Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells

The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A)+ hnRNA in intact HeLa cells, as revealed by a novel in situ RNA- protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the greater than 42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A)+ hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in Cs2SO4 density gradients. These results establish that poly(A)+ hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo. This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modulating hnRNA structure and processing in the cell.     

Detection of a cellular polypeptide associated with adenovirus-coded VA RNA using in vitro labeling of proteins cross-linked to RNA.

Ultraviolet light induced RNA-protein cross-linking for identification of polypeptides interacting with RNA in intact cells (Wagenmakers et al. 1980), is limited by the intensity of the label in the proteins or in residual nucleotides remaining attached to the proteins after RNase treatment of the RNA-protein complexes. Here we report a method, where th cross-linked RNA-protein complexes are treated with RNase T1 and the T1-oligonucleotides covalently linked to the proteins are labeled in the 5' terminus using gamma-32P-ATP and T4 polynucleotide kinase. The cross-linked proteins can then readily be identified owing to the incorporated 32P label. As examples, proteins associated with polyadenylated mRNA, hnRNA and adenoviral VA RNA were identified. A protein with a molecular weight of approximately 50,000 is found associated with adenovirus-coded VA RNA. This was confirmed by binding assays, in which labeled VAI RNA is incubated with proteins from uninfected and adenovirus infected HeLa cells immobilized on nitrocellulose sheets.     

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.     

Non-random localization of ribonucleoprotein (RNP) structures within an adenovirus mRNA precursor.

Heterogeneous nuclear protein complexes (hnRNP) containing the precursor RNA from the adenovirus early region 2 were analysed to determine the specificity of protein-RNA interaction. RNA precursor sequences were present in isolated hnRNP complexes and endogenous 30S particles. At least 20-40 bases long fragments were protected when RNase A was used to remove unprotected RNA sequences in hnRNA complexes. Similarly around 40 bases of RNA were protected in 30S particles. These sequences represent discrete regions of the adenovirus genome. Especially sequences complementary to the EcoRI-F fragment encoding the first leader and the major intron for the DNA binding protein (DBP) RNA precursor, were analysed in detail. Tentatively, sequences resistant to RNase A were located in the middle of the intron and at the splice-donor junction of the first leader of the DBP precursor RNA. The same sequences were identified irrespective whether hnRNP complexes or 30S particles were used suggesting that 30S particles originate from hnRNP complexes. A 38.000 dalton protein appears to be in direct contact with RNA sequences complementary to the EcoRI-F fragment.     

Small nuclear RNA and VA RNA in nuclear ribonucleoprotein fibrils from adenovirus-2 infected HeLa cells

The small RNA of hnRNP¹ were studied in HeLa cells infected with adenovirus-2. At 15 h post-infection, when 50–60 % of the hnRNA was of viral origin, all the small nuclear RNA of hnRNP from non-infected cells were present in hnRNP from infected cells. The small, virus-encoded VA RNA could not be detected by staining like the snRNA but only after labeling. It represented less than 1 % of the small nuclear RNA in hnRNP. The low level of VA RNA in hnRNP as compared to that of the small nuclear RNA does not favor the hypothesis of a similar function for these 2 classes of small RNA.     

hnRNP proteins interact with nascent Adenovirus RNA

By using Adenovirus 2 infected HeLa cells labeled during very brief pulses of (³H)Uridine, we have shown that nascent chains of heterogenous nuclear RNA (hnRNA) were already associated with proteins to form ribonucleoprotein particles (hnRNP). It was also shown that the small Ad2 specific VA RNA was not associated with these hnRNP.     

Ribonucleoprotein organization of eukaryotic RNA. XXX. Evidence that U1 small nuclear RNA is a ribonucleoprotein when base-paired with pre-messenger RNA in vivo

U1 small nuclear RNA is thought to be involved in messenger RNA splicing by binding to complementary sequences in pre-mRNA. We have investigated intermolecular base-pairing between pre-mRNA (hnRNA) and U1 small nuclear RNA by psoralen crosslinking in situ, with emphasis on ribonucleoprotein structure. HeLa cells were pulse-labeled with [3H]uridine under conditions in which hnRNA is preferentially labeled. Isolated nuclei were treated with aminomethyltrioxsalen , which produces interstrand crosslinks at sites of base-pairing between hnRNA and U1 RNA. hnRNA-ribonucleoprotein (hnRNP) particles were isolated in sucrose gradients containing 50% formamide, to dissociate non-crosslinked U1 RNA, and then analyzed by immunoaffinity chromatography using a human autoantibody that is specific for the ribonucleoprotein form of U1 RNA (anti-U1 RNP). After psoralen crosslinking, pulse-labeled hnRNA in hnRNP particles reproducibly bound to anti-U1 RNP. The amount of hnRNA bound to anti-U1 RNP was reduced 80 to 85% when psoralen crosslinking of nuclei was omitted, or if the crosslinks between U1 RNA and hnRNA were photo-reversed prior to immunoaffinity chromatography. Analysis of the proteins bound to anti-U1 RNP after crosslink reversal revealed polypeptides having molecular weights similar to those previously described for U1 RNP. These proteins did not bind to control, non-immune human immunoglobulin G. These results indicate that the subset of nuclear U1 RNA that is base-paired with hnRNA at a given time in the cell is a ribonucleoprotein. This raises the possibility that these proteins, as well as U1 RNA itself, may participate in pre-mRNA splice site recognition by U1 RNP.     

Cross-linked informofers.

The proteins of 30S RNP particles containing pre-mRNA (hnRNA) were cross-linked with bifunctional reagents (dimethyl-suberimidate and dimethyl-3,3'-dithiobispropionimidate). Further treatment with 1 or 2 M NaCl dissociates all RNA from protein. However, a significant part of protein particles--informofers being cross-linked survived high salt treatment. Their sedimentation coefficients were close to those of original particles. No RNA could be detected in the informofers even after labeling the cells with a precursor for a long period of time. Sodium dodecylsulfate or urea dissociated cross-linked informofers into oligomeric polypeptides. They could be dissociated by beta-mercaptoethanol treatment if a reversible cross-linked reagent had been used. The resulting polypeptides were represented by informatin. RNP particles (30S RNP or poly-particles) were reconstituted upon mixing of cross-linked informofers with pre-mRNA and removal of 2 M NaCl.     

Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.     

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 core proteins of 35S hnRNP complexes. Characterization of nine different species

Ribonucleoprotein complexes (hnRNP) containing fragments of heterogeneous nuclear (hn)RNA and sedimenting at 35-40 S were isolated from the nuclei of HeLa S3 cells using the pH 8.0/diffusion technique. These hnRNP complexes are thought to be part of the hnRNA processing apparatus. The major protein components (core proteins) were identified by their constant ratios in native particles and in 35S hnRNP particles reconstituted in vitro. All of the core proteins, with one exception, show an increase in Mr on sodium dodecylsulfate (NaDodSO4)/polyacrylamide gels containing 8 M urea, indicative of secondary structure elements resistant to denaturation by NaDodSO4. The nine core proteins found by us are: A1 [Mr(NaDodSO4) 31 X 10(3)/Mr (urea) 38 X 10(3), apparent isoelectric point, pIapp 9.3], A2 (32.5 X 10(3)/39 X 10(3), 8.4), B1a (35.5 X 10(3)/41 X 10(3), 8.8), B1b (35.5 X 10(3)/44 X 10(3), 8.3), B1c (35.5 X 10(3)/43 X 10(3), 5.7) B2 (37 X 10(3)/42 X 10(3), 9.15), C1 (39 X 10(3)/46 X 10(3), 9.2), C2 (40.5 X 10(3)/45 X 10(3), 5.55) and C3 (38.5 X 10(3)/37 X 10(3), 4.8). Individual proteins were electroeluted from two-dimensional gels and their amino acid composition determined. Difference indices were calculated and show a group of closely related basic proteins (A1, A2, B1a, B1b, B2, C1), two related slightly acidic proteins (B1c, C2) and a distinct acidic member (C3). Two-dimensional analysis of tryptic fragments and one-dimensional separation of peptides after V8 protease treatment support these data. Peptide mapping of the proteins A1 and A2 from bovine and human cells yields identical fragments indicating a high degree of cross-species conservation. An additional protein (D4: 44 X 10(3)/55 X 10(3), greater than 9.5) was found, which preferentially associates with heavier, oligomeric hnRNP structures. Only traces of actin are present in the 35S hnRNP fraction. All core proteins are modified by charge. A large part of the charge isomers arises by phosphorylation, which has been shown by labeling with 32PO4 in vivo and with [gamma-32P]ATP in vitro. In vitro the phosphate transfer is mediated by an endogenous protein kinase associated with the 35S hnRNP complexes. The major core protein A1 exists in two conformeric forms (A1 and A1x) of which only A1x serves as phosphate acceptor in vivo.     

Control of translation in adenovirus-infected cells.

The initiation of protein synthesis in adenovirus-infected cells is regulated during the late phase in two ways, which may be related. The overall translation rate is maintained by a small viral RNA, VA RNAI, which prevents the phosphorylation of initiation factor eIF-2 by a double-stranded RNA-activated protein kinase, DAI. In addition, the relative efficiency of translation of host cell and viral mRNA populations is regulated in the infected cell during the late phase such that viral mRNAs are selectively utilized. Three viral elements have been implicated in this process: the 5' leader present on most late viral mRNAs; the late protein, 100K; and VA RNA. This article reviews the mechanisms underlying these translational control phenomena.