Aquatic silk proteins in Chironomus: A review

Silk proteins secreted by salivary glands in the dipteran insect, Chironomus play a significant role as proteinaceous adhesives for construction of underwater housing nests by larvae. To date, only three Chironomus species, C. tentans Fabricius, C. pallidivittatus Malloch and C. riparius Meigen have been explored for characterization of their aquatic silk protein. Genes coding for silk proteins are located on specific chromosomal ‘puffs’ called Balbiani rings as well as non-Balbiani ring regions.  Expression of these genes is closely regulated by developmental and hormonal alterations and environmental factors. Furthermore, pilot studies have postulated that silk proteins probably occur in diverse size classes grouped into large (~1000 kDa), intermediate (100-200 kDa) and small (≤100 kDa). Barring few preliminary reports that date back to the 1990s, the physical and bioproperties of silk from chironomid midges remain largely unknown, leading to paucity of updated information. This review was therefore aimed to compile existing literature database and to highlight the wide possibilities for commercialization of midge larval silk as a novel biopolymer

Nuclear export of single native mRNA molecules observed via light sheet fluorescence microscopy and transcriptional regulation of BR2.1 during heat-shock

Eucaryotes store most of their genetic information in the nucleus. Parts of this information encode the amino acid sequence of proteins. To synthesize a protein according to the nucleotide sequence, first the corresponding DNA-sequence is transcribed by RNA-Polymerase II to mRNA. Subsequently ribosomes translate the mRNA into the correct amino acid sequence. In eucaryotes the ribosomes are localized in the cytoplasm and are separated from the nucleus by the nuclear envelope. On the one hand separation of transcription and translation enables eucaryotes to process the transcript post-transcriptionally, on the other it requires a transport of the mRNA from the nucleoplasm into the cytoplasm. The nucleoplasm is interconnected with the cytoplasm by nuclear pore complexes. Most of the nucleo-cytoplasmic trafficking is facilitated through the nuclear pore complexes. Messenger RNA is exported into the cytoplasm through the nuclear pore complexes, too. During transcription the nascent mRNA is bound by several proteins which are essential e.g. for mRNA processing and export. The complex of the mRNA and its associated proteins is called an mRNP-particle. Fully processed mRNP-particles are able to cross the permeability barrier of nuclear pore complexes. In this thesis the kinetics of the mRNA-export were measured in salivary gland cells of C. tentans at the single molecule level. Therefore, mRNA was labeled by Hrp36, which was bacterially expressed and subsequently covalently linked to a fluorescent dye. Hrp36 associates cotranscriptionally with the nascent mRNA and is part of the mRNP-particle. After microinjection, labeled Hrp36 is transported into the nucleus, via its endogenous M9-shuttle domain. As all mRNP-particles, also the labeled ones, diffuse through the nucleus after transcription is finished and can be imaged by advanced fluorescence microscopy. In this thesis it is shown that the kinetics of the mRNA-export across the nuclear prore complexes follow a broad distribution in the range of 20ms to seconds. Furthermore, only 30% of all mRNP-particles are exported after they engaged an NPC. Fitting the mRNA-export kinetics with a bimodal gamma distribution revealed average export times of t1exp = 76ms, which is governed by multiple rate limiting steps and t2exp = 158ms, which is governed by just a single rate limiting step. Therefore, the translocation of the mRNA across the nuclear pore complex is not rate limiting for protein-biosynthesis which takes on average several minutes. Trajectory analysis of export events =300ms, showed that the mRNA were localized mainly in the nuclear basket during the export process. Here proteins are localized which are crucial for the mRNP-particle quality control. These proteins bind mRNP-particles, which are only partially processed, and thereby inhibit their translocation through the nuclear pore complex until their processing is completed. Assuming that the general reaction scheme is the same for all mRNP-particles and considering the fact that these slow export events show only a single rate limiting reactions step, this export events presumably correspond to mRNP-particles, whose processing were not finished. In addition to the mRNP-particle export kinetics, the Dbp5 interaction kinetics with the nuclear pore complexes were measured. Dbp5isaRNA-helicase, which is essential form RNP- particle export. It is assumed that Dbp5 removes the transport receptors from the mRNA via its helicase activity and thereby inhibit the translocation of mRNA back into the nucleus. The interaction kinetics of Dbp5 showed two interaction times (t1Dbp5 200Hz = 26ms & t2Dbp5 200Hz = 240ms). Due to the low number of observations, the interaction times gained by fitting the data with a bimodal gamma distribution showed a high uncertainty This makes a comparison of this results with the observed mRNA-export kinetics not advisable. In the second part of the thesis a so far unknown regulation mechanism of transcription was studied. First hints to this mechanism were observed by a control experiment during the examination of the mRNA-export kinetics. Transcription can be subdivided into the four stages of initiation, early elongation, stable elongation and termination. It was previously believed that after transition into stable elongation the transcription process is either completed or terminated prematurely. The results of this thesis give evidence that the transcription process in salivary gland cells of C. tentans can be halted temporally at the stage of stable elongation by applying a heat-shock to the larvae. The halted transcription processes can be resumed after heat-shock is released. Since RNA-polymerase II is highly conserved throughout eucaryotes, it seems very likely that this regulatory mechanism is not limited to C. tentans . The transcription halt during stable elongation described here, shows that eucaryotes have a more direct and far-ranging access to transcription as believed. This direct control of transcription significantly increases the temporal dynamic of transcriptional regulation

Characterization of black fly (Diptera: Simuliidae) silk proteins

Black fly (Simuliidae) silk is produced by the larvae and pharate pupae and is used for anchorage and cocoon production. There exists limited information on simuliid silks, including protein composition and genetic sequences encoding such proteins. The present study aimed to expand what is known about simuliid silks by examining the silks of several simuliid species and by making comparisons to the silk of non-biting midges (Chironomidae). Silk glands were dissected out of larval and pupal simuliids, and protein contents were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized with silver stain. Protein contents were compared by mass in kilodaltons (kDa) between life stages and among species. Polymerase chain reaction (PCR) was used to expand upon known gene sequence information, and to determine the presence of genes homologous to chironomid silk. SDS-PAGE of cocoons revealed the presence of a 56 kDa and a 67 kDa protein. Silk gland contained as many as 28 different proteins ranging from 319 kDa to 8 kDa. Protein profiles vary among species, and group into large (>200), intermediate(>100), and small (<100) protein classes as is found in chironomids. It is likely that silk evolved in a common ancestor of simuliids and chironomid

Hrp59, an hnRNP M protein in Chironomus and Drosophila, binds to exonic splicing enhancers and is required for expression of a subset of mRNAs

Here, we study an insect hnRNP M protein, referred to as Hrp59. Hrp59 is relatively abundant, has a modular domain organization containing three RNA-binding domains, is dynamically recruited to transcribed genes, and binds to premRNA cotranscriptionally. Using the Balbiani ring system of Chironomus, we show that Hrp59 accompanies the mRNA from the gene to the nuclear envelope, and is released from the mRNA at the nuclear pore. The association of Hrp59 with transcribed genes is not proportional to the amount of synthesized RNA, and in vivo Hrp59 binds preferentially to a subset of mRNAs, including its own mRNA. By coimmunoprecipitation of Hrp59–RNA complexes and microarray hybridization against Drosophila whole-genome arrays, we identify the preferred mRNA targets of Hrp59 in vivo and show that Hrp59 is required for the expression of these target mRNAs. We also show that Hrp59 binds preferentially to exonic splicing enhancers and our results provide new insights into the role of hnRNP M in splicing regulation

Unexpected homology between inducible cell wall protein QID74 of filamentous fungi and BR3 salivary protein of the insect Chironomus

A gene, qid74, of mycoparasitic filamentous fungus Trichoderma harzianum and its allies encodes a cell wall protein that is induced by replacing glucose in the culture medium with chitin (simulated mycoparasitism conditions). Because no trace of this gene can be detected in related species such as Gibberella fujikuroi and Saccharomyces cerevisiae, the qid74 gene appears to have arisen de novo within the genus Trichoderma. Qid74 protein, 687 residues long, is now seen as highly conserved tandem repeats of the 59- residue-long unit. This unit itself, however, may have arisen as tandem repeats of the shorter 13-residue-long basic unit. Within the genus Trichoderma, the amino acid sequence of Qid74 proteins has been conserved in toto. The most striking is the fact that Qid74 shares 25.3% sequence identity with the carboxyl-terminal half of the 1,572-residue-long BR3 protein of the dipteran insect Chironomus tentans. BR3 protein is secreted by the salivary gland of each aquatic larva of Chironomus to form a tube to house itself. Furthermore, the consensus sequence derived from these 59-residue-long repeating units resembles those of epidermal growth factor-like domains found in divergent invertebrate and vertebrate proteins as to the positions of critical cysteine residues and homology of residues surrounding these cysteines.Comisión Interministerial de la Ciencia y la Tecnología BIO 94-0289European Commission TS3-CT92-014

Forty years of the 93D puff of Drosophila melanogaster

The 93D puff of Drosophila melanogaster became attractive in 1970 because of its singular inducibility by benzamide and has since then remained a major point of focus in my laboratory. Studies on this locus in my and several other laboratories during the past four decades have revealed that (i) this locus is developmentally active, (ii) it is a member of the heat shock gene family but selectively inducible by amides, (iii) the 93D or heat shock RNA omega (hsrω) gene produces multiple nuclear and cytoplasmic large non-coding RNAs (hsrω-n, hsrω-pre-c and hsrω-c), (iv) a variety of RNA-processing proteins, especially the hnRNPs, associate with its >10 kb nuclear (hsrω-n) transcript to form the nucleoplasmic omega speckles, (v) its genomic architecture and hnRNP-binding properties with the nuclear transcript are conserved in different species although the primary base sequence has diverged rapidly, (vi) heat shock causes the omega speckles to disappear and all the omega speckle associated proteins and the hsrω-n transcript to accumulate at the 93D locus, (vii) the hsrω-n transcript directly or indirectly affects the localization/stability/activity of a variety of proteins including hnRNPs, Sxl, Hsp83, CBP, DIAP1, JNK-signalling members, proteasome constituents, lamin C, ISWI, HP1 and poly(ADP)-ribose polymerase and (viii) a balanced level of its transcripts is essential for the orderly relocation of various proteins, including hnRNPs, RNA pol II and HP1, to developmentally active chromosome regions during recovery from heat stress. In view of such multitudes of interactions, it appears that large non-coding RNAs like those produced by the hsrω gene may function as hubs to coordinate multiple cellular networks and thus play important roles in maintenance of cellular homeostasis

Mechanisms of mRNA export

AbstractRelease of properly processed and assembled mRNPs from the actively transcribing genes, movement of the mRNPs through the interchromatin and interaction with the Nuclear Pore Complexes, leading to cytoplasmic export, are essential steps of eukaryotic gene expression. Here, we review these intranuclear gene expression steps