Fibroin mRNA Research Articles

Gene regulation involved in the formation of long-term memory

My former research focused on silk fibroin gene transcription. The in vivo transcription initiation site of the fibroin gene, which is similar to the site corresponding to the 5'-terminal of mature fibroin mRNA, was determined. By developing a cell-free transcription system prepared from silk glands, it was found that the upstream region of the fibroin gene is responsible for efficient transcription initiation, which has enhancer-like features. More recent research has switched my focus to cellular neurobiology to understand the molecular mechanisms of long-term memory at the level of gene expression in terms of cell differentiation. I first developed an experimental system to analyze promoter activity in primary cultured neuronal cells. Particularly focusing on the transcription regulation of the brain-derived neurotrophic factor (BDNF) gene (Bdnf), I found that the interaction of the cAMP response element-binding protein (CREB) with the CRE sequence is important for the activity-dependent activation of the Bdnf promoter. In addition, this activity-dependent transcriptional regulation occurs in cultured neurons stimulated with excitatory GABAergic inputs, which plays a critical role in promoting the step of neuronal differentiation. Finally, I found that stimulation of the G-protein coupled receptor (GPCR) effectively activates Bdnf promoter IV through selective activation of the calcineurin pathway, irrespective of the type of GPCR if the protein kinase A or C pathway is activated. This induction mechanism appears important to understand intracellular mechanisms evoked via simultaneous neurotransmission of excitatory and modulatory inputs into neurons of the brain.

Ras1(CA) overexpression in the posterior silk gland improves silk yield.

Sericulture has been greatly advanced by applying hybrid breeding techniques to the domesticated silkworm, Bombyx mori, but has reached a plateau during the last decades. For the first time, we report improved silk yield in a GAL4/UAS transgenic silkworm. Overexpression of the Ras1(CA) oncogene specifically in the posterior silk gland improved fibroin production and silk yield by 60%, while increasing food consumption by only 20%. Ras activation by Ras1(CA) overexpression in the posterior silk gland enhanced phosphorylation levels of Ras downstream effector proteins, up-regulated fibroin mRNA levels, increased total DNA content, and stimulated endoreplication. Moreover, Ras1 activation increased cell and nuclei sizes, enriched subcellular organelles related to protein synthesis, and stimulated ribosome biogenesis for mRNA translation. We conclude that Ras1 activation increases cell size and protein synthesis in the posterior silk gland, leading to silk yield improvement.

Expression of the Japanese oak silkworm Antheraea yamamai fibroin gene in the domesticated silkworm Bombyx mori

Abstract To understand the evolutionary conservation of the gene expression mechanism and secretion machinery between Antheraea and Bombyx fibroins, we introduced the genomic A. yamamai fibroin gene into the domesticated silkworm, B. mori. The spliced A. yamamai fibroin mRNA appeared only in the posterior region of the silk gland of the transgenic silkworm, suggesting that the functions of the fibroin promoter region and the splicing machinery are conserved between these two species. The A. yamamai fibroin protein was detected in the lumen of the silk gland of the transgenic silkworm, albeit at lower levels compared with the B. mori‐type fibroin. We found a strong degeneration of the posterior region of the silk gland of the transgenic silkworm. As a result, the cocoon shell weight was much lower in the transgenic silkworm than in the non‐transgenic line. These results indicate that the promoter function and splicing machinery are well conserved between A. yamamai and B. mori but that the secretion mechanism of fibroin is diversified between the two.

Analysis of the Conserved N-Terminal Domains in Major Ampullate Spider Silk Proteins

Major ampullate silk, also known as dragline silk, is one of the strongest biomaterials known. This silk is composed of two proteins, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2). Only partial cDNA sequences have been obtained for these proteins, and these sequences are toward the C-terminus. Thus, the N-terminal domains have never been characterized for either protein. Here we report the sequence of the N-terminal region of major ampullate silk proteins from three spider species: Argiope trifasciata, Latrodectus geometricus, and Nephila inaurata madagascariensis. The amino acid sequences are inferred from genomic DNA clones. Northern blotting experiments suggest that the predicted 5' end of the transcripts are present in fibroin mRNA. The presence of more than one Met codon in the N-terminal region indicates the possibility of translation of both a long and a short isoform. The size of the short isoform is consistent with the published, cDNA based, N-terminal sequence found in flagelliform silk. Analyses comparing the level of identity of all known spider silk N-termini show that the N-terminus is the most conserved part of silk proteins. Two DNA sequence motifs identified upstream of the putative transcription start site are potential silk fibroin promoter elements.

Differential expression of the fibroin gene in developmental stages of silkworm, Antheraea mylitta (Saturniidae)

Fibroin gene expression during the larval developmental stages of the Saturniid silkworm, Antheraea mylitta, was analyzed. Northern blot analysis of larval silk gland total RNA using the fibroin gene as a probe showed that fibroin is expressed in the intermoult stages and repressed during the moulting stages. Abundance of fibroin transcripts gradually increased from the third to fifth intermoult stage, reaching a peak in the fifth intermoult. Transcripts declined during the early spinning stage. Western blot analysis of fibroin protein production with anti-fibroin antibody confirmed the differential fibroin expression, in accordance with fibroin mRNA synthesis. Dot blot hybridization of genomic DNA isolated from each larval developmental stage with the labelled fibroin gene showed that at the genomic level, the relative concentration of the fibroin gene was constant throughout the developmental stages. Our data confirm that fibroin gene expression in A. mylitta, like in B. mori, is transcriptionally controlled and shows differential temporal variations.

Discontinuous translation and mRNA structure of the coding region.

An analysis of the published data of the electrophoresis of the products of silk fibroin exhibiting discontinuities or pauses in the translational process showed that about 60 times of pauses occur during the translation of the mRNA from silk fibroin. In addition, we estimated that silk fibroin gene as a whole (15 kb) is composed of about 60 alternate, crystalline-noncrystalline, arrays of nucleotide sequence elements each connected by the boundary sequence, on the basis of the nucleotide sequence of the partial cDNA clone of the heavy chain gene of Bombyx mori available in the literature. The coincidence of the total number of pauses with that of the alternate arrays suggests that one translational pausing site exists in each of the unit (alternate array + boundary) nucleotide sequence in the silk fibroin mRNA template.

Features of the cell-free translation of a spider fibroin mRNA.

The massive production of fibroin by the large ampullate glands of the spider, Nephila clavipes, serves as a model system in which to study the synthesis and control of a large secretory protein. Their tissue-specific product, fibroin, produced during the entire adult life of the female, is approximately 320 kilodaltons, and rich in glycine and alanine. Highly purified fibroin mRNA from the glands has been translated in a rabbit reticulocyte cell-free system with variable supplements. The translational products analyzed by SDS-PAGE display two features, tRNA modulation and discontinuous pauses during elongation. tRNA complements exert their effects both in the translational efficiency and in the size of the peptides generated. The pauses observed during translation generate subsets of smaller discrete peptides, visualized in the gels as ladders of variable relative intensities, appearing exclusively and concomitantly with the fibroin. Definitive linkage between the discrete peptides and fibroin synthesis process has been established by their selective labeling with specific radioactive amino acids.

Specifie codon usage pattern and its implications on the secondary structure of silk fibroin mRNA

We have identified two distinctive regions of the repetitive unit nucleotide sequence of fibroin mRNA of Bombyx mori. The codon usage for the major amino acids, glycine, alanine and serine is distinctly different in these two regions, indicating that it is determined by the fibroin mRNA or gene structure but not by the tRNA population. Comparative computer analyses of nucleotide substitutions in the unit sequence suggest that selection has operated on the codon usage to optimize the secondary structure characteristic of the fibroin mRNA.

Low temperature causes accumulation of unspliced fibroin mRNA precursor molecules in silkworm larvae.

Silk fibroin premessenger mRNA is a large (17 kb) molecule containing a single intron. Previous S1 mapping studies yielded evidence of processing cleavages at sites within the intron, in addition to the major cleavages at the intron/exon boundaries. We have performed S1 mapping experiments using RNA from animals which had been incubated at low temperature. These experiments show a marked accumulation of full-length fibroin mRNA precursor relative to controls, and a reduction in the abundance of molecules lacking portions of the intron. These results demonstrate a partial uncoupling of transcription and splicing in vivo.

The Effect of 20-Hydroxyecdysone on the Synthesis of Fibroin mRNA in the Cultured Posterior Silk Gland of Bombyx mori

The activity of total RNA synthesis including fibroin mRNA in the cultured posterior silk gland of Bombyx mori was dependent on the developmental stage of larva from which the silk gland was dissected out. The majority of the RNA synthesized in the posterior silk gland cultured for 24 hr was ribosomal RNA and transfer RNA at all investigated stages. The synthesis of fibroin mRNA primarily decrease by the application of the molting hormone, 20-hydroxyecdysone, whereas the hormone stimulated the synthesis of both ribosomal and transfer RNAs.

Developmental variations of a nonfibroin mRNA of Bombyx mori silkgland, encoding for a low-molecular-weight silk protein

The characterization of a new silk protein mRNA (P25 mRNA) in posterior silkgland cells (PSG) and the developmental variations of its cell molecular concentration versus that of fibroin mRNA are described. A 80% pure P25 cDNA was obtained by class separation of total nonfibroin cDNA from PSG and used to identify the mRNA in blotted PSG mRNA as a single 1100 nucleotide long species. When purified from agarose gel and translated in a reticulocyte cell-free system, P25 mRNA yielded a 25-kD polypeptide (P25), identical to a 25-kD protein of the cocoon in terms of p I value and partial peptide mapping pattern. Moreover, this protein comigrated with an abundant polypeptide of the posterior silkgland (PSG) and of the middle silkgland (MSG). When tritiated leucine was injected in vivo, labeled P25 showed up in the PSG after a 2-hr pulse but appeared in the MSG only after 24 hr of labeling. Since MSG cells were found to be devoid of P25 mRNA, we concluded that P25 is exclusively synthesized in the PSG, that it accumulates in the MSG lumen and that it is spun out in the same way as fibroin. Specific probes were used to measure the concentrations of P25 mRNA and also fibroin mRNA in PSG total RNA by hybridization with an excess of cDNA. Both species are highly degraded in the few hours following the physiological arrest of feeding which precedes the fourth molting period. Their subsequent accumulation during the fifth intermolt is triggered by food uptake and proceeds in such a way that a constant 1:1 molar ratio is maintained during the period of silk secretion.

Gene expression of two fibroin alleles in the hybrid silkworm, J-131/Nd(2).

Fibroin gene expression in the posterior silk gland of normal strain (J-131/J-131), Nd strain (Nd(2)/Nd(2)) and their hybrid (J-131/Nd(2)) silkworms was compared on the levels of fibroin and fibroin mRNA production. The syntheses of both fibroin and fibroin mRNA in the Nd(2) and the hybrid silkworms were greatly reduced when compared with those of the normal silkworm. This result indicates that the Nd(2) gene represses dominantly fibroin gene expression, presumably at the level of transcription of fibroin gene. Estimation of gene products (fibroin and fibroin mRNA) from two fibroin alleles, Fnor and SNd, of the hybrid silkworm, J-131/Nd(2), demonstrates that the expression of the fibroin allele SNd derived from Nd(2) mutant strain is more severely repressed than that of the fibroin allele Fnor derived from J-131 strain.

Effect of tRNA pool balance on rate and uniformity of elongation during translation of fibroin mRNA in a reticulocyte cell-free system

Unsuccessful attempts to synthesize complete fibroin chains in vitro were previously made in heterologous cell-free system [3]. In the present work, we succeeded to obtain complete translation of purified fibroin mRNA in a rabbit reticulocyte lysate. Whilst this work was being completed [1], similar results were published by Lizardi et al. [4]. The synthesis of full-sized molecules of fibroin (M.W. 360,000) was achieved by adding tRNA from the posterior silk gland to the cell-free system. With tRNA from other sources, both the translation rate and the amount of complete fibroin chains dropped. This effect of tRNA is situated at the elongation levels. Analysis of cell-free synthesized products by polyacrylamide gel electrophoresis shows that smaller discrete polypeptides are accumulated after 120 minutes of incubation. These polypeptides correspond to growing fibroin chains. This pattern of translation products suggests that elongation might decelerate at specific sites of the fibroin mRNA. These results show that a tRNA pool adjusted to mRNA codon frequency is required to obtain the maximal average elongation rate. A stochastic model based on random acceptance of tRNA at the ribosomal A site for the codon-anticodon recognition process can explain this phenomenon. It can also explain the occurrence of the unfinished discrete fibroin polypeptides during in vitro translation.

Does quantitative tRNA adaptation to codon content in mRNA optimize the ribosomal translation efficiency? Proposal for a translation system model

Neither a dynamic nor an energetic approach of the translation process has taken into account that intracellular levels of iso-tRNA species are adapted or adjusted to the codon frequency of mRNA being decoded (Bombyx mori silk gland, rabbit reticulocyte). A critical study of available experimental data suggests that the average elongation rate of a protein is maximized in the presence of an adapted tRNA population, usually an homologous tRNA. In addition, the amount of synthesized protein parallels that of corresponding mRNA. Other evidences--including in vitro and in vivo elongation assays with fibroin mRNA--show that individual elongation rates are not uniform. Pauses occur at certain sites of the mRNA chain. The relative lifetime of these pauses depends on the tRNA pool used. Finally, it appears that translation accuracy also depends on the balanced tRNA population. We propose to explain these different effects by using a codon-anticodon recognition model, called "trial and error system" based on a stochastic processing of the ribosome. Accordingly, various acylated tRNA species which surround a ribosome randomly encounter the receptor A site. Every trapped tRNA species is tested for a proper pairing with the codon to be recognized at the level of a comparator or discriminator function. If the pairing is correct, transpeptidation becomes irreversible. If not, the aminoacyl-tRNA is rejected and another randomly trapped tRNA is processed in turn. Mathematical analysis of this model shows that the mean number of trials used for translating the whole sequence of a mRNA is minimized when the proportion of different iso-tRNA species is correlated with the square root of codon frequency. Quantitations of reticulocyte tRNA support such a parabolic relation. Our translation system model brings some light into the role of tRNA adaptation for optimizing translation efficiency, i.e. maximizing both speed and accuracy. Some consequences of the model are discussed.

Expression du génome de la cellule séricigène de Bombyx mori au dernier stade larvaire

Poly (A)-containing RNA was isolated from the posterior silkgland of Bombyx mori just after the fourth molt, when the gland is not producing fibroin (stage V0), and at the middle of the fifth larval instar, when the fibroin is massively synthesized (stage V6). The hybridization kinetics of these mRNA with their complementary DNA (cDNA) were remarkably different. The mRNA from stage V0 consisted of two abundance classes, comprising 38 and 2 915 different species, respectively. At stage V6, fibroin mRNA (FmRNA) was separated from the rest of the poly (A)-containing RNA (F-mRNA) by centrifugation on sucrose gradients, and both preparations were analyzed separately. The kinetic complexity of FmRNA was very low as compared to its actual size. This result agrees with the existence of a short repetitive sequence accounting for 60 p. 100 of the molecule. Stage V6 F-mRNA was resolved into four classes containing 1, 20, 319 and about 2 600 species, respectively. We carried out cross-hybridization with each of the two cDNA classes from stage V0 and stage V6 F-mRNA. They demonstrated that almost all the sequences present at stage V0 were also present at stage V6, but that their abundance class distribution was modified. Our data show that the specialization of the silkgland cell for fibroin production is characterized by massive accumulation of FmRNA and another unknown species, and that stage variations of mRNA populations are related to relative quantitative variations rather than to qualitative ones.

Adaptation des tRNA et optimisation de la traduction

The intracellular level of each tRNA species is adjusted to the codon frequency of the mRNA being decoded. This was first observed in such highly differentiated cells as the silk gland of Bombyx mori, which produces fibroin and sericin, and the rabbit reticulocyte. tRNA adaptation also occurs in other cell types from E. coli to mammalian cells. Regardless of the mechanism regulating tRNA biosynthesis, we believe that tRNA adaptation is the basic step optimizing chain elongation at the ribosomal level. We propose the system of trial and error as a working model for the ribosome. This model clarifies the correlations between iso-accepting tRNA levels and codon frequencies, as well as the effect of tRNA pool balance on mean elongation rate and non-uniform individual elongation rate (depending on whether codons are rare or abundant) for fibroin mRNA translated in a reticulocyte cell-free system.

Internal structure of the silk fibroin gene of Bombyx mori. I The fibroin gene consists of a homogeneous alternating array of repetitious crystalline and amorphous coding sequences.

The DNA sequence orgainzation of the protein encoding region of the gene for silk fibroin has been analyzed. The accompanying paper (Manningm R. F., and Gage, L. P. (1980) J. Biol. Chem. 255, 9451-9457) shows that the total length of the gene, and its protein, as well as the pattern of restriction sites in the gene is highly polymorphic among inbred stocks of Bombyx mori, In this paper, those features of fibroin gene structure which are invariant among these alleles are presented. Fibroin is composed primarily of relatively short "crystalline" and "amorphous" peptides of known sequence whose arrangement in the protein is unknown. Knowledge of the codons most commonly used in fibroin mRNA allowed utilization of particular restriction inzymes as a means for determing the nature and organization of crystalline and amorphous coding sequences in the fibroin gene. Three restriction endonucleases were identified that cleve sequences coding for amorphous region peptides. Their cleavage pattern revelaed that the repetitive coding sequence of the gene core (approximately 15 kilobases) is divided into at least 10 large crystalline coding domains interrupted by smaller amorphous coding domains. Many restriction endoncleases do not cleave the fibroin core at all, three of them with four gase recognition sequences. Specific deductions as to codon usage and repetitive sequence homogeneity in the gene follow from these results. One novel finding is the rigorous exclusion of the glycine codon GGA prior to serine codons even though this glycine codon is used frequently prior to alanine codons. The sequence homogeneity and the regularly alternating arrangement of crystalline and amorphous coding sequences of the gene are discussed in terms of the function of fibroin protein and the evolution of highly repetitive DNA.

Isolation of giant silk fibroin polysomes and fibroin mRNP particles using a novel ribonuclease inhibitor, hydroxystilbamidine.

Hydroxystilbamidine isethionate, a dye capable of binding to both DNA and RNA, has been found to be a powerful inhibitor of cellular ribonucleases. A procedure has been developed that, with the aid of this compound, permits the preparative isolation of giant silk fibroin polyribosomes from the posterior silk gland of Bombyx mori. The polyribosomes contain approximately 45-112 ribosomal particles, as judged by electron microscopy. Treatment of giant fibroin polyribosomes with EDTA releases a particle that sediments at 125S. This mRNP particle contains biologically active silk fibroin mRNA, as judged by cell-free translation in an mRNA-dependent reticulocyte cell-free system.

Repeated turn-off and turn-on of fibroin gene transcription during silk gland development of Bombyx mori

To measure the rates of fibroin mRNA synthesis in the posterior silk gland of Bombyx mori through the fourth feeding, fourth molting, and early fifth feeding stages, a saturation hybridization of 3H-RNA pulse labeled for 10 or 30 min to filter-bound fibroin DNA has been carried out. A high rate, 13 molecules/gene/min, of mRNA synthesis at 4 hr before the apolysis stage decreased precipitously to 0.44 and less than 0.084 molecule/gene/min at 4 and 7 hr after the apolysis, respectively. Such low rates, in the range of less than 0.006 to 0.084 molecule/gene/min, are observed for about 30 hr, from 7 hr after the apolysis to 5 hr after the fourth ecdysis, and are followed by a rapid elevation to 2.9 and 14 molecules/gene/min at 10 and 16 hr after the ecdysis, respectively. The rates of fibroin mRNA synthesis, if any, in the middle silk gland (fibroin nonproducer cells) are always less than 0.008 molecule/gene/min. These rates of fibroin mRNA synthesis are totally different from those of synthesis of ribosomal RNA and heterogeneous nuclear RNA of high molecular weight. From these results, we propose the possibility that fibroin gene expression is regulated at the transcriptional level through the repeated turn-off and turn-on of the gene during silk gland development.

Flimsy Cocoon Mutant of <I>Bombyx mori</I> Larva Produces a Reduced Amount of Fibroin mRNA

The proportions of fibroin mRNA synthesized in the posterior silk gland of the flimsy cocoon mutant (flc) of Bombyx mori at days 3, 4 and 5 of the fifth instar were 1.2 %, 0.7 % and 1.8 % of the labeled total RNA, re-spectively, whereas those in heterozygous larva (+/flc) at the same stages were 1.6 %, 3.2 % and 8.5 %, respectively. The total amount of fibroin mRNA that accumulated in one larva at day 5 of flc was less than 10 μg and that of +/flc was about 110 μg. The intracellular distribution of the mRNA also was measured. The proportion in the nuclear fraction was 0.5 % of the total labeled RNA for flc and 0.8 % of that for +/flc; in the cytoplasmic fraction the pro-portion was 0.2 % for flc and 2.4 % for +/flc. Thus, the reduced amount of fibroin mRNA in the flc mutant is considered to be due to the degradation of the synthesized mRNA rather than to a reduction in the synthesis of mRNA. It is believed that this degradation causes abnormal fibroin production. The primary action site of the flc gene is not known.