Small nuclear ribonucleoproteins of Drosophila: identification of U1 RNA-associated proteins and their behavior during heat shock.

In Drosophila, two nuclear proteins of approximately 26,000 and 14,000 molecular weight are recognized by a human autoimmune antibody for mammalian ribonucleoprotein (RNP) particles that contain U1 small nuclear RNA. The antibody-selected Drosophila RNP contains, in addition to these two proteins, a single RNA species that has been identified as U1 by hybridization with a cloned Drosophila U1 DNA probe. Small nuclear RNP isolated from human cells under the same conditions as used for Drosophila and selected by the anti-U1 RNP-specific antibody contains eight proteins, two of which are similar in molecular weight to the two Drosophila U1 RNP proteins. Thus, even though the nucleotide sequences of Drosophila and human U1 RNA are about 72% homologous, and the corresponding RNPs are both recognized by the same human autoantibody, Drosophila U1 RNP appears to have a simpler protein complement than its mammalian counterpart. The two Drosophila U1 RNA-associated proteins are synthesized at normal or slightly increased rates during the heat shock response and are incorporated into antibody-recognizable RNP complexes. This raises the possibility that U1 RNP is an indispensable nuclear element for cell survival during heat shock.     

Characterization of the prosome from Drosophila and its similarity to the cytoplasmic structures formed by the low molecular weight heat-shock proteins.

We have identified and characterized a ribonucleoprotein structure from the cytoplasm of Drosophila melanogaster tissue culture cells which is equivalent to the prosome, a recently described ribonucleoprotein particle of duck and mouse cells. During the recovery period following heat shock, the low mol. wt. heat-shock proteins form cytoplasmic ribonucleoprotein particles which co-purify with the Drosophila prosome. Both ribonucleoprotein particles share several structural properties but their protein constituents differ in their metabolism and cellular localization during the heat treatment. We also report the partial nucleotide sequences of several small RNA species associated with the Drosophila prosome. One of them has a strong sequence homology with the U6 mammalian small nuclear RNA.     

GAL4/UAS系统在果蝇Tis11基因RNA干扰中的应用

TTP在哺乳动物许多关键基因表达的转录后水平上起调控作用,Tis11是TTP蛋白在果蝇中的同源物.目前还没有现成的可用于研究Tis11功能的基因敲除或敲低的果蝇.为了获得肌动蛋白启动子或者热激蛋白启动子驱动表达Tis11 mRNA干扰序列的具有较高干扰效率的Tis11基因干扰果蝇,将肌动蛋白启动子或者热激启动子驱动表达的GAL4果蝇品系与融合有Tis11 mRNA干扰序列的UAS品系杂交,收集同时带有GAL4基因和UAS序列的子一代果蝇.提取所收集果蝇的总RNA,将其中的mRNA逆转录成cDNA,并设计检测Tis11基因的特异性引物,然后通过Real-time PCR检测Tis11 mRNA的表达情况.结果显示所收集的能表达Tis11基因干扰序列的子一代果蝇与不能表达Tis11基因干扰序列的对照果蝇相比,其体内Tis11 mRNA的表达水平下降明显.收集的果蝇其体内所表达的干扰序列对Tis11 mRNA干扰效果显著,我们成功获得了Tis11基因的RNA干扰果蝇.     

Regulated expression of a Drosophila melanogaster heat shock locus after stable integration in a Drosophila hydei cell line.

DNA-mediated cotransformation has been used to transfer a Drosophila melanogaster heat shock locus into cultured Drosophila hydei cells by use of the copia-based selectable vector pCV2gpt and of pMH10A, a cloned 87A7 heat shock locus encoding a mutant heat shock protein (hsp). Transformed lines contain between 50 and 200 copies of both plasmids, each separately organized as a head-to-tail concatemer which is stably maintained in the transformed lines. Exposure of the cotransformants to heat shock temperatures induces the regulated expression of the hsp RNA and the mutant hsp in all the lines analyzed.     

Specific protection from phenocopy induction by heat shock

Mild heat treatments applied to whole animals or cell cultures of Drosophila prior to lethal heat shocks result in increased survival and protection against phenocopy induction. The optimal condition for the preliminary mild heat treatment is that which induces the synthesis of heat-shock proteins but does not turn off the protein synthesis that is in progress. Recovery of protein synthesis but not RNA synthesis following a drastic heat shock is much enhanced by the pretreatments. The results suggest that the protection for survival and against phenocopy induction is due to storage of messenger RNA.     

Regulation of HSP70 synthesis by messenger RNA degradation.

When Drosophila cells are heat shocked, hsp70 messenger RNA (mRNA) is stable and is translated at high efficiencies. During recovery from heat shock, hsp70 synthesis is repressed and its messenger RNA (mRNA) is degraded in a highly regulated fashion. Dramatic differences in the timing of repression and degradation are observed after heat treatments of different severities. The 3' untranslated region (UTR) of the hsp70 mRNA was sufficient to transfer this regulated degradation to heterologous mRNAs. Altering the translational efficiency of the message or changing its natural translation-termination site did not alter its pattern of regulation, although in some cases it changed the absolute rate of degradation. We have previously shown that hsp70 mRNA is very unstable when it is expressed at normal growth temperatures (from a metallothionein promoter). We report here that the 3' untranslated region of the hsp70 mRNA is responsible for this instability as well. We postulate that a mechanism for degrading hsp70 mRNA pre-exists in Drosophila cells, that it is inactivated by heat shock and that it is the reactivation of this mechanism that is responsible for hsp70 repression during recovery. This degradation system may be the same as that used by other unstable mRNAs.     

RNA Polymerase B (or II) in heat induced puffs of Drosophila polytene chromosomes

Using indirect immunofluorescence visualization techniques we investigated the distribution of RNA polymerase B (or II) and histone H1 at heat shock puff loci in Drosophila melanogaster polytene chromosomes at different times during and after heat shock. After heat treatments of from 5 to 45 min, the heat shock puff displayed intense fluorescence when stained for RNA polymerase B, but relatively little fluorescence when stained for histone H1. Returning heat shocked larvae to room temperature resulted in the appearance of a distinctive pattern of RNA polymerase-associated fluorescence in the heat shock puff at 87C, presumably reflecting events associated with the inactivation and regression of this puff. Large differences observed in the apparent RNA polymerase B content of puffs of similar size suggest that the interaction of RNA polymerase B with chromosomal loci does not depend on simply the state of condensation or decondensation of the chromatin.     

GTP-induced conformational changes in translin: a comparison between human and Drosophila proteins

Human translin is a conserved protein, unique in its ability to bind both RNA and DNA. Interestingly, GTP binding has been implicated as a regulator of RNA/DNA binding function of mouse translin (TB-RBP). We cloned and overexpressed the translin orthologue from Drosophila melanogaster and compared its DNA/RNA binding properties in relation to GTP effects with that of human protein. Human translin exhibits a stable octameric state and binds ssDNA/RNA/dsDNA targets, all of which get attenuated when GTP is added. Conversely, Drosophila translin exhibits a stable dimeric state that assembles into a suboctameric (tetramer/hexamer) form and fails to bind ssDNA and RNA targets. Interestingly enough, CD spectral analyses, partial protease digestion profile revealed GTP-specific conformational changes in human translin, whereas the same were largely missing in Drosophila protein. Isothermal calorimetry delineated specific heat changes associated with GTP binding in human translin, which invoked subunit "loosening" in its octamers; the same effect was absent in Drosophila protein. We propose that GTP acts as a specific molecular "switch" that modulates the nucleic acid binding function selectively in human translin, perhaps by affecting its octameric configuration.