hnRNA Processing Research Articles

Fine structural analyses of pancreatic acinar cell nuclei from mice fed on genetically modified soybean

We carried out ultrastructural morphometrical and immunocytochemical analyses on pancreatic acinar cell nuclei from mice fed on genetically modified (GM) soybean, in order to investigate possible structural and molecular modifications of nucleoplasmic and nucleolar constituents. We found a significant lowering of nucleoplasmic and nucleolar splicing factors as well as a perichromatin granule accumulation in GM-fed mice, suggestive of reduced post-transcriptional hnRNA processing and/or nuclear export. This is in accordance to already described zymogen synthesis and processing modifications in the same animals.

The control of prostaglandin endoperoxide H-Synthase-2 expression in the human chorion laeve at term.

Prostaglandin endoperoxide H synthase-2 (PGHS-2), the key enzyme of prostaglandin biosynthesis in gestational tissues, is expressed in the chorion laeve at term. We have determined the mechanisms that control the level of PGHS-2 mRNA in the chorion membrane in order to assess the significance of chorion-derived prostaglandins in term labor. Chorion membranes were collected after elective cesarean delivery (CD, n = 21) and after spontaneous labor (SL, n = 20) at term. The PGHS-2 gene transcription rate was measured by transcriptional run-on, and PGHS-2 mRNA and heterogeneous RNA (hnRNA) abundance was determined by quantitative real-time reverse transcriptase polymerase chain reaction. PGHS-2 mRNA stability, PGHS-2 hnRNA processing rate, and the short-term dynamics of the two RNA species were characterized in 0-24-hour-long tissue incubations. The transcriptional activity of the PGHS-2 gene predicted (P <.02) the abundance of PGHS-2 mRNA and hnRNA in individual tissues. PGHS-2 gene activity and hnRNA processing rate were not different in the CD and SL groups. PGHS-2 mRNA was constitutively stable before and after labor, and its abundance spontaneously increased sixfold in tissues incubated for 24 hours. At the same time, PGHS-2 gene activity decreased by 80% within 2 hours and rebounded to 60% of its initial level by 24 hours. PGHS-2 mRNA is highly stable, and its abundance is transcriptionally controlled in the chorion laeve at term. Labor is not associated with changing PGHS-2 gene activity. Endogenous factors drive PGHS-2 gene transcription in the chorion, and the stable PGHS-2 mRNA accumulates in the tissue at term. This accumulation has little or no impact on the timing of labor.

Cytometric methods to analyze thermal effects

Publisher Summary An investigation of thermal effects on cells is of critical interest to at least three diverse fields: (1) studies of the regulation of gene expression in the responses of cells to environmental stress; (2) the application of hyperthermia as a single or combined modality in the treatment of cancer; and (3) the identification of thermal artifacts (or effects) in studies of the biological effects of physical agents such as radiofrequency radiation and ultrasound. Hyperthermia in the 41°–45°C range causes a plethora of effects on most cellular organelles and functions. These effects range from the interaction of the plasma membrane with the extracellular matrix to protein aggregation with the nuclear matrix. Functional changes are observed in cytosol and the nucleoplasm. Several cytometric methods are used to assay the effects of hyperthermia on cells. The first assay, the amount of protein that coisolates with nuclei or nuclear matrices, measures a parameter that correlates with numerous heat effects on nuclear function such as inhibition of DNA repair, DNA synthesis, transcription, and processing of hnRNA. This parameter correlates very well with cell death in many cell lines, particularly those that do not die by apoptosis. The next parameter is the expression of heat-shock proteins whose nuclear localization and delocalization correlates with the development of thermotolerance and heat resistance. Another assay measures intracellular prooxidant capacity. One of the effects that can be induced by the exposure of cells to hyperthermia is an imbalance between prooxidant and antioxidant status either by a reduced antioxidant function or an increased prooxidant function, or both.

Control of transgenesis in higher cells: the procell transposon Tn10 TetR mRNA has several major hairpins and can be unstable in eucells.

The TetR regulatory gene from the transposon Tn10 has some excellent characteristics for transgenic control in higher cells. However, we experienced severe problems with mRNA instability for this gene in eucells (CHO cells). This may be connected with the existence within the Tn10 TetR mRNA of several sizeable hairpins. They resemble canonical RNase E sites for mRNA destabilisation in procells and possibly also in eucells. Two of the hairpins also included sequences resembling eucell hnRNA polyadenylation or processing signals. The TetR counterpart from the plasmid RA1 appears to have less of the hairpin secondary structure; perhaps because of this, it did not present these mRNA instability problems in CHO cells, and it may prove a valuable alternative for transgene control in gene therapy and biotechnology.

Nuclear matrix as a target for hyperthermic killing of cancer cells.

The nuclear matrix organizes nuclear DNA into operational domains in which DNA is undergoing replication, transcription or is inactive. The proteins of the nuclear matrix are among the most thermal labile proteins in the cell, undergoing denaturation at temperatures as low as 43-45 degrees C, i.e. relevant temperatures for the clinical treatment of cancer. Heat shock-induced protein denaturation results in the aggregation of proteins to the nuclear matrix. Protein aggregation with the nuclear matrix is associated with the disruption of many nuclear matrix-dependent functions (e.g. DNA replication, DNA transcription, hnRNA processing, DNA repair, etc.) and cell death. Heat shock proteins are believed to bind denatured proteins and either prevents aggregation or render aggregates more readily dissociable. While many studies suggest a role for Hsp70 in heat resistance, we have recently found that nuclear localization/delocalization of Hsp70 and its rate of synthesis, but not its amount, correlate with a tumor cell's ability to proliferate at 41.1 degrees C. These results imply that not only is the nuclear matrix a target for the lethal effects of heat, but it also is a target for the protective, chaperoning and/or enhanced recovery effects of heat shock proteins.

Major internal nuclear matrix proteins are common to different human cell types.

The nuclear matrix may be involved in the structural and functional organization of the cell nucleus. However, we still do not understand the molecular basis of the intranuclear fibrogranular network that is part of the nuclear matrix. We recently described a method to identify internal nuclear matrix proteins [Mattern et al. (1996): J Cell Biochem 62:275-289], which was done by comparing two nuclear matrix preparations: one with and one without the internal structure by using quantitative two-dimensional gel electrophoresis. In the present study, we use the same approach to compare the nuclear matrix proteins of four different human cell types to investigate whether they have a similar internal nuclear matrix protein composition. Major nuclear matrix proteins present in all these cell types likely represent the base of the internal nuclear matrix. We demonstrate that the 25 most abundant internal nuclear matrix proteins are common to all four cell types. Together, these common proteins represent more than 75% of the total internal nuclear matrix protein mass in each cell type. This set of proteins includes B23 and most hnRNP proteins. The quantity of most of these proteins is very similar in the four cell types. The fact that the internal nuclear matrix consists mainly of hnRNP proteins, which may be involved in transcription, transport, and processing of hnRNA, supports the idea that the internal nuclear matrix is the result of these processes.

The nuclear matrix: a target for heat shock effects and a determinant for stress response.

The nuclear matrix organizes nuclear DNA into operational DNA domains for replication, transcription, and repair. The proteins of the nuclear matrix are among the most thermal labile proteins in the cell, undergoing denaturation at 43 degrees C to 45 degrees C. Heat-shock-induced protein denaturation results in the aggregation of proteins to the nuclear matrix. As many as 100 protein changes have been observed as a result of this aggregation. Protein aggregation with the nuclear matrix is associated with the disruption of nuclear matrix-dependent DNA replication, DNA transcription, hnRNA processing, and DNA repair. Disruptions of these processes lead to cell death. Nuclear matrix protein changes affect these processes by inhibiting DNA supercoiling ability and inhibiting the access to matrix-associated DNA. Heat-shock proteins are believed to bind denatured proteins and either prevent aggregation or render aggregates more readily dissociable. The nuclear matrix appears to be a target for the detrimental effects of heat shock and hsp70 serves to protect against such effects. However, the nuclear matrix may be involved in the pre- and post-heat shock expression of hsp70. We have found a heat-inducible MAR covering the promoter region of murine hsp70.3, implying that changes in matrix association are needed for hsp70 expression. However, the hsp70.1, 70.3, and hsc70t gene family is organized as an active gene with respect to the nuclear matrix. Thus, it may be that heat-inducible genes have a unique matrix-dependent organization. The work presented in this review implies that the nuclear matrix is a target for the lethal effects of heat and is also a determinant in the protective expression of heat-shock genes.

Posttranscriptional regulation of gene expression in liver regeneration: role of mRNA stability

The rentry of hepatocytes and nonparenchymal cells from the normal quiescent G0 phase into the cell cycle during liver regeneration after 70% partial hepatectomy results in the discrete modulation of mRNA transcripts for many different genes. The modulation of steady-state levels of transcripts for genes involved in hepatocyte growth and replication during liver regeneration indicates that gene expression is regulated not only transcriptionally but also posttranscriptionally. In fact, posttranscriptional control appears to be the primary mechanism of regulating gene expression after the first 3 h after partial hepatectomy. Alteration in transcript stability is a key posttranscriptional regulatory mechanism used by the regenerating liver to modulate the steady-state transcript levels of multiple genes. Even genes that are transcriptionally activated during liver regeneration exhibit posttranscriptional control at the level of transcript stability. Moreover, the abundance of mRNA binding proteins, as well as translational activity and rate of poly(A) tail removal, are modulated and appear to influence transcript stability during liver regeneration. However, alteration of transcript stability is not the sole posttranscriptional mechanism regulating steady-state levels. Posttranscriptional control also occurs at the level of alternative splicing, stabilization of heterogeneous nuclear (hn) RNA, and hnRNA processing. Moreover, the role of nucleocytoplasmic transport of mature mRNA during liver regeneration is still undefined. Thus, during liver regeneration gene expression is regulated at multiple levels after the initial synthesis of hnRNA. By understanding the role of posttranscriptional mechanisms in regulating steady-state transcript levels in an in vivo model of normal growth, we will begin to appreciate its role in the genesis of abnormal growth.

Both RNA-Binding Domains in Heterogeneous Nuclear Ribonucleoprotein A1 Contribute Toward Single-Stranded-RNA Binding

Heterogenous nuclear ribonucleoproteins (hnRNPs) such as hnRNP A1 are tightly associated with heterogenous nuclear RNAs (hnRNAs) within eukaryotic nuclei and are thought to be involved in hnRNA processing and splice site selection. The NH2-terminal two-thirds of hnRNP A1 contains two 92-amino acid RNA binding domains (RBDs) that are arranged in tandem and are more than 30% homologous with each other. Following this region is a flexible glycine-rich COOH-terminal domain. We have studied the nucleic acid binding properties of the two isolated RBDs (residues 1-92 and 93-184, respectively) and of A1 fragments corresponding to residues 1-184 and 1-196 (i.e., the latter fragment is called UP1) in order to evaluate their relative contributions to A1 binding. We have determined that the individual RBDs of A1 bind poly[r(epsilon A)], a fluorescent single-stranded RNA (ssRNA), with a surprisingly low apparent association constant of only 1.5 x 10(4) M-1 (1-92) and 4.5 x 10(4) M-1 (93-184), respectively. We hypothesize that this low affinity represents a basal level of binding that is common to most RBD-containing proteins. Oligonucleotide binding studies suggest the interaction site size for the 93-184 fragment is approximately 4 nucleotides or less and salt sensitivity studies indicate that only about 27% of the free energy of binding of this RBD derives from ionic interactions. Since the affinity of the 1-184 fragment is at least 10-fold above that of either of its component RBDs, both must contribute to binding. This conclusion is further supported by the increased occluded site size of 1-184 (n = 14 +/- 2), as compared to its 93-184 RBD (n = 6 +/- 1), and by the biphasic binding that was observed for the UP1:poly(U) interaction at pH 6.0. Our finding that the affinity of the 1-184 fragment is 1000-fold less than the product of the affinities of its 1-92 and 93-184 RBDs is consistent with these domains being joined by a flexible linker. By comparing the affinities of the 1-184 fragment with that for A1, we conclude that together the two RBDs in A1 account for only 53% of the free energy of A1 binding. Comparative binding studies with UP1 demonstrate that the short region spanning residues 185-->195 represents an important determinant of the binding affinity of A1 and, since this region contains a site of dimethylation, it may provide a mechanism for regulating the affinity of A1 for specific nucleic acid targets.

Processing of gonadotropin-releasing hormone gene transcripts in the rat brain.

The precursor of gonadotropin-releasing hormone (GnRH) and the 56-amino acid GnRH-associated peptide is encoded in an mRNA of about 560 bases in length. This mRNA derives from an approximately 4300-base pair-long gene consisting of four relatively short exons (denoted 1, 2, 3, and 4) and three large introns (A, B, and C). In this study, we characterized the order by which the three introns are spliced from the primary transcript and processing intermediates to give rise to a mature mRNA and evaluated the potential role of gene transcription and pre-mRNA processing in the control of proGnRH mRNA levels in vivo. Nuclear and cytoplasmic RNA fractions isolated from rat preoptic area-anterior hypothalamus (POA-AH) and basal olfactory area (located rostral to the POA) were analyzed by 1) solution hybridization-RNase protection mapping using several RNA probes directed at various regions of the proGnRH gene and 2) reverse transcription-polymerase chain reaction using several oligonucleotide primers. Both types of analysis showed that proGnRH pre-mRNA processing begins with the splicing of intron B from the primary gene transcript. Hence, intron B is the ideal target for studying proGnRH primary transcript by in situ hybridization. Subsequent splicing of introns A and C appeared to take place in two alternative, although not equally prevalent pathways. Quantitative analysis indicated that the proGnRH hnRNA species constituted, on a mole basis, about 20% of the total gene transcripts in the POA-AH. The primary transcript alone constituted about 10% of the total gene transcripts in the POA-AH and as much as 20% in the basal olfactory area. The prospect of blockade of proGnRH hnRNA processing by means of hybridization with endogenous antisense RNAs (transcribed from the SH gene on the opposite strand of the same DNA locus) did not prove to be likely, as the SH transcripts were present at very low levels compared to any of the proGnRH RNA species. We conclude that the relatively large pool of proGnRH hnRNA may reflect a high rate of gene transcription and/or slow RNA processing.

Autoantibodies as Probes in Cell and Molecular Biology

ConclusionsHuman sera containing autoantibodies that specifically recognize important cellular antigens are a key tool being used for studying cellular processes (summarized in Table I). Human autoantisera have been used as essential research probes that allowed for the identification of many important cellular proteins and for the elucidation of several biochemical processes. As has been shown in this Minireview, in certain instances, such as the processing of hnRNA to mRNA, human autoantisera have allowed for cellular events to be investigated in incredible detail and an exhaustive literature exists that summarizes these experimental studies. Human autoantibodies have been used for the identification and purification of the snRNP particles and for investigations into the assembly and workings of spliceosomes. However, studies of mRNA processing are more the exception than the rule. In most instances, human autoantibodies are only beginning to be realized as powerful experimental tools. Human autoantiser...

Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to influence the structure of hnRNA and participate in the processing of hnRNA to mRNA. The hnRNP U protein is an abundant nucleoplasmic phosphoprotein that is the largest of the major hnRNP proteins (120 kDa by SDS-PAGE). HnRNP U binds pre-mRNA in vivo and binds both RNA and ssDNA in vitro. Here we describe the cloning and sequencing of a cDNA encoding the hnRNP U protein, the determination of its amino acid sequence and the delineation of a region in this protein that confers RNA binding. The predicted amino acid sequence of hnRNP U contains 806 amino acids (88,939 Daltons), and shows no extensive homology to any known proteins. The N-terminus is rich in acidic residues and the C-terminus is glycine-rich. In addition, a glutamine-rich stretch, a putative NTP binding site and a putative nuclear localization signal are present. It could not be defined from the sequence what segment of the protein confers its RNA binding activity. We identified an RNA binding activity within the C-terminal glycine-rich 112 amino acids. This region, designated U protein glycine-rich RNA binding region (U-gly), can by itself bind RNA. Furthermore, fusion of U-gly to a heterologous bacterial protein (maltose binding protein) converts this fusion protein into an RNA binding protein. A 26 amino acid peptide within U-gly is necessary for the RNA binding activity of the U protein. Interestingly, this peptide contains a cluster of RGG repeats with characteristic spacing and this motif is found also in several other RNA binding proteins. We have termed this region the RGG box and propose that it is an RNA binding motif and a predictor of RNA binding activity.

Thalassemia: genotypes and phenotypes

The large degree of phenotypic heterogeneity of thalassemia can now be related to the underlying genomic defects. This information has accumulated rapidly over the last years through the recent advances in molecular technology. The list of main types of thalassemia (α or β) that can be differentiated includes several gene deletions (complete or partial) and point mutations (or very short deletions). These occur within the genes or across the flanking DNA sequences and apparently interfere with the expression of these genes. From a quantitative point of view, the severity of the condition is directly related to the amount of functional globin chain mRNA which is made available to the ribosomes; this may vary from zero (gene deletions, frameshift, non-sense mutations or mutations at the splice-junction nucleotides) to very little (mostly hnRNA processing mutants) or to slightly subnormal (transcriptional mutants, mutations resulting in cryptic site activation or in defective cleavage of the poly-A tail). A few hyper-unstable globin chains also produce a thalassemic phenotype. This pattern is straightforward in the α-thalassemias. In the β-thalassemias, the decreased β-chain synthesis reflects the available mRNA, but the phenotypic expression depends also on the ability of the patient to reactivate γ-chain synthesis and complement the red cell content with hemoglobin F.

Thalassemia: genotypes and phenotypes

The large degree of phenotypic heterogeneity of thalassemia can now be related to the underlying genomic defects. This information has accumulated rapidly over the last years through the recent advances in molecular technology. The list of main types of thalassemia (alpha or beta) that can be differentiated includes several gene deletions (complete or partial) and point mutations (or very short deletions). These occur within the genes or across the flanking DNA sequences and apparently interfere with the expression of these genes. From a quantitative point of view, the severity of the condition is directly related to the amount of functional globin chain mRNA which is made available to the ribosomes; this may vary from zero (gene deletions, frameshift, non-sense mutations or mutations at the splice-junction nucleotides) to very little (mostly hnRNA processing mutants) or to slightly subnormal (transcriptional mutants, mutations resulting in cryptic site activation or in defective cleavage of the poly-A tail). A few hyper-unstable globin chains also produce a thalassemic phenotype. This pattern is straightforward in the alpha-thalassemias. In the beta-thalassemias, the decreased beta-chain synthesis reflects the available mRNA, but the phenotypic expression depends also on the ability of the patient to reactivate gamma-chain synthesis and complement the red cell content with hemoglobin F.

Molecular organization of RNP complexes containing P11 antigen in heat‐shocked and non‐heat‐shocked Drosophila cells

Immunofluorescence analysis of polytene chromosomes of Drosophila melanogaster using the monoclonal antibody P11 has shown that after heat-shock the 38-kDa P11 antigen almost exclusively localizes at heat-shock puff 93D where it is part of giant puff-specific RNP granules. The biochemical experiments reported here show that, independent of growth temperature, the P11 antigen is a component of nuclear 10S RNP particles. The P11-containing 10S snRNPs can be stabilized in CsCl with 20 mM Mg2+ and possess a buoyant density of rho = 1.4 g/cm3. Sucrose gradient analysis of nuclear RNP extracts of heat-shocked Schneider's S-3 tissue culture cells shows that, after a 37 degree C heat-shock, the 10S RNPs associate with large RNP complexes sedimenting at 170-220S. The change in distribution is a temperature-dependent process with intermediate forms at 29 degrees C and 33 degrees C. In thermotolerant cells this observed change in distribution is strongly reduced. DEAE-Sephacel column chromatography and sucrose gradient analysis of nuclear RNP, followed by Northern blot analysis using 93D-specific probes of the TaqI repeat and immunoblotting experiments, show that the P11-containing 10S snRNPs are distinct from the RNP complexes formed by the 93D transcripts, suggesting an indirect association after heat-shock. Our experiments demonstrate that, despite the fact that a 37 degrees C heat-shock does not affect the overall integrity of nuclear RNP, it imposes changes on the general organization and interaction of the nuclear RNP population, resulting in the formation of large nuclear RNP aggregates and complexes. Such changes may be important for the survival strategy of the cell and for hnRNA processing and storage events which are effected by heat-shock.

Mammalian single-stranded DNA binding protein UP I is derived from the hnRNP core protein A1.

Antibodies induced against mammalian single-stranded DNA binding protein (ssDBP) UP I were shown to be cross-reactive with most of the basic hnRNP core proteins, the main constituents of 40S hnRNP particles. This suggested a structural relationship between both groups of proteins. Using the anti-ssDBP antibodies, a cDNA clone (pRP10) was isolated from a human liver cDNA library in plasmid expression vector pEX1. By DNA sequencing this clone was shown to encode in its 949 bp insert the last 72 carboxy terminal amino acids of the ssDBP UP I. Thereafter, an open reading frame continued for another 124 amino acids followed by a UAA (ochre) stop codon. Direct amino acid sequencing of a V8 protease peptide from hnRNP core protein A1 showed that this peptide contained at its amino terminus the last 11 amino acids of UP I followed by 19 amino acids which are encoded by the open reading frame of cDNA clone pRP10 immediately following the UP I sequence. This proves that ssDBP UP I arises by proteolysis from hnRNP core protein A1. This finding must lead to a re-evaluation of the possible physiological role of UP I and related ssDBPs. The formerly assumed function in DNA replication, although not completely ruled out, should be reconsidered in the light of a possible alternative or complementary function in hnRNA processing where UP I could either be a simple degradation product of core protein A1 (as a consequence of controlling the levels of active A1) or may continue to function as an RNA binding protein which has lost the ability to interact with the other core proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

SV40 large T-antigen and transformation related protein p53 are associated in situ with nuclear RNP structures containing hnRNA of transformed cells

The localization of SV40 large T-antigen (T-Ag) and the cellular protein p53 in the nuclei of mouse and human SV40-transformed cells and of a methylcholanthrene-transformed mouse cell line, was studied. Their detection by ultrastructural immunocytochemistry with specific monoclonal antibodies employed two complementary methods used in parallel. These consisted of indirect immunoperoxidase labelling carried out before embedment on Triton-permeabilized cells, or indirect immunogold labelling applied to thin sections of cells embedded in Lowicryl K4M. The results indicate that in SV40-transformed cells both proteins are chiefly localized on peri- and interchromatin RNP fibrils. This shows that they occur in structures involved in the synthesis and processing of hnRNA. The nucleoli and chromatin did not appear to be labelled. In methylcholanthrene-transformed cells the protein p53 (in the absence of large T-Ag) was also detected on peri- and interchomatin fibrils. Taken together with recent results which demonstrated that, during lytic infection, T-Ag was associated chiefly with cellular chromatin (Harper F, Florentin, Y & Puvion E, Exp cell res 161 (1985) 434) [33], our experiments provide evidence that the transforming function of SV40 large T-Ag is dissociable from its function in SV40 lytic infection in terms of its subnuclear distribution.

5′-terminal caps of snRNAs are reactive with antibodies specific for 2,2,7-trimethylguanosine in whole cells and nuclear matrices: Double-label immunofluorescent studies with anti-m 3G antibodies and with anti-RNP and anti-Sm autoantibodies

Antibodies specific for 2,2,7-trimethylguanosine (m 3G), which do not cross-react with m 7G-capped RNA molecules were used to study, by immunofluorescence microscopy, the reactivity of the m 3G-containing cap structures of the snRNAs U1 to U5 in situ. In interphase cells, immunofluorescent sites were restricted to the nucleus, whilst nucleoli were free of fluorescence. This indicates that the 5′ termini of most of the nucleoplasmic snRNAs are not protected by an m 3G cap-recognizing protein and that the snRNA caps are not necessarily required for the binding of snRNPs to subnuclear structures. The snRNAs in the nucleoplasm appeared as distinct units in the light microscope, and this allowed the comparison of the distribution of snRNP proteins by double label studies with anti-RNP or anti-Sm antibodies within the same cell. The three antibody classes produced superimposable fluorescent patterns. Taking into account that the various IgGs react with antigenic sites on snRNAs or snRNP proteins not shared by all the snRNP species, these data suggest that U1 snRNP particles are distributed in the same way as the other snRNPs in the nucleus. Qualitatively the same results were obtained with DNase-treated nuclear matrices indicating that intact snRNPs are part of the nuclear matrix. Our data are consistent with proposals that the various snRNPs may be involved in processing of hnRNA and that this may take place at the nuclear matrix.

Immunocytochemical identification of nuclear structures containing snRNPs in isolated rat liver cells

Small nuclear ribonucleoproteins (snRNPs) containing U1, U2, U4, U5, and U6 small nuclear RNAs were detected by ultrastructural immunocytochemistry in the nuclei of isolated rat hepatocytes using Fab fragments of anti-Sm and anti-RNP autoantibodies. Their localization was carried out in normal cells and in cells treated with two drugs, the adenosine analog DRB and CdCl2, which alter the number and distribution of nuclear RNP components. It was found that more precise determination of the distribution of these small RNAs could be obtained by using two complementary procedures in parallel rather than either one alone. They consisted of an indirect immunoperoxidase labeling carried out before embedment and an indirect immunogold labeling applied to thin sections of cells embedded in Lowicryl K4M. The results indicate that snRNPs are associated with all extranucleolar perichromatin fibrils and granules and interchromatin fibrils, which confirms that they occur in structures involved in the synthesis and processing of hnRNA. The snRNPs are not associated with nucleolar perichromatin granules induced by DRB, which confirms that there may be two kinds of perichromatin granules. The snRNPs are also associated with the still enigmatic interchromatin granules which apparently do not contain hnRNA but at least in DRB-treated cells, also contain ribosomal RNA.

Nonpackaging and packaging proteins of hnRNA in Drosophila melanogaster

Monoclonal antibodies have previously been raised against chromosomal proteins of Drosophila. Using a biochemical fractionation method for the isolation of large hnRNA-containing structures (hnRNP) of Drosophila tissue culture cells, we show that seven of these antibodies recognize different antigens, and that these antigens are associated with RNA. Analysis of the sedimentation behavior of antigen-containing structures in sucrose gradients reveals that the antigens are differentially distributed with respect both to one another and to pulse-labeled RNA. We demonstrate that the antigens are minor components of hnRNP and are different from the major Drosophila hnRNP packaging proteins, which we have also identified. The antigens are probably involved in the processing of hnRNA in the nucleus.