Stress-induced nuclear bodies are sites of accumulation of pre-mRNA processing factors.

Heterogeneous nuclear ribonucleoprotein (hnRNP) HAP (hnRNP A1 interacting protein) is a multifunctional protein with roles in RNA metabolism, transcription, and nuclear structure. After stress treatments, HAP is recruited to a small number of nuclear bodies, usually adjacent to the nucleoli, which consist of clusters of perichromatin granules and are depots of transcripts synthesized before stress. In this article we show that HAP bodies are sites of accumulation for a subset of RNA processing factors and are related to Sam68 nuclear bodies (SNBs) detectable in unstressed cells. Indeed, HAP and Sam68 are both present in SNBs and in HAP bodies, that we rename "stress-induced SNBs." The determinants required for the redistribution of HAP lie between residue 580 and 788. Different portions of this region direct the recruitment of the green fluorescent protein to stress-induced SNBs, suggesting an interaction of HAP with different components of the bodies. With the use of the 580-725 region as bait in a two-hybrid screening, we have selected SRp30c and 9G8, two members of the SR family of splicing factors. Splicing factors are differentially affected by heat shock: SRp30c and SF2/ASF are efficiently recruited to stress-induced SNBs, whereas the distribution of SC35 is not perturbed. We propose that the differential sequestration of splicing factors could affect processing of specific transcripts. Accordingly, the formation of stress-induced SNBs is accompanied by a change in the splicing pattern of the adenovirus E1A transcripts.

Structure and dynamics of hnRNP-labelled nuclear bodies induced by stress treatments.

We have previously described HAP, a novel hnRNP protein that is identical both to SAF-B, a component of the nuclear scaffold, and to HET, a transcriptional regulator of the gene for heat shock protein 27. After heat shock, HAP is recruited to a few nuclear bodies. Here we report the characterisation of these bodies, which are distinct from other nuclear components such as coiled bodies and speckles. The formation of HAP bodies is part of a general cell response to stress agents, such as heat shock and cadmium sulfate, which also affect the distribution of hnRNP protein M. Electron microscopy demonstrates that in untreated cells, similar to other hnRNP proteins, HAP is associated to perichromatin fibrils. Instead, in heat shocked cells the protein is preferentially associated to clusters of perichromatin granules, which correspond to the HAP bodies observed in confocal microscopy. Inside such clusters, perichromatin granules eventually merge into a highly packaged 'core'. HAP and hnRNP M mark different districts of these structures. HAP is associated to perichromatin granules surrounding the core, while hnRNP M is mostly detected within the core. BrU incorporation experiments demonstrate that no transcription occurs within the stress-induced clusters of perichromatin granules, which are depots for RNAs synthesised both before and after heat shock.

Emerin presence in platelets.

Emerin is an almost ubiquitous protein which is abnormal in X-linked Emery-Dreifuss muscular dystrophy (EMD), a syndrome characterized by muscle weakness, joint contractures and cardiac arrhythmia. Emerin is localized in the cells at the nuclear rim and its function is still unknown. In some models, emerin has also been described in the cytoplasm; however, its presence outside the nucleus is still matter of debate. We report the presence of emerin in circulating normal human platelets and its absence in platelets from X-linked EMD patients. Since platelets are cytoplasmic fragments derived from megakaryocytes, the presence of emerin in platelets confirms cytoplasmic localization of this protein, probably related to specific functions. We found also that emerin is present in the cytoplasm of megakaryocytes, while it is absent in circulating granulocytes.

A novel hnRNP protein (HAP/SAF-B) enters a subset of hnRNP complexes and relocates in nuclear granules in response to heat shock.

A two-hybrid screening in yeast for proteins interacting with the human hnRNP A1, yielded a nuclear protein of 917 amino acids that we termed hnRNP A1 associated protein (HAP). HAP contains an RNA binding domain (RBD) flanked by a negatively charged domain and by an S/K-R/E-rich region. In in vitro pull-down assays, HAP interacts with hnRNP A1, through its S/K-R/E-rich region, and with several other hnRNPs. HAP was found to be identical to the previously described Scaffold Attachment Factor B (SAF-B) and to HET, a transcriptional regulator of the Heat Shock Protein 27 gene. We show that HAP is a bona fide hnRNP protein, since anti-HAP antibodies immunoprecipitate from HeLa cell nucleoplasm the complete set of hnRNP proteins. Unlike most hnRNP proteins, the subnuclear distribution of HAP is profoundly modified in heat-shocked HeLa cells. Heat-shock treatment at 42 degrees C causes a transcription-dependent recruitment of HAP to a few large nuclear granules that exactly coincide with sites of accumulation of Heat Shock Factor 1 (HSF1). The recruitment of HAP to the granules is temporally delayed with respect to HSF1 and persists for a longer time during recovery at 37 degrees C. The hnRNP complexes immunoprecipitated from nucleoplasm of heat-shocked cells with anti-HAP antibodies have an altered protein composition with respect to canonical complexes. Altogether our results suggest an involvement of HAP in the cellular response to heat shock, possibly at the RNA metabolism level.

Immunocytochemical detection of emerin within the nuclear matrix.

Emerin, the protein whose production is altered in the X-linked form of Emery-Dreifuss muscular distrophy, has been hypothesized to be associated with the nuclear matrix on the basis of biochemical studies. In addition, immunocytochemical data reported its localization at the nuclear periphery, on the nuclear lamina, in sections of several normal tissues. We investigated the association of emerin with the nuclear matrix, by using cultured cells (SaOS-2, MG63 and HeLa-S3) and their in situ extracted matrix as a model, and immunocytochemical methods, both at the light and electron microscope level. Our results show a normal presence of emerin in the cultured cells and the specific persistence of emerin on the lamina of the in situ extracted nuclear matrix. This suggests a tight binding between emerin and the nuclear lamina independently from the interactions between the C-terminal hydrophobic domain of the protein and the inner nuclear membrane.

Oral exfoliative cytology for the non-invasive diagnosis in X-linked Emery-Dreifuss muscular dystrophy patients and carriers.

Emery-Dreifuss muscular dystrophy (EMD) is an inherited myopathy characterised by muscle contractures, progressive muscle wasting and weakness, with humeroperoneal distribution. Cardiac arrhythmia and heart conduction block are also important characteristics of this disease. The X-linked form of EMD is caused by the absence of emerin, encoded by the STA gene (Xq28). Emerin is normally localized in muscle and other tissues at the nuclear rim. Currently, muscle and skin biopsies are used for the immunohistochemical diagnosis. We demonstrate that emerin is present in the cheek oral mucosa, in the exfoliating epithelial cells, and we propose the collection of these cells as a new method for the diagnosis of X-linked EMD patients and the detection of carriers by immunofluorescence techniques: smears from healthy subjects contained about 98% emerin-positive cells, those from X-linked EMD patients contained none and those from carriers contained about 45%. The technique is completely non-invasive, simple, repeatable and inexpensive.

Human defunctionalized colon: a histopathological and pharmacological study of muscularis propria in resection specimens.

Despite the regression of "diversion colitis," temporary functional disorders after bowel continuity restoration could be caused by changes in the smooth muscle of excluded segments; however, studies on the muscularis propria have yielded contradictory results. This study was aimed at evaluating possible histopathological changes in muscular layers and motility of the defunctionalized human colon. Ten patients with defunctionalized colorectum (group A) and 10 controls (group B) underwent restorative or primary resection surgery. Strips were taken proximal to the colostomy (specimens A1) and the defunctionalized segment (specimens A2), and from the proximal (specimens B1) and distal extremity (specimens B2) of resected colons. Measurements of the thickness of the muscularis propria and of the volume density of the myenteric plexus, as well as of spontaneous motility and responses to electrical and pharmacological stimulation were taken. The muscularis propria was thicker in A2 than in A1 specimens (P = 0.004) and in B2 than in B1 specimens (P = 0.007). No differences were recorded either in the myenteric plexus volume density or in colonic motility. No differences were recorded in intergroup comparisons. As no structural or functional changes related to defunctionalization were found, clinical disorders after colorectal restoration could rather result from underlying colonic pathology and/or incomplete distal colon resection.

X-linked Emery-Dreifuss muscular dystrophy can be diagnosed from skin biopsy or blood sample.

We have raised an anti-emerin polyclonal antibody against a fusion protein encompassing most of the hydrophilic portion of emerin. Using this antibody, we have analyzed emerin expression in Emery-Dreifuss muscular dystrophy (EDMD) patients and controls, by immunocytochemistry, in skeletal muscle and skin, and by immunoblot, in peripheral blood mononuclear cells and lymphoblasts. Emerin was localized on the surfaces of nuclei in control skeletal muscle and skin but was absent or reduced in patient skeletal muscle, was absent from the skin of patients, and was expressed only in a few nuclei in a patient's mother. Immunoblot of peripheral blood cells from EDMD patients showed absence of the emerin band, altered-size emerin, or a protein of normal molecular mass but slightly reduced quantity. The diagnosis of X-linked EDMD is normally confirmed by genetic analysis of the STA gene coding for emerin. We propose immunocytochemical evaluation of emerin expression in skin biopsies as a sensitive and more convenient tool for diagnosing X-linked EDMD and, in particular, for distinguishing it from the autosomal dominant form. This technique may be applied to suspected EDMD patients, especially sporadic cases or those with incomplete clinical phenotype, and also suspected carriers. Immunoblot of peripheral blood cells is also useful, but it may not unequivocally identify carriers and some patients.

Heart-specific localization of emerin: new insights into Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is an X-linked inherited disease characterized by early contracture of the elbows, Achilles tendons and post-cervical muscles, slow progressive muscle wasting and weakness and cardiomyopathy presenting with arrhythmia and atrial paralysis: heart block can eventually lead to sudden death. The EDMD geneencodes a novel ubiquitous protein, emerin, which decorates the nuclear rim of many cell types. Amino acid sequence homology and cellular localization suggested that emerin is a member of the nuclear lamina-associated protein family. These findings did not explain the role of emerin nor account for the skeletal muscle- and heart-specific clinical manifestations associated with the disorder. Now we report that emerin localizes to the inner nuclear membrane, via its hydrophobic C-terminal domain, but that in heart and cultured cardiomyocytes it is also associated with the intercalated discs. We propose a general role for emerin in membrane anchorage to the cytoskeleton. In the nuclear envelope emerin plays a ubiquitous and dispensable role in association of the nuclear membrane with the lamina. In heart its specific localization to desmosomes and fasciae adherentes could account for the characteristic conduction defects described in patients.

Identification of autoantibodies to the I protein of the heterogeneous nuclear ribonucleoprotein complex in patients with systemic sclerosis.

OBJECTIVE: To assess the presence of autoantibodies to the 1 protein (polypyrimidine-tract binding protein) of the heterogeneous nuclear RNPs (hnRNP) in different connective tissue diseases. Antibodies to other hnRNP proteins (A1, A2, and B) have been previously found in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD). METHODS: Sera from 101 patients with various connective tissue diseases and 25 normal controls were investigated by enzyme-linked immunosorbent assay and immunoblotting, for their reactivity to highly purified recombinant hnRNP I. Moreover, reactivity to cellular hnRNP I protein was investigated by immunoblotting using a partially purified preparation of hnRNP proteins (including A1, A2, B, and I), and by indirect immunofluorescence. For the analysis of the fluorescence pattern, affinity-purified antibodies to hnRNP I; obtained from a selected patient, were tested on HEp-2 cells. RESULTS: By immunoblotting, antibodies reacting to recombinant hnRNP I were found in 22 of 40 patients with systemic sclerosis (SSc), 3 of 32 with RA, 0 of 23 with SLE, and 0 of 6 with MCTD. Antibodies to recombinant hnRNP I were more frequently found in patients with pre-SSc or limited SSc (15 of 24) than in those with intermediate or diffuse SSc (7 of 16). In indirect immunofluorescence studies, affinity-purified anti-hnRNP I autoantibodies gave a diffuse nucleoplasmic staining. Using an hnRNP preparation from nuclear extracts, anti-hnRNP I reactivity was detectable in SSc sera, while it was not detectable in RA, SLE, and MCTD sera reacting with hnRNP A/B proteins. CONCLUSIONS: Human autoimmune sera show distinct patterns of anti-hnRNP reactivity, i.e., anti-A/B in SLE and RA sera, and anti-I in SSc sera. This suggests that A/B proteins and the I protein may be involved in different dynamic hnRNP complexes that elicit different autoimmune responses. From a clinical perspective, anti-hnRNP I antibodies are frequently associated with pre-SSc features, suggesting an early appearance of these antibodies during the course of the disease.

Effect of fedotozine on human distal colon.

Fedotozine was rested in colonic strips removed during surgery from patients suffering from different diseases of the colon; the effects were compared to those of morphine and of the selective opiate agonist U-69593. Fedotozine did not affect the spontaneous motility of human colonic strips, unless very high concentrations were used. Fedotozine (10(-6)-3 x 10(-4) M) induced a concentration-dependent reduction of the excitatory effect induced by field stimulation, an effect which was partially mimicked by compound U-69593 and by morphine but not inhibited by naloxone. The cumulative dose-response curve to exogenous acetylcholine was inhibited by fedotozine (3 x 10(-4) M), whereas morphine had no effect up to 3 x 10(-4) M. In colonic strips incubated with [3H]-choline, fedotozine (10(-5)-10(-4) M) induced an erratic decrease of acetylcholine-release induced by electric stimulation. In our experimental model, the inhibitory effect of fedotozine does not seem to be related to opioid receptor activation.

hnRNP A1 selectively interacts through its Gly-rich domain with different RNA-binding proteins.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are abundant nuclear polypeptides, most likely involved in different steps of pre-mRNA processing. Protein A1 (34 kDa), a prominent member of the hnRNP family, seems to act by modulating the RNA secondary structure and by antagonizing some splicing factors (SR proteins) in splice-site selection and exon skipping/inclusion. A role of A1 in the nucleo-cytoplasmic transport of RNA has also been proposed. These activities might depend not only on the RNA-binding properties of the protein but also on specific protein-protein interactions. Here we report that A1 can indeed selectively interact, in vitro, both with itself and with other hnRNP basic "core" proteins. Such selective binding is mediated exclusively by the Gly-rich C-terminal domain, where a novel protein-binding motif constituted by hydrophobic repeats can be envisaged. The same domain is necessary and sufficient to promote specific interaction in vivo, as assayed by the yeast two-hybrid assay. Moreover, an in vitro interaction with some SR proteins was also observed. These observations suggest that diverse and specific protein-protein interactions might contribute to the different functions of the hnRNP A1 protein in mRNA maturation.

Human hnRNP protein A1: a model polypeptide for a structural and genetic investigation of a broad family of RNA binding proteins.

The hnRNP fiber is the substrate on which pre-mRNA processing occurs. The protein moiety of the fiber (hnRNP proteins) constitutes a broad family of RNA binding proteins that revealed, upon molecular analysis, a number of interesting features. Heterogeneous nuclear ribonucleoprotein A1 is a major component of the human hnRNP complex. In recent years this protein has attracted great attention because of several emerging evidences of its direct involvement in pre-mRNA processing and it has become one of the best characterized RNA binding proteins. Detailed knowledge of the structure of protein A1 has laid the basis for the understanding of its function, and for this reason A1 can be considered as a model polypeptide for the investigation of a large number of RNA binding proteins. In this work we report recent findings regarding the binding properties of protein A1 as well as new data on the gene structure of A1 and of its closely related hnRNP protein A2. Our results show that a single A1 molecule contains the determinants for simultaneous binding of two single-stranded nucleic acid molecules and we demonstrate that the glycine-rich domain of A1, isolated from the rest of the molecule, is capable of sustaining protein-protein interactions. These features probably account for the reannealing activity of the protein and for its capacity to modulate the binding of snRNPs to intron sequences in vitro. Comparison of A1 and A2 gene sequences revealed a remarkable conservation of the overall structural organization, suggesting important functions for the different structural elements.

Human hnRNP protein A1 gene expression. Structural and functional characterization of the promoter.

hnRNP protein A1 (34 kDa, pl 9.5) is a prominent member of the family of proteins (hnRNP proteins) that associate with the nascent transcripts of RNA polymerase II and that accompany the hnRNA through the maturation process and the export to the cytoplasm. New evidence suggests an active and specific role for some of these proteins, including protein A1, in splicing and transport. Contrary to the other hnRNP proteins, the intracellular level of protein A1 was reported to change as a function of proliferation state and cell type. In this work we analyse the A1 gene expression in different cells under different growth and differentiation conditions. Proliferation dependent expression was observed in lymphocytes and fibroblasts while purified neurons express high A1 mRNA levels both in the proliferative (before birth) and in the quiescent (after birth) state. Transformed cell lines exhibit very high (proliferation independent) A1 mRNA levels compared to differentiated tissues. A structural and functional characterization of the A1 gene promoter was carried out by means of DNase I footprinting and CAT assays. The observed promoter features can account for both elevated and regulated mRNA transcription. At least 12 control elements are contained in the 734 nucleotides upstream of the transcription start site. Assays with the deleted and/or mutated promoter indicate a co-operation of multiple transcriptional elements, distributed over the entire promoter, in determining the overall activity and the response to proliferative stimuli (serum).

Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity.

In HeLa cells metabolically labeled in vivo with [32P] orthophosphate in the presence of okadaic acid the concentration of phosphorylated A1 protein was increased significantly as compared to controls. Purified recombinant hnRNP protein A1 served as an excellent substrate in vitro for the catalytic subunit of cAMP-dependent protein kinase (PKA) and for casein kinase II (CKII). Thin layer electrophoresis of A1 acid hydrolysates showed the protein to be phosphorylated exclusively on serine residue by both kinases. V8 phosphopeptide maps revealed that the target site(s) of in vitro phosphorylation are located in the C-terminal region of A1. Phosphoamino acid sequence analysis and site directed mutagenesis identified Ser 199 as the sole phosphoamino acid in the protein phosphorylated by PKA. Phosphorylation introduced by PKA resulted in the suppression of the ability of protein A1 to promote strand annealing in vitro, without any detectable effect on its nucleic acid binding capacity. This finding indicates that phosphorylation of a single serine residue in the C-terminal domain may significantly alter the properties of protein A1.

Interaction of hnRNP A1 with snRNPs and pre-mRNAs: evidence for a possible role of A1 RNA annealing activity in the first steps of spliceosome assembly.

The in vitro interaction of recombinant hnRNP A1 with purified snRNPs and with pre-mRNAs was investigated. We show that protein A1 can stably bind U2 and U4 snRNP but not U1. Oligo-RNAse H cleavage of U2 nucleotides involved in base pairing with the branch site, totally eliminates the A1-U2 interaction. RNase T1 protection and immunoprecipitation experiments demonstrate that recombinant protein A1 specifically binds the 3'-end regions of both beta-globin and Ad-2 introns. However, while on the beta-globin intron only binding to the polypyrimidine tract was observed, on the Ad-2 intron a 32 nt fragment encompassing the branch point and the AG splice-site dinucleotide was bound and protected. Such protection was drastically reduced in the presence of U2 snRNP. Altogether these results indicate that protein A1 can establish a different pattern of association with different pre-mRNAs and support the hypothesis that this protein could play a role in the annealing of U2 to the branch site and hence in the early events of pre-splicing complex assembly.

Antibodies to hn-RNP protein A1 in systemic lupus erythematosus: clinical association with Raynaud's phenomenon and esophageal dysmotility.

Antibodies to recombinant hn-RNP protein A1 were found by ELISA in sera from 26 out of 67 unselected patients with systemic lupus erythematosus. A higher number of anti-A1 positive patients had Raynaud's phenomenon (50% vs 7%) and esophageal dysmotility (42% vs 5%) than the anti-A1 negative patients. All 8 patients with both Raynaud's phenomenon and esophageal dysmotility had a positive anti-A1 assay. No association was found with other clinical findings, nor with disease activity and treatment regimes. Anti-A1 antibodies did not correlate with anti-RNP and anti-Sm antibodies, which were present in 30% and 12% of the anti-A1 positive cases and in 22% and 7% of the anti-A1 negative cases, respectively. Our results indicate that antibodies to hn-RNP protein A1 may be associated with a subset of SLE patients with clinical features overlapping those of progressive systemic sclerosis and quite distinct from the group identified by anti-RNP antibodies.

Modeling by homology of RNA binding domain in A1 hnRNP protein.

Eukaryotic nuclear RNA binding proteins share a common sequence motif thought to be implicated in RNA binding. One of the two domains present in A1 hnRNP protein, has been modelled by homology in order to make a prediction of the main features of the RNA binding site. Acylphosphatase (EC 3.6.1.7) was selected as template for the modeling experiment. The predicted RNA binding site is a beta-sheet containing the two RNP consensus sequences as well as lysines and arginines conserved among the family.

Recombinant hnRNP protein A1 and its N-terminal domain show preferential affinity for oligodeoxynucleotides homologous to intron/exon acceptor sites.

The reported binding preference of human hnRNP protein A1 for the 3'-splice site of some introns (Swanson and Dreyfuss (1988) EMBO J. 7, 3519-3529; Mayrand and Pederson (1990) Nucleic Acids Res. 18, 3307-3318) was tested by assaying in vitro the binding of purified recombinant A1 protein (expressed in bacteria) to synthetic oligodeoxynucleotides (21-mers) of suitable sequence. In such a minimal system we find preferential binding of protein A1 to oligodeoxynucleotide sequences corresponding to the 3'-splice site of IVS1 of human beta-globin pre-mRNA and of IVS1 of Adenovirus type 2 major late transcript. Mutation studies demonstrate that the binding specificity is dependent on the known critical domains of this intron region, the AG splice site dinucleotide and polypyrimidine tract, and resides entirely in the short oligonucleotide sequence. Moreover specific binding does not require the presence of other hnRNP proteins or of snRNP particles. Studies with a truncated recombinant protein demonstrated that the minimal protein sequence determinants for A1 recognition of 3'-splice acceptor site reside entirely in the N-terminal 195 aa of the unmodified protein.

Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein.

The human hnRNP core protein A1 (34 kd) is encoded by a 4.6 kb gene split into 10 exons. Here we show that the A1 gene can be differentially spliced by the addition of an extra exon. The new transcript encodes a minor protein of the hnRNP complex, here defined A1B protein, with a calculated mol. wt of 38 kd, that coincides with a protein previously designated as B2 by some authors. In vitro translation of the mRNAs selected by hybridization with A1 cDNA produced two proteins of 34 and 38 kd; Northern blot analysis of poly(A)+ RNA from HeLa cells revealed that the abundance of the A1B mRNA was approximately 5% that of A1. The A1B protein was detected by Western blotting with an anti-A1 monoclonal antibody both in enriched preparations of basic hnRNP proteins and in 40S hnRNP particles. The A1B protein exhibits a significantly higher affinity than A1 for ssDNA. The recombinant A1B protein, expressed in Escherichia coli, shows the same electrophoretic mobility and charge as the cellular one.