Heterogeneous nuclear ribonucleoproteins C1 and C2 associate with the RNA component of human telomerase

Here we demonstrate that heterogeneous nuclear ribonucleoproteins (hnRNPs) C1 and C2 can associate directly with the integral RNA component of mammalian telomerase. The binding site for hnRNPs C1 and C2 maps to a 6-base uridylate tract located directly 5' to the template region in the human telomerase RNA (TR) and a 4-base uridylate tract directly 3' to the template in the mouse TR. Telomerase activity is precipitated with antibodies specific to hnRNPs C1 and C2 from cells expressing wild-type human TR but not a variant of the human TR lacking the hnRNPs C1 and C2 binding site, indicating that hnRNPs C1 and C2 require the 6-base uridylate tract within the human TR to associate with the telomerase holoenzyme. In addition, we demonstrate that binding of hnRNPs C1 and C2 to telomerase correlates with the ability of telomerase to access the telomere. Although correlative, these data do suggest that the binding of hnRNPs C1 and C2 to telomerase may be important for the ability of telomerase to function on telomeres. The C proteins of the hnRNP particle are also capable of colocalizing with telomere binding proteins, suggesting that the C proteins may associate with telomeres in vivo. Therefore, human telomerase is capable of associating with core members of the hnRNP family of RNA binding proteins through a direct and sequence-specific interaction with the human TR. This is also the first account describing the precise mapping of a sequence in the human TR that is required to associate with an auxiliary component of the human telomerase holoenzyme.     

Long C terminal splice variant CaV2.2 identified in presynaptic membrane by mass spectrometric analysis

Ca_V2.2 voltage-gated calcium ­channels play a key role in the gating of transmitter release at presynaptic terminals. Recently we used mass spectrometry (MS) to analyze the protein complex associated with Ca_V2.2 in purified presynaptic terminal membranes. A number of known and new Ca_V2.2-associated proteins were identified, but not the channel itself. Here we set out to explore this anomaly. As previously, we used antibody Ab571 to capture the channel from purified synaptosome membrane lysate. We prepared a brain membrane lysate enriched for presynaptic active zones using standard methods to fractionate purified synaptosomes. These were osmotically lysed to generate a fraction enriched in presynaptic surface membranes. The lysate was solubilized in modified RIPA buffer and was passed over anti-Ca_V2.2 antibody covalently bonded to immunoprecipitation beads. Captured complexes on the beads were then stripped of weakly-bound proteins by exposure to high salt to enrich the channel fraction. Proteins remaining bound to the sample were recovered in high concentration urea and the sample was subjected to standard enzyme digestion and MS analysis. We identified 12 distinct Ca_V2.2 peptides, but no other ion channel peptides, in the lysate-exposed bead sample but no other ion channel peptides were recovered. Interestingly one of the channel peptides was derived from the alternatively spliced, long-C terminal region. Hence, confidence in identification of Ca_V2.2 was beyond reasonable doubt. The identification of the long-splice Ca_V2.2 provides compelling evidence that this variant is targeted to the presynaptic terminal, as we and others have suggested.     

The internal ribosome entry site-mediated translation of antiapoptotic protein XIAP is modulated by the heterogeneous nuclear ribonucleoproteins C1 and C2

The X-chromosome-linked inhibitor of apoptosis, XIAP, is the most powerful and ubiquitous intrinsic inhibitor of apoptosis. We have shown previously that the translation of XIAP is controlled by a potent internal ribosome entry site (IRES) element. IRES-mediated translation of XIAP is increased in response to cellular stress, suggesting the critical role for IRES translation during cellular stress. Here, we demonstrate that heterogeneous nuclear ribonucleoproteins C1 and C2 (hnRNPC1 and -C2) are part of the RNP complex that forms on XIAP IRES. Furthermore, the cellular levels of hnRNPC1 and -C2 parallel the activity of XIAP IRES and the overexpression of hnRNPC1 and -C2 specifically enhanced translation of XIAP IRES, suggesting that hnRNPC1 and -C2 may modulate XIAP expression. Given the central role of XIAP in the regulation of apoptosis these results are important for our understanding of the control of apoptosis.     

U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing

Two different experimental approaches have provided evidence that both U2 and U1 snRNPs function in pre-mRNA splicing. When the U2 snRNPs in a nuclear extract are selectively degraded using ribonuclease H and either of two deoxyoligonucleotides complementary to U2 RNA, splicing activity is abolished. Mixing an extract in which U2 has been degraded with one in which U1 has been degraded recovers activity. Use of anti-(U2)RNP autoantibodies demonstrates that U2 snRNPs associate with the precursor RNA during in vitro splicing. At 60 min, but not at 0 min, into the reaction intron fragments that include the branch-point sequence are immunoprecipitated by anti-(U2)RNP. At all times, U1 snRNPs bind the 5' splice site of the pre-mRNA. Possible interactions of the U2 snRNP with the U1 snRNP and with the pre-mRNA during splicing are considered.     

Heterogeneous nuclear ribonucleoproteins as regulators of gene expression through interactions with the human thymidine kinase promoter

In search for nuclear proteins that interact with the human thymidine kinase (htk) promoter, we discovered that p37AUF, a hnRNP C-like protein, and hnRNP A1, both members of the heterogeneous ribonucleoprotein family, can bind with high affinity to an ATTT sequence motif contained within the cell cycle regulatory unit (CCRU). We report here that over-expression of p37AUF stimulates gene expression mediated by the htk promoter in a promoter-sequence specific manner, whereas hnRNP A1 suppresses it. Both recombinant p37AUF and hnRNP A1 can bind the htk CCRU, suggesting that their binding to the DNA target does not require additional cellular components. We further discovered that hnRNP K is a potent suppressor of htk mediated gene activity. However, its mechanism of action is mediated through protein-protein interaction, since hnRNP K itself cannot bind the htk CCRU but can competitively inhibit the binding of other hnRNPs. The binding site for the hnRNPs on the htk CCRU is not required for S-phase induction of the htk promoter. However, in stable but not transient transfectants, the mutation of the hnRNP binding site results in 5- to 10-fold reduction of htk mediated gene activity in synchronized and exponentially growing cells. Collectively, these findings support emerging evidence that hnRNPs, in addition to their traditional role in RNA biogenesis, could be regulators of gene expression through direct DNA binding or interaction with other proteins.     

Heterogeneous nuclear ribonucleoproteins F and H/H' show differential expression in normal and selected cancer tissues

The heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H/H', containing the quasi-RNA recognition motif (qRRM) domains, are implicated in several steps of pre-mRNA processing and in cellular differentiation. We have compared a set of tissues and found striking differences in their levels of expression as well as in the nuclear versus the cytoplasmic distribution. Generally, hnRNP F is broadly expressed in many tissues with extremely strong expression in the prostate gland while hnRNP H/H' shows a more restricted degree of expression with low expression in some tissues, for example, liver, exocrine acini of the pancreas, thyroid gland and heart. At the cellular level, hnRNP F is, with few exceptions, predominantly expressed in the cytoplasm while hnRNP H/H' is more abundant in the nuclei. A quite pronounced heterogeneous expression pattern is seen in the proximal tubules of the kidney where hnRNP F is present at moderate cytoplasmic levels while hnRNP H/H' is undetectable, whereas both proteins are more evenly expressed in distal tubules and collecting ducts. Generally, tumor tissues reveal a broad expression of hnRNP F in the nuclei as well as in the cytoplasm while hnRNP H/H' is expressed at higher levels in the nuclei than in the cytoplasm. Up-regulation of hnRNP H/H' is found in a few tissues that normally express low cytoplasmic levels of hnRNP H/H', for example, adenocarcinoma of the pancreas, hepatocellular carcinoma and gastric carcinoma. hnRNP F is down-regulated in hepatocellular carcinoma and up-regulated in gastric carcinoma. The present study indicates the important potential role of this subset of hnRNPs on the gene expression in many tissues.     

Steroid epimers studied by different mass spectrometric methods

The combined use of different mass spectrometric ionization methods and MS/MS techniques provide the possibility to differentiate between stereoisomers or epimers. In this paper the mass spectral decomposition of 11alpha- and 11beta-substituted estrans was studied. Distinctive stereochemical effects have been observed in their fast atom bombardment product ion spectra. In the electron ionisation (EI) mode, the 5alpha- and 5beta-hydroxylated compounds showed significant differences in the abundance of water elimination. Mass spectrometry has proved to be an effective tool when stereoisomer steroids are compared.     

Phosphorylation of rat liver heterogeneous nuclear ribonucleoproteins A2 and C can be modulated by calmodulin.

It was previously reported that the phosphorylation of three proteins of 36, 40 to 42, and 50 kDa by casein kinase 2 is inhibited by calmodulin in nuclear extracts from rat liver cells (R. Bosser, R. Aligué, D. Guerini, N. Agell, E. Carafoli, and O. Bachs, J. Biol. Chem. 268:15477-15483, 1993). By immunoblotting, peptide mapping, and endogenous phosphorylation experiments, the 36- and 40- to 42-kDa proteins have been identified as the A2 and C proteins, respectively, of the heterogeneous nuclear ribonucleoprotein particles. To better understand the mechanism by which calmodulin inhibits the phosphorylation of these proteins, they were purified by using single-stranded DNA chromatography, and the effect of calmodulin on their phosphorylation by casein kinase 2 was analyzed. Results revealed that whereas calmodulin inhibited the phosphorylation of purified A2 and C proteins in a Ca(2+)-dependent manner, it did not affect the casein kinase 2 phosphorylation of a different protein substrate, i.e., beta-casein. These results indicate that the effect of calmodulin was not on casein kinase 2 activity but on specific protein substrates. The finding that the A2 and C proteins can bind to a calmodulin-Sepharose column in a Ca(2+)-dependent manner suggests that this association could prevent the phosphorylation of the proteins by casein kinase 2. Immunoelectron microscopy studies have revealed that such interactions could also occur in vivo, since calmodulin and A2 and C proteins colocalize on the ribonucleoprotein particles in rat liver cell nuclei.     

Separation and identification of F2-isoprostane regioisomers and diastereomers by novel liquid chromatographic/mass spectrometric methods

Isoprostanes are isomers of prostaglandins that are generated from free radical-initiated autoxidation of arachidonic acid. Quantification of F(2)-isoprostanes is regarded as the "gold standard" to assess oxidative stress in various human diseases. There are 32 possible racemic isoprostane isomers that exist as four sets of regioisomers. Each regioisomer is composed of eight diastereomers. We report liquid chromatographic/mass spectrometric methods to separate and identify F(2)-isoprostane stereoisomers. These methods have been applied to the analysis of F(2)-isoprostanes derived from tissues of rats exposed to an oxidative stress and are useful to assess the relative formation of various regioisomers and stereoisomers generated in vitro and in vivo. The delineation of the more abundant isomers formed will allow for studies to examine the biological relevance of selected compounds in vivo.     

Candidate autoantigens identified by mass spectrometry in early rheumatoid arthritis are chaperones and citrullinated glycolytic enzymes

Introduction  

The aim of our study was to identify new early rheumatoid arthritis (RA) autoantibodies.     

Ultrastructural distribution of nuclear ribonucleoproteins as visualized by immunocytochemistry on thin sections

The ultrastructural distribution of nuclear ribonucleoproteins (RNP) has been investigated by incubation of thin sections of mouse or rat liver, embedded in Lowicryl K4M or prepared by cryoultramicrotomy, with antibodies specific for RNP. The antibodies were localized by means of a protein A-colloidal gold complex. Anti-small nuclear (sn)RNP antibodies, specific for determinants of the nucleoplasmic snRNP species containing U1, U2, U4, U5, and U6 RNAs, were found associated preferentially with perichromatin fibrils, interchromatin granules, and coiled bodies. This indicates an early association of snRNP with structural constituents containing newly synthesized heterogeneous nuclear RNA. It also suggests a possible structural role of some snRNPs in nuclear architecture. Antibodies against the core proteins of heterogeneous nuclear RNP particles associate preferentially with the border regions of condensed chromatin, and in particular with perichromatin fibrils and some perichromatin granules. These results are discussed in view of recent knowledge about the possible role of nucleoplasmic RNP-containing components in the functions of the cell nucleus.     

Disclosure of new and recurrent microbial metabolites by mass spectrometric methods

The application of modern mass spectrometry methods (SI-CID-MS/MS; MS ⁿ ) in the disclosure of new and recurrent microbial metabolites is discussed. Spray ion (SI) sources coupled to different kinds of mass analyzers enable the determination of molecular weights and chemical formulas of given samples even in mixtures. Diagnostic fragment formation by collision-induced dissociation (CID-MS/MS) and MS ⁿ experiments using ion trap mass analyzers are shown as another indispensable source of structural information. Due to the development of benchtop-type mass spectrometers coupled to high-performance liquid chromatography (HPLC), MS can be practised in almost every laboratory as a powerful tool in natural product analysis. Examples are given for special MS applications in identification of bioactive metabolites from screening strains. Journal of Industrial Microbiology & Biotechnology (2001) 27, 136–143. Received 21 September 1999/ Accepted in revised form 19 January 2000     

Human U1 and U2 small nuclear ribonucleoproteins contain common and unique polypeptides.

Immunoprecipitation of human small nuclear ribonucleoproteins (snRNPs) containing the small nuclear RNAs U1, U2, U4, U5, and U6 with two antibodies produced in certain patients suffering from systemic lupus erythematosus was used to identify the polypeptides present on human U1 and U2 snRNPs. U1 and U2 snRNPs contain both common and unique polypeptides; visualization of the differences was possible through the use of non-methionine protein labeling and partial fractionation of snRNP populations. To facilitate comparisons with results from other laboratories, we have designated the snRNP polypeptides by their molecular weights. Four small polypeptides, P8, P9, P10, and P12, of 8,000 to 12,000 daltons, are each present in equal amounts on both U1 and U2 snRNPs. U1 snRNPs also contain a unique 30,000-dalton polypeptide, P30, whereas U2 snRNPs contain a unique 27,000-dalton, methionine-deficient polypeptide, P27. A closely migrating pair of polypeptides, P23 and P22, of 23,000 and 21,500 daltons, respectively, is present on both snRNPs; U2 snRNPs are enriched in the former, and U1 snRNPs are enriched in the latter.     

Fractionation and characterization of human small nuclear ribonucleoproteins containing U1 and U2 RNAs

Human small nuclear ribonucleoproteins (snRNPs) containing U1 and U2 snRNAs have been isolated from cultured cells by nonimmunological methods. The U1 snRNP population remained immunoprecipitable by systemic lupus erythematosis anti-RNP and anti-Sm antibodies throughout fractionation and contained polypeptides of molecular weights corresponding to those defined as U1 snRNP polypeptides by immunoprecipitation of crude extracts. The purified assemblies contained U1 RNA and nine snRNP polypeptides of molecular weights 67,000 (P67), 30,000 (P30), 23,000 (P23), 21,500 (P22), 17,500 (P18), 12,300 (P12), 10,200 (P10), 9,100 (P9), and 8,500 (P8). P67, P30, and P18 were unique to U1 snRNPs. The U2 snRNP population remained immunoprecipitable by the systemic lupus erythematosis anti-Sm antibody throughout fractionation. The purified U2 assemblies contained six polypeptides of molecular weights corresponding to those defined by immunoprecipitation to be common to U1 and U2 snRNPs including P23, P22, P12, P10, P9, and P8. In addition, U2 snRNPs contained a unique polypeptide of 27,000 Da.     

Heterogeneous Nuclear Ribonucleoprotein C1/C2 Controls the Metastatic Potential of Glioblastoma by Regulating PDCD4

MicroRNAs (miRNAs) have been implicated in the pathogenesis and progression of brain tumors. miR-21 is one of the most highly overexpressed miRNAs in glioblastoma multiforme (GBM), and its level of expression correlates with the tumor grade. Programmed cell death 4 (PDCD4) is a well-known miR-21 target and is frequently downregulated in glioblastomas in accordance with increased miR-21 expression. Downregulation of miR-21 or overexpression of PDCD4 can inhibit metastasis. Here, we investigate the role of heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC) in the metastatic potential of the glioblastoma cell line T98G. hnRNPC bound directly to primary miR-21 (pri-miR-21) and promoted miR-21 expression in T98G cells. Silencing of hnRNPC lowered miR-21 levels, in turn increasing the expression of PDCD4, suppressing Akt and p70S6K activation, and inhibiting migratory and invasive activities. Silencing of hnRNPC reduced cell proliferation and enhanced etoposide-induced apoptosis. In support of a role for hnRNPC in the invasiveness of GBM, highly aggressive U87MG cells showed higher hnRNPC expression levels and hnRNPC abundance in tissue arrays and also showed elevated levels as a function of brain tumor grade. Taken together, our data indicate that hnRNPC controls the aggressiveness of GBM cells through the regulation of PDCD4, underscoring the potential usefulness of hnRNPC as a prognostic and therapeutic marker of GBM.     

Binding of an RNA trafficking response element to heterogeneous nuclear ribonucleoproteins A1 and A2

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin. hnRNP A2 bound A2RE in the latter site with a K(d) near 50 nm, whereas the K(d) for hnRNP A1 was above 10 microm. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 microm for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis.     

Heterogeneous nuclear ribonucleoprotein D/AUF1 interacts with heterogeneous nuclear ribonucleoprotein L

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is one of the principal pre-mRNA-binding proteins found in human cells. The hnRNP L protein is fairly abundant. However,it is not restricted to the nucleus, and instead shuttles between the nucleus and the cytoplasm. It is composed of 558 amino acid residues and harbours four loosely conserved RNP-consensus RNA-binding domains.In an attempt to characterize the interaction occurring between cellular proteins and hnRNP L, yeast two-hybrid screening was conducted using a HeLa cDNA library. Some of the cDNA clones were found to harbour a partial human hnRNP D/AUF1 cDNA (GeneBank accession number NM_031369). In this study,we determined that hnRNP L interacts specifically with the hnRNP D/AUF1 in the yeast two-hybrid system. This interaction was verified via an in vitro pull-down assay.