Demonstration of a 7-nm RNP fiber as the basic structural element in a premessenger RNP particle

Balbiani ring granules in Chironomus salivary glands represent premessenger ribonucleoprotein (RNP) particles, each containing a 35- to 40-kb message for a secretory polypeptide. Their gross structure can be described as an RNP ribbon bent into a toroid. We now demonstrate that an unfolded, thin RNP fiber is observed after low salt treatment of isolated Balbiani ring granules. Moreover, the thin RNP fiber, 7 nm in diameter, can be revealed as the main structural element in Balbiani ring granules studied in situ in 3-D with electron microscope tomography. It is proposed that the thin RNP fiber consists of a premessenger RNA molecule coiled around a filamentous core of polymeric proteins, which has functional implications for processes such as assembly of RNP, intranuclear degradation of RNA, and delivery of RNA through the nuclear pores.     

Higher order structure of Balbiani ring premessenger RNP particles depends on certain RNase A sensitive sites

Specific premessenger ribonucleoprotein (RNP) particles, the Balbiani ring (BR) granules from Chironomus tentans salivary glands, were treated with RNase A to study the effect of RNA strand breaks on the higher order structure of the particles. Isolated, radioactively labeled BR granules, known to sediment at 300 S, were digested with RNase A and centrifuged in sucrose gradients. The fractionated particles were subsequently analyzed using electron microscopy and caesium chloride centrifugation. At a low RNase concentration, most of the 300 S particles disintegrated completely, and no metastable degradation products were observed. At intermediate RNase concentrations, no 300 S particles were left, but a minor fraction of the BR granules had unfolded and sedimented at 160 S. These granules could represent particles modified during the RNase treatment or represent a more slowly degrading subfraction of the particles. At a high RNase concentration, no RNP particles at all remained in the gradient. The rapid disintegration of the majority of the BR granules was investigated further by electrophoretic analysis of RNA in the remaining particles. During the RNase treatment BR granules, still sedimenting at 300 S, accumulated strand breaks; in fact, as many as 50 to 100 nicks in the 37 kb RNA could be tolerated. It was concluded from RNA analyses that the disintegration of the BR granules was not dependent on any single nick in the RNA, nor on the accumulation of a certain number of nicks, but rather on one or a few critical strand breaks. We propose that there are organizing sequences essential for particle integrity; once these sequences are nicked, the premessenger RNP particles are rapidly and completely degraded.     

RNP, Sm and SS-B antigens from calf thymus: molecular stability upon enzymatic digestion

RNP, Sm and SS-B nuclear antigens from calf thymus were studied with respect to the size distribution on sucrose gradients as well as to the molecular integrity and related structural changes when they were subjected to enzymatic digestions under different conditions. Making a difference with RNP particles, the Sm size distribution is concentration dependent, a property in accordance with the complexity of the Sm particles in comparison with the RNPs. The use of combined effects of temperature, endogenous proteases and RNase A, allowed us to gain insight into the limits of stability of the three antigenic particles. Following treatments in the absence of RNAse A, the degradation products (32-38 Kd molecular weight) of the 70 Kd RNP polypeptide remain stable and associated with other molecules within the RNP particle. It was also found that the phosphate groups of the SS-B protein moiety are only accessible to alkaline phosphatase if the RNA of the SS-B particle is degraded by the action of RNAse A.     

Osmium tetroxide/p-phenylenediamine staining of nucleoli and Balbiani Rings in Chironomus salivary glands.

This paper deals with the application of the osmium tetroxide fixation followed by p-phenylenediamine treatment to salivary gland cells from Chironomus larvae. After this procedure, cytoplasm, nucleoli and Balbiani rings show a high degree of staining both in light and electron microscopy, while chromatin remains unstained. Ethanol fixation followed by osmium tetroxide/p-phenylenediamine does not modify the above mentioned staining pattern. Under these conditions, extractive procedures for lipids do not affect the osmiophilia of nucleoli and Balbiani rings, while RNase or trichloroacetic acid treatment decreaes the staining degree of these structures. In osmium tetroxide/p-phenylenediamine treated salivary glands, the highest contrast within nuclei is seen to occur in the pars granulosa from normal or segregated nucleoli, as well as in Balbiani ring granules, which appear either as hollow granules or with a bipartite or horseshoe-like structure.     

Osmium tetroxide/p-phenylenediamine staining of nucleoli and balbiani rings in Chironomus salivary glands

Summary This paper deals with the application of the osmium tetroxide fixation followed by p-phenylenediamine treatment to salivary gland cells from Chironomus larvae. After this procedure, cytoplasm, nucleoli and Balbiani rings show a high degree of staining both in light and electron microscopy, while chromatin remains unstained. Ethanol fixation followed by osmium tetroxide/p-phenylenediamine does not modify the above mentioned staining pattern. Under these conditions, extractive procedures for lipids do not affect the osmiophilia of nucleoli and Balbiani rings, while RNase or trichloroacetic acid treatment decreaes the staining degree of these structures. In osmium tetroxide/p-phenylenediamine treated salivary glands, the highest contrast within nuclei is seen to occur in the pars granulosa from normal or segregated nucleoli, as well as in Balbiani ring granules, which appear either as hollow granules or with a bipartite or horseshoe-like structure.     

Structural analyses of the 7SK ribonucleoprotein (RNP), the most abundant human small RNP of unknown function.

The human 7SK ribonucleoprotein (RNP) has been analyzed to determine its RNA secondary structure and protein constituents. HeLa cell 7SK RNA alone and within its RNP have been probed by chemical modification and enzymatic cleavage, and sites of modification or cleavage have been mapped by primer extension. The resulting secondary structure suggests that structural determinants necessary for capping (a 5' stem followed by the sequence AUPuUPuC) and nuclear migration (the sequence AUPuUPuC) of 7SK RNA may be similar to those for U6 small nuclear RNA (snRNA). It also supports existence of a 3' stem structure which could serve to self-prime cDNA synthesis during pseudogene formation. Oligonucleotide-directed RNase H digestion indicated regions of 7SK RNA capable of base pairing with other nucleic acids. Antisense 2'-O-methyl RNA oligonucleotides were used to affinity select the 7SK RNP from an in vivo 35S-labeled cell sonic extract and identify eight associated proteins of 83, 48, 45, 43, 42, 21, 18, and 13 kDa. 7SK RNA has extensive sequence complementarity to U4 snRNA, within the U4/U6 base pairing domain, and also to U11 snRNA. The possibility that the 7SK RNP is an unrecognized component of the pre-mRNA processing machinery is discussed.     

Studies on the Function of Intracellular Ribonucleases : II. The Interaction of Ribonucleoprotein and Enzymes

To determine the possible significance of in vivo or in vitro enzyme action in ribonucleoprotein systems, rat liver microsomes and ribonucleoprotein particles (RNP) prepared from them by deoxycholate treatment were incubated for 1 hour at 37°C. with crystalline pancreatic ribonuclease (RNase) or various RNase-free crystalline proteolytic enzymes. The extent of the degradation of the RNA of the microsomes and RNP was determined and the protein degradation estimated in both cases. With either microsomes or RNP, RNase (0.5 to 1.0 mg. per ml.) degraded from 75 to 95 per cent of the RNA, with little protein breakdown being apparent when microsomes were used but with significant protein degradation in the RNP. When microsomes were treated with proteolytic enzymes approximately 40 to 50 per cent of the original microsomal protein became nonsedimentable while at the same time 60 to 80 per cent of the RNA was also found to be non-sedimentable. Of the non-sedimentable RNA, approximately one-third was in the form of acid-precipitable RNA while the remainder was in the form of acid-soluble nucleotides. When RNP was treated with proteolytic enzymes, about 95 per cent of the RNA could no longer be sedimented. About half of this appeared as acid-precipitable RNA and half as acid-soluble nucleotides. Both microsomes and RNP contained significant RNase activity with RNP exhibiting about 10 times the specific activity of microsomes. Some of the characteristics of this RNase activity were determined and the results with proteolytic enzymes interpreted in light of this activity.     

Protein Interactions in Xenopus Germ Plasm RNP Particles

Hermes is an RNA-binding protein that we have previously reported to be found in the ribonucleoprotein (RNP) particles of Xenopus germ plasm, where it is associated with various RNAs, including that encoding the germ line determinant Nanos1. To further define the composition of these RNPs, we performed a screen for Hermes-binding partners using the yeast two-hybrid system. We have identified and validated four proteins that interact with Hermes in germ plasm: two isoforms of Xvelo1 (a homologue of zebrafish Bucky ball) and Rbm24b and Rbm42b, both RNA-binding proteins containing the RRM motif. GFP-Xvelo fusion proteins and their endogenous counterparts, identified with antisera, were found to localize with Hermes in the germ plasm particles of large oocytes and eggs. Only the larger Xvelo isoform was naturally found in the Balbiani body of previtellogenic oocytes. Bimolecular fluorescence complementation (BiFC) experiments confirmed that Hermes and the Xvelo variants interact in germ plasm, as do Rbm24b and 42b. Depletion of the shorter Xvelo variant with antisense oligonucleotides caused a decrease in the size of germ plasm aggregates and loosening of associated mitochondria from these structures. This suggests that the short Xvelo variant, or less likely its RNA, has a role in organizing and maintaining the integrity of germ plasm in Xenopus oocytes. While GFP fusion proteins for Rbm24b and 42b did not localize into germ plasm as specifically as Hermes or Xvelo, BiFC analysis indicated that both interact with Hermes in germ plasm RNPs. They are very stable in the face of RNA depletion, but additive effects of combinations of antisense oligos suggest they may have a role in germ plasm structure and may influence the ability of Hermes protein to effectively enter RNP particles.     

Isolation of informoferes from rat liver : Effects of α-amanitin and actinomycin D

RNP particles carrying rapidly labelled RNA (informoferes) were isolated from rat liver nuclei by extraction with isotonic and 0.3 M salt buffer at pH 8 either with or without ultrasonic treatment. The importance of preliminary extraction of the nuclei with the isotonic salt buffer at a lower pH of 7 or in the presence of 50 mM EDTA is demonstrated. Administration of α-amanitin or of actinomycin D, in doses inhibiting the labelling of the heterogeneous RNA with RNA precursors in the range of 60–85%, reduces the amount of informoferes recovered. The informoferes isolated from treated animals are highly depleted in HnRNA. They still contain, however, low molecular RNA species with a slower turnover than the HnRNA. The polypeptide pattern observed after acrylamide gel electrophoresis of the informofere proteins is qualitatively changed in the treated preparations, whereas the ratio of protein to RNA decreases. In the presence of RNase inhibitor the informoferes are recovered in form of polymer structures. α-Amanitin and actinomycin D significantly reduce the amount of polymers recovered.     

Exclusion of mRNPs and ribosomal particles from a thin zone beneath the nuclear envelope revealed upon inhibition of transport

We have studied the nucleocytoplasmic transport of a specific messenger RNP (mRNP) particle, named Balbiani ring (BR) granule, and ribosomal RNP (rRNP) particles in the salivary glands of the dipteran Chironomus tentans. The passage of the RNPs through the nuclear pore complex (NPC) was inhibited with the nucleoporin-binding wheat germ agglutinin, and the effects were examined by electron microscopy. BR mRNPs bound to the nuclear basket increased in number, while BR mRNPs translocating through the central channel decreased, suggesting that the initiation of translocation proper had been inhibited. The rRNPs accumulated heavily in nucleoplasm, while no or very few rRNPs were recorded within nuclear baskets. Thus, the transport of rRNPs had been blocked prior to the entry into the baskets. Remarkably, the rRNPs had been excluded both from baskets and the space in between the baskets. We propose that normally basket fibrils move freely and repel RNPs from the exclusion zone unless the particles have affinity for and bind to nucleoporins within the baskets.