Fractionation of constituents of ribonucleoproteins containing heterogeneous nuclear ribonucleic acid.

A method of fractionation of hnRNP constituents adaptable to large-scale preparation is presented. It is based on differential resistance to salt dissociation of the two classes of units of hnRNP, the 30--50S monoparticles and the heterogeneous complexes. The monoparticle proteins were released from hnRNP by 0.4 M NaCl. They were separated from the salt-resistant RNP corresponding to the heterogeneous complexes in three steps: chromatography on DEAE-cellulose, high-speed centrifugation, and Bio-Gel chromatography. The latter chromatography permitted a first fractionation of monoparticle proteins according to molecular weight. Such fractions may serve for purification of individual proteins of molecular weight below 80 000. After the two first steps, two fractions of salt-resistant RNP were obtained. In addition to heterogeneous RNA up to 30 S, small nuclear RNAs were detected which represented 6% of total RNA. The protein pattern was complex, and no clear-cut segregation of groups of proteins could be observed between the two fractions. They were both highly enriched in phosphoproteins as compared to nomoparticle proteins. In another fraction corresponding to the void volume of Bio-Gel chromatography, one-third of the RNA was small nuclear RNA. It is suggested that this fraction contains snRNP in addition to free proteins of molecular weight above 80 000 and to salt-resistant RNP similar to those described above but of small size.     

Distinct RNP complexes of shuttling hnRNP proteins with pre-mRNA and mRNA: candidate intermediates in formation and export of mRNA

Nascent pre-mRNAs associate with hnRNP proteins in hnRNP complexes, the natural substrates for mRNA processing. Several lines of evidence indicate that hnRNP complexes undergo substantial remodeling during mRNA formation and export. Here we report the isolation of three distinct types of pre-mRNP and mRNP complexes from HeLa cells associated with hnRNP A1, a shuttling hnRNP protein. Based on their RNA and protein compositions, these complexes are likely to represent distinct stages in the nucleocytoplasmic shuttling pathway of hnRNP A1 with its bound RNAs. In the cytoplasm, A1 is associated with its nuclear import receptor (transportin), the cytoplasmic poly(A)-binding protein, and mRNA. In the nucleus, A1 is found in two distinct types of complexes that are differently associated with nuclear structures. One class contains pre-mRNA and mRNA and is identical to previously described hnRNP complexes. The other class behaves as freely diffusible nuclear mRNPs (nmRNPs) at late nuclear stages of maturation and possibly associated with nuclear mRNA export. These nmRNPs differ from hnRNPs in that while they contain shuttling hnRNP proteins, the mRNA export factor REF, and mRNA, they do not contain nonshuttling hnRNP proteins or pre-mRNA. Importantly, nmRNPs also contain proteins not found in hnRNP complexes. These include the alternatively spliced isoforms D01 and D02 of the hnRNP D proteins, the E0 isoform of the hnRNP E proteins, and LRP130, a previously reported protein with unknown function that appears to have a novel type of RNA-binding domain. The characteristics of these complexes indicate that they result from RNP remodeling associated with mRNA maturation and delineate specific changes in RNP protein composition during formation and transport of mRNA in vivo.     

Heterogeneous nuclear RNA-protein fibers in chromatin-depleted nuclei

The heterogeneous nuclear RNA-protein (hnRNP) fibers in HeLa cell nuclei are visualized by a nuclear subfractionation technique which removes 96% of the chromatin in a single step and 99% in a two-step elution but leaves the bulk of the hnRNA complexed with the remnant nuclear structure or lamina. Both steady-state and newly synthesized (approximately 15-s label) hnRNA are associated with the remnant nuclei to about the same extent. This association does not appear to depend on the presence of chromatin and exists in addition to any possible association of hnRNP with chromatin itself. Electron microscopy of partially purified nuclear hnRNA complexes shows that the hnRNP fibers form a ribonucleoprotein network throughout the nucleus, whose integrity is dependent on the RNA. Autoradiography confirms that hnRNA is a constituent of the fibers. The RNA network visualized in these remnant nuclei may be similar to RNA networks seen in intact cells. The hnRNA molecules appear to be associated with the nuclear lamina, at least in part, by unusual hnRNA sequences. More than half of the recovered poly(A) and double-stranded hnRNA regions remains associated with the nuclear structures or the laminae after digestion with RNase and elution with 0.4 M ammonium sulfate. In contrast, the majority of oligo(A), another ribonuclease resistant segment, is released together with most of the partially digested but still acid-precipitable single- stranded hnRNA and the hnRNP proteins not eluted by the ammonium sulfate alone. These special RNA regions appear to be tightly bound and may serve as points of attachment of the hnRNA to nuclear substructures. It is suggested that hnRNA metabolism does not take place in a soluble nucleoplasmic compartment but on organized structures firmly bound to the nuclear structure.     

hnRNA and its attachment to a nuclear protein matrix

In this study, DNA-depleted nuclear protein matrices are isolated from HeLa S3 cells. These nuclear matrices consist of peripheral laminae, residual nucleoli, and internal fibrillar structures. High molecular weight, heterogeneous nuclear RNA (hnRNA) is quantitatively associated with these structures and can be released intact only by affecting the integrity of the matrices. It is, therefore, concluded that hnRNA is part of a highly organized nuclear structure. By irradiation of intact cells or isolated nuclear matrices with ultraviolet light, proteins tightly associated with hnRNA can be induced to cross-link with the RNA. Performing the cross-linking in vivo is an extra guarantee that only hnRNA-protein (hnRNP) complexes existing in the intact cell are covalently linked. Such hnRNP complexes were isolated and purified under conditions that completely dissociate nonspecific RNA-protein complexes. By comparison of the hnRNP found in nuclear matrices and the published data on the composition of hnRNP particles, it was found that the so-called hnRNP "packaging" proteins (32,000-38,000 mol wt) were not efficiently cross-linked to hnRNA by UV irradiation. They were, however, present in the matrix preparations, bound to hnRNA, because they were released from nuclear matrices after ribonuclease treatment of these structures. On the other hand, two major hnRNPs (41,500 and 43,000 mol wt) were efficiently cross-linked to hnRNA. These proteins were not released by ribonuclease treatment, which suggests that they are involved in the binding of hnRNA to the nuclear matrix.     

Isolation and characterization of hnRNA-snRNA-protein complexes from Morris hepatoma cells

Of the RNA labelled after incubation of hepatoma cells with radioactive precursors for 20 and 150 min. 35% and 70%, respectively, can be isolated from nuclei by two consecutive extractions with 0.14 M NaCl at pH 8. The isolated RNA is complexed with nuclear proteins forming structures with sedimentation coefficients of less than 30 S to greater than 100 S. Similar complexes from rat liver isolated under the same experimental conditions show coefficients of 30-40 S. The RNA-associated proteins are similar, on the basis of sodium dodecyl sulphate/polyacrylamide gel electrophoresis, to the respective proteins of other cell types. The presence on these RNP complexes of six discrete small nuclear RNAs (snRNA) has been established. Experiments with a reversible inhibitor of RNA synthesis, D-galactosamine, demonstrated, differences in the turnover of hnRNA and snRNA. The half-lives of the six snRNA species has been determined, varying from 32 h for snRNA species a, b and d, to 22 h for snRNA species e and f and to 13 h for snRNA species c. Treatment of the nuclear extracts with 0.7 M and 1 M NaCl results in dissociation of hnRNA from the 'core' and other polypeptides, whereas snRNA remains complexed with polypeptides of Mr 54 000-59 000. Incubation of the nuclear extracts at 0 C with low doses of pancreatic R Nase (up to 1.5 micrograms/ml), which renders approximately 80% of the hnRNA acid-soluble and cleaves most of the snRNA, results in conversion of the high-molecular-weight hnRNPs to 30-S structures, without disrupting the 30-S RNP. Treatment of the nuclear extracts with higher doses of RNase (3 micrograms/ml) leads to disruption of the 30-S RNP and release of the hnRNA-associated proteins, underlining the importance of hnRNA-protein interaction for the retainment of the hnRNP structures.     

The distribution of two hnRNP-associated proteins defined by a monoclonal antibody is altered in heat-shocked HeLa cells

A monoclonal antibody obtained after mice were immunized with hnRNP purified from HeLa cells recognizes two polypeptides of Mr 35,000 and 37,000. By immunocytofluorescence, these antigens can be visualized only in cells previously heat shocked at 45 degrees C for 5 or 10 min, although they are present at the same level in unstressed and stressed cells. The signal, which is mostly concentrated in the interchromatin space, where hnRNP fibrils are located, does not accumulate with time and disappears 4 to 5 h after heat shock. Discrimination between the two types of hnRNP substructures, the 30-50 S monoparticles and the nuclear matrix fibrils, based on differential sensitivity to salt or ribonuclease treatment, showed that in unstressed cells the antigens behave as monoparticle proteins. In contrast, in heat-shocked cells, most 35-37K antigens behave as nuclear matrix proteins. Thus, heat shock seems to induce a rapid and reversible switch of these two antigens from hnRNP monoparticles to the nuclear matrix. The data demonstrate that heat shock, which was previously shown not to alter the overall RNA: protein packaging ratio of hnRNP, induces subtle modifications of their substructure. Such modifications might be of importance since heat shock is known for instance to affect pre-mRNA processing.     

Intranuclear distribution of SV40 large T-antigen and transformation-related protein p53 in abortively infected cells

The intranuclear localization of SV40 T-antigen (T-Ag) and the cellular protein p53 was studied in SV40 abortively infected baby mouse kidney cells using two complementary methods of ultrastructural immunocytochemistry in combination with preferential staining of nuclear RNP components and electron microscope autoradiography. Both proteins were revealed in association with peri- and interchromatin RNP fibrils containing the newly synthesized hnRNA. In addition, T-Ag and p53 remained bound, at least in part, to the residual internal nuclear matrix following nuclease and salt extractions of infected cells. The localization of T-Ag was different in SV40 lytically infected monkey kidney cells since, in addition to hnRNP fibrils, the viral protein was also associated with cellular chromatin. However, when lytic infection was performed in conditions of blocked viral DNA replication, T-Ag was no longer associated with the cellular chromatin but remained bound to the hnRNP fibrils. We conclude that the transforming and lytic functions of T-Ag can be distinguished by different subnuclear distributions. The significance of the association of T-Ag and p53 with hnRNP fibrils and the internal nuclear matrix is discussed in relation to the role of these structures in the control of cellular mRNA biogenesis.     

Differential nuclear localization and nuclear matrix association of the splicing factors PSF and PTB

A monoclonal antibody raised against nuclear matrix proteins detected a protein of basic pI in human nuclear matrix protein samples of various cellular origin. The ubiquitously occurring (common) nuclear matrix protein was identified as splicing factor PSF (PTB associated splicing factor). The interaction between the splicing factors PSF and PTB/hnRNP I was confirmed by co-immunoprecipitation from nuclear salt extracts. However, the nuclear localization of PSF and PTB and their distribution in subnuclear fractions differed markedly. Isolated nuclear matrices contained the bulk of PSF, but only minor amounts of PTB. In confocal microscopy both proteins appeared in speckles, the majority of which did not co-localize. Removing a large fraction of the soluble PTB structures by salt extraction revealed some colocalization of the more stable PTB fraction with PSF. These PTB/PSF complexes as well as the observed PSF-PTB interaction may reflect the previously reported presence of PTB and PSF in spliceosomal complexes during RNA processing. The present data, however, point to different cellular distribution and nuclear matrix association of the majority of PSF and PTB.     

Nuclear matrix-like filaments and fibrogranular complexes form through the rearrangement of specific nuclear ribonucleoproteins

The behavior of nuclear pre-mRNA-binding proteins after their nuclease and/or salt-induced release from RNA was investigated. After RNase digestion or salt extraction, two proteins that initially exist as tetramers (A2)(3)B1 in isolated heterogeneous nuclear ribonucleoprotein (hnRNP) complexes quantitatively reassociated to form regular helical filaments ranging in length from 100 nm to >10 microm. In highly magnified preparations prepared for scanning transmission electron microscopy, single filaments have diameters near 18 nm. In conventional negatively stained preparations viewed at low magnification, the diameters of the thinnest filaments range from 7 to 10 nm. At protein concentrations of >0.1 mg/ml, the filaments rapidly aggregated to form thicker filamentous networks that look like the fibrogranular structures termed the "nuclear matrix." Like the residual material seen in nuclear matrix preparations, the hnRNP filaments were insoluble in 2 M NaCl. Filament formation is associated with, and may be dependent on, disulfide bridge formation between the hnRNP proteins. The reducing agent 2-mercaptoethanol significantly attenuates filament assembly, and the residual material that forms is ultrastructurally distinct from the 7- to 10-nm fibers. In addition to the protein rearrangement leading to filament formation, nearly one-third of the protein present in chromatin-clarified nuclear extracts was converted to salt-insoluble material within 1 min of digestion with RNase. These observations are consistent with the possibility that the residual material termed the nuclear matrix may be enriched in, if not formed by, denatured proteins that function in pre-mRNA packaging, processing, and transport.     

Complexes of nuclear matrix DNA with proteins tightly bound to DNA contain a specific small-size RNA of a novel type

Analysis of DNA-protein structures composed of nuclear matrix attached DNA and the most tightly bound proteins was performed. Although the previously described non-histone proteins (1) were present the buoyant density of the complex was the same as that of pure DNA. RNA inaccessible to RNase in 0.4 M NaCl but digestible in low ionic strength buffer was detected. This RNA is not a nascent one. It turned out to be homogeneous and represent a novel type of small nuclear RNA. Partial sequence of this RNA is presented.     

SUMO modification of heterogeneous nuclear ribonucleoproteins

Small ubiquitin-related modifiers (SUMOs) are proteins that are posttranslationally conjugated to other cellular proteins, particularly those that localize and function in the nucleus. Enzymes regulating SUMO modification localize in part to nuclear pore complexes (NPCs), indicating that modification of some proteins may occur as they are translocated between the nucleus and the cytoplasm. Substrates that are regulated by SUMO modification at NPCs, however, have not been previously identified. Among the most abundant cargos transported through NPCs are the heterogeneous nuclear ribonucleoproteins (hnRNPs). HnRNPs are involved in various aspects of mRNA biogenesis, including regulation of pre-mRNA splicing and nuclear export. Here, we demonstrate that two subsets of hnRNPs, the hnRNP C and M proteins, are substrates for SUMO modification. We demonstrate that the hnRNP C proteins are modified by SUMO at a single lysine residue, K237, and that SUMO modification at this site decreases their binding to nucleic acids. We also show that Nup358, a SUMO E3 ligase associated with the cytoplasmic fibrils of NPCs, enhances the SUMO modification of the hnRNP C and M proteins. Based on our findings, we propose that SUMO modification of the hnRNP C and M proteins may occur at NPCs and facilitate the nucleocytoplasmic transport of mRNAs.     

Evidence that a nuclear matrix protein participates in premessenger RNA splicing

The role of nuclear matrix proteins in premessenger RNA splicing has been investigated using antibodies raised against isolated rat liver nuclear matrix and cross-reactive with a 65-kDa HeLa cell nuclear matrix protein (IGA-65). IGA-65 is an internal nuclear matrix component which can be solubilized as a component of nuclear splicing extracts, by the action of endogenous ribonucleases, EDTA, and DTT during extract preparation. Preincubation of splicing extract with antibodies against IGA-65 (anti-IGA-65) inhibited in vitro splicing of exogenous adenovirus precursor RNA. Furthermore, assembly of precursor RNA into active spliceosome complexes was inhibited by pretreatment of extracts with anti-IGA-65, suggesting a role for IGA-65 during early spliceosome assembly. The IGA-65 present in splicing extracts was distinguishable from known U-snRNP and hnRNP proteins on protein gels. Furthermore, electrophoresis of splicing extract on native gels indicated that IGA-65 was present in protein complexes different from those containing U-snRNPs or hnRNP C protein. The data support identification of complexes containing IGA-65 as nuclear factors involved in pre-mRNA splicing and, by extension, suggest a role for the nuclear matrix during processing in vivo.     

Cell cycle-regulated phosphorylation of the pre-mRNA-binding (heterogeneous nuclear ribonucleoprotein) C proteins.

Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes, the structures that contain heterogeneous nuclear RNA and its associated proteins, constitute one of the most abundant components of the eukaryotic nucleus. hnRNPs appear to play important roles in the processing, and possibly also in the transport, of mRNA. hnRNP C proteins (C1, M(r) of 41,000; C2, M(r) of 43,000 [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis]) are among the most abundant pre-mRNA-binding proteins, and they bind tenaciously to sequences relevant to pre-mRNA processing, including the polypyrimidine stretch of introns (when it is uridine rich). C proteins are found in the nucleus during the interphase, but during mitosis they disperse throughout the cell. They have been shown previously to be phosphorylated in vivo, and they can be phosphorylated in vitro by a casein kinase type II. We have identified and partially purified at least two additional C protein kinases. One of these, termed Cs kinase, caused a distinct mobility shift of C proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These phosphorylated C proteins, the Cs proteins, were the prevalent forms of C proteins during mitosis, and Cs kinase activity was also increased in extracts prepared from mitotic cells. Thus, hnRNP C proteins undergo cell cycle-dependent phosphorylation by a cell cycle-regulated protein kinase. Cs kinase activity appears to be distinct from the well-characterized mitosis-specific histone H1 kinase activity. Several additional hnRNP proteins are also phosphorylated during mitosis and are thus also potential substrates for Cs kinase. These novel phosphorylations may be important in regulating the assembly and disassembly of hnRNP complexes and in the function or cellular localization of RNA-binding proteins.     

Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.