ooplasms during the episode of extensive cytoplasmic rearrangement that occurs after fertilization, suggesting they are associated with a structural framework. The role of the cytoskeletal framework (CF) in determining the spatial distribution of maternal mRNA was tested by subjecting Triton X-100 extracted (Styela plicata) eggs and early embryos to in situ hybridization with poly(U) and cloned DNA probes. Grain counts indicated that substantial proportions of the egg poly(A)⁺RNA, histone mRNA, and actin mRNA were present in the CF and that there was no alteration in the extent of mRNA-CF interactions during the period between fertilization and the two-cell stage. Analysis of grain distributions indicated that poly(A)⁺RNA, histone mRNA, and actin mRNA were concentrated in the same regions of detergent-extracted eggs as they are in intact eggs. The proportions and spatial distribution of these RNAs in the CF were not affected when the actin cytoskeleton was destabilized by cytochalasin B or DNAse I. The data suggest that maternal mRNA is associated with the CF, that this association is responsible for mRNA rearrangement during ooplasmic segregation, and that mRNA-CF interactions are not dependent on the integrity of the actin cytoskeleton.     

Translation of maternal histone mRNAs in sea urchin embryos: a test of control by 5' cap methylation

Recent results have demonstrated the occurrence of mRNA cap methylation in the sea urchin embryo following fertilization. It has been suggested that this methylation event is responsible for the translational activation of maternal histone mRNAs in these embryos. We have used aphidicolin, an effective inhibitor of both DNA synthesis and cap methylation in cleavage stage sea urchin embryos, to examine the relationship between cap methylation and translation. At 5 micrograms/ml, a dose which rapidly abolishes DNA replication and blocks cleavage, we note no effect on recruitment or translation of maternal alpha-subtype histone mRNAs. This suggests that a postfertilization cap methylation event is not critical to the process of regulation of the translation of stored alpha-subtype histone mRNAs.     

Messenger RNA in the cytoskeletal framework: analysis by in situ hybridization

The possibility of an association of mRNA with the cytoskeletal framework (CF) of ascidian (Styela plicata) follicle cells was examined in this study. The approach was to extract the follicle cells with Triton X-100 and determine whether mRNA persisted in the insoluble residue by two methods, in situ hybridization with poly(U) and actin DNA probes and the incorporation of radioactive isotopes into RNA. Triton X-100 extraction of follicle cells yielded a filamentous CF containing approximately 70% of the total poly (A) but only 9% of the total lipid, 23% of the total protein, and 28% of the total RNA. In situ hybridization with a poly (U) probe indicated that approximately 70% of the poly (A) was associated with the CF. In situ hybridization with a cloned actin DNA probe indicated that approximately 60% of the actin mRNA was associated with the CF. Autoradiography of detergent- extracted follicle cells, which had been labeled with [3H]uridine or [3H]adenosine, indicated that greater than 90% of the newly synthesized poly (A)+RNA was preserved in the CF. Thus more newly synthesized mRNA than steady-state mRNA may be present in the Triton X-100 insoluble fraction. It is concluded that a significant proportion of the mRNA complement of ascidian follicle cells is associated with the CF.     

Polyadenylation state of abundant mRNAs during Drosophila development

We have used a two-dimensional gel analysis of cell-free translation products to determine whether individual mRNAs present in Drosophila melanogaster embryos, larvae, pupae, and adults are predominantly polyadenylated or nonadenylated. While the majority of the embryonic mRNAs we detected exist mainly in the polyadenylated form, these mRNAs become more evenly distributed between the poly(A)+ and poly(A)- RNA fractions during postembryonic development. Although DNA:RNA hybridization experiments have indicated that Drosophila RNA populations contain a large group of rare class mRNAs restricted to the poly(A)- RNA compartment, this is not true for the 150 more abundant mRNA species analyzed by our methods. The histone mRNAs are the only abundant mRNA species which appear to be exclusively in the poly(A)- RNA class.     

Changes in relaxin precursor mRNA levels in the rat ovary during pregnancy

Levels of preprorelaxin mRNA in the rat ovary during pregnancy were determined by cell-free translation and by hybridization analyses with cloned preprorelaxin cDNA. Translation of poly(A+) RNA from rat ovaries taken at different stages of pregnancy resulted in the incorporation of [35S]cysteine into two peptides, of Mr 17,500 and 20,500, that were specifically bound by anti-relaxin IgG. Both peptides also were demonstrated by translation of ovarian poly(A+) RNA that was hybrid-selected with cloned preprorelaxin cDNA, the sequence of which corresponds to the Mr 20,500 peptide. The origin of the Mr 17,500 putative precursor is not presently known. Preprorelaxin mRNA translational activities corresponded to previously reported concentrations of relaxin in rat ovaries during pregnancy. The results of hybridization analyses, both by Northern blotting of poly(A+) RNA and dot blotting of unfractionated RNA, agreed with those of translation assays. Preprorelaxin mRNA activity/concentration was low in early pregnancy, rose markedly and reached a plateau on days 15-20 (about 1-2% of total translation activity), and then fell to low levels again by day 23, the time of parturition. These findings indicate that the concentration of relaxin in the rat ovary is directly dependent on preprorelaxin mRNA levels.     

Histone messenger RNA synthesis and accumulation during early development in the echiuroid worm, Urechis caupo

RNA isolated from Urechis caupo mature oocytes and embryos was analyzed for the presence of histone messenger RNAs (mRNAs) by in vitro translation and by filter blot hybridization to determine the contribution of maternal and newly transcribed histone mRNAs to the pattern of histone synthesis during early development. Histone mRNAs were not detected in mature oocyte RNA which suggests that relatively few if any maternal histone mRNAs are sequestered in the mature oocytes. Histone mRNAs were detected in cleavage-stage RNA and increased in amount from midcleavage through late gastrula stages. The in vitro translation analysis also demonstrated that the amount of H1 histone mRNA in late cleavage- and early blastula-stage embryos exceeds that of the individual core histone mRNAs. The disproportionate accumulation of individual histone mRNAs correlates with the noncoordinate synthesis of H1 and core histones which occurs during early embryogenesis.     

Cellular localization of induced human interferon-beta mRNA by non-radioactive in situ hybridization

Induced interferon-beta (IFN-beta) mRNA was localized in human FS-4 fibroblasts by in situ hybridization using biotinylated probes. The hybridization sites were detected by incubation with a nick-translated genomic DNA probe (1.8 kb) via streptavidin-colloidal gold followed by silver contrast enhancement. The positive signals were observed by reflection-contrast light microscopy. IFN-beta mRNA was transiently induced by poly r(I): r(C) in fibroblasts 2-4 h after induction. Induction in the presence of cycloheximide and actinomycin D (superinduction conditions) exhibited an enhanced level of IFN-beta mRNA with a maximum at 4-8 h. The kinetics of the IFN-beta mRNA expression in the cytoplasm as revealed by in situ hybridization proved to be compatible with the results of Northern blotting experiments of total cellular RNA.     

The mobilization of maternal histone messenger RNA after fertilization of the sea urchin egg

The extent of protein, RNA and DNA synthesis in early cleavage stages of the sea urchin embryo (Parechinus angulosus) was determined. A histone mRNA specific cDNA was used in hybridization experiments to investigate the cytoplasmic localization of maternal histone mRNA in the unfertilized sea urchin egg and first cleavage stage embryo. In the unfertilized egg histone mRNA was localized exclusively in ribonucleoprotein particles with none in ribosomes or polyribosomes. This distribution changed after fertilization, in particular, coupled with the first cleavage telophase there was a significant transfer of histone mRNA from the ribonucleoprotein fraction to the polyribosomes. The results indicate mRNA specific translational control mechanisms.     

The construction and partial characterization of plasmids containing complementary DNA sequences to human calcitonin precursor polyprotein.

(1) Total poly(A)-containing RNA isolated from human thyroid medullary carcinoma tissue was shown to direct the synthesis in the wheat germ cell-free system of a major (Mr 21000) and several minor forms of human calcitonin precursor polyproteins. Evidence for processing of these precursor(s) by the wheat germ cell-free system is also presented. (2) A small complementary DNA (cDNA) plasmid library has been constructed in the PstI site of the plasmid pAT153, using total human thyroid medullary carcinoma poly(A)-containing RNA as the starting material. (3) Plasmids containing abundant cDNA sequences were selected by hybridization in situ, and two of these (ph T-B3 and phT-B6) were characterized by hybridization--translation and restriction analysis. Each was shown to contain human calcitonin precursor polyprotein cDNA sequences. (4) RNA blotting techniques demonstrate that the human calcitonin precursor polyprotein is encoded within a mRNA containing 1000 bases. (5) The results demonstrate that human calcitonin is synthesized as a precursor polyprotein.     

Isolation of poly (A)+ mRNA for downstream reactions from some medicinal and aromatic plants

In the present protocol for extraction of RNA, hexadecyltrimethylammoniumbromide (CTAB) and insoluble polyvinylpyrrolidone were used followed by LiCl precipitation, CsCl ultracentrifugation and finally poly (A)+ mRNA was isolated with the help of oligo(dT)-cellulose columns. The isolated poly (A)+ mRNA was found to be suitable for cDNA-AFLP and suppression subtractive hybridization applications. It is a modified and consolidated protocol based on previously described methods for isolated steps and works better for medicinal and aromatic plants. High yield of poly (A)+ mRNA coupled with its amenability for downstream reactions like RT-PCR, northern blotting and cDNA synthesis for library construction is a key feature of the present protocol.     

Polyadenylic acid-containing of rna in unfertilized and fertilized eggs of the rabbit.

Molecular hybridization between ³H-polyuridylic acid and unlabeled RNA prepared from unfertilized rabbit eggs and 10-h postfertilization stage rabbit embryos has been used to measure the amount and subcellular localization of adenylated maternal RNA. The results reported indicate that there is poly (A)-containing RNA (putative messenger RNA) in unfertilized rabbit eggs. The amount of poly (A) in the RNA in rabbit eggs does not increase immediately after fertilization and is located primarily in the ribosomal fraction of the cell. The rate of protein synthesis in fertilized eggs is insensitive to α-amanitin at concentrations which inhibit RNA synthesis. These results suggest that maternal mRNA makes an important contribution to protein synthesis in early stages of cleavage in the rabbit embryo.