The diverse involvement of heterogeneous nuclear ribonucleoprotein K in mitochondrial response to insulin

Heterogeneous nuclear ribonucleoprotein K (hnRNP K protein) is an RNA/DNA-binding protein that acts in several compartments, including mitochondria. It integrates cellular signaling cascades with multiple processes of gene expression mechanisms. Our studies demonstrate that: (1) insulin activates the import of hnRNP K protein into mitochondria in vitro and in vivo; (2) overexpression of hnRNP K protein modulates insulin-activated mitochondrial gene expression; and (3) insulin treatment stimulates binding of hnRNP K protein to mitochondrial DNA. Based on these and our previously reported results we conclude that hnRNP K protein may be a mediator of mitochondrial response to insulin.     

African swine fever virus protein p30 interaction with heterogeneous nuclear ribonucleoprotein K (hnRNP-K) during infection

Heterogeneous nuclear ribonucleoprotein K (hnRNP-K) was identified as interacting cellular protein with the abundant immediate early protein p30 from African swine fever virus (ASFV) in a macrophage cDNA library screening. The interacting regions of hnRNP-K with p30 were established within residues 35-197, which represent KH1 and KH2 domains responsible for RNA binding. Colocalization of hnRNP-K and p30 was observed mainly in the nucleus, but not in the cytoplasm of infected cells and infection modified hnRNP-K subcellular distribution and decreased the incorporation of 5-fluorouridine into nascent RNA. Since similar effects were observed in cells transiently expressing p30, this interaction provides new insights into p30 function and could represent a possible additional mechanism by which ASFV downregulates host cell mRNA translation.     

Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity electrophoresis

The phosphorylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is thought to play an important role in cell regulation and signal transduction. However, the relationship between hnRNP K phosphorylation and cellular events has only been indirectly examined, and the phosphorylated forms of endogenous hnRNP K have not been biochemically characterized in detail. In this study, we extensively examined the phosphorylated forms of endogenous hnRNP K by direct protein-chemical characterization using phosphate-affinity electrophoresis followed by immunoblotting and MS. Phosphate-affinity electrophoresis enabled us to sensitively detect and separate the phosphorylated forms of hnRNP K. When we used 2-DE with phosphate-affinity SDS-PAGE in the second dimension, the nuclear fraction contained more than 20 spots of endogenous hnRNP K on the 2-D map. We determined that the multiple forms of hnRNP K were produced mainly by alternative splicing of the single hnRNP K gene and phosphorylation of Ser116 and/or Ser284. Furthermore, the subcellular localization of these proteins revealed by the 2-D gel correlated with their phosphorylation states and alternative splicing patterns. The results also indicated that the multiple forms of hnRNP K were differentially modulated in response to external stimulation with bacterial lipopolysaccharide or serum.     

Limited nuclease digestion of heterogeneous nuclear ribonucleoprotein in nuclei.

We have used limited nuclease digestion of nuclei to probe the structure of nuclear ribonucleoprotein (nRNP). Analysis of [³H]uridine-labeled heterogeneous nuclear RNA isolated from nuclease digested nuclei revealed preferential generation of discrete bands of RNA ranging in size from 1.5 × 10⁵ to 6 × 10⁵ daltons. The nuclease digestion pattern of nRNP differed from the nuclease digestion pattern obtained with chromatin in that the RNA bands generated in these experiments were transient, appearing only early in the course of digestion, and no stable nRNP monomer size was evident. Therefore, although nRNP may be organized in a regular configuration, nRNP structure differs considerably from the repeating subunit structure of chromatin.     

Detection of beta-globin mRNA precursor in heterogeneous nuclear ribonucleoprotein associated with U1-RNP by using anti-RNP antibody

Heterogeneous nuclear RNA-ribonucleoprotein (hnRNP) fractions were isolated from Friend erythroleukemia cells and separated by 15-45% sucrose gradient centrifugation. The distribution of small nuclear RNAs (snRNAs) in hnRNP fractions indicated that the snRNAs are associated with hnRNP particles. HnRNP fractions were incubated with normal IgG or anti-U1 RNP IgG, and the resulting immunocomplexes were isolated by binding to a protein A-Sepharose column. HnRNP was found in bound fractions only when anti-U1 RNP IgG was used. By Northern hybridization of RNA extracted from the immunocomplexes with a beta-globin genomic DNA probe, 15S beta-globin mRNA precursors and 10S mature mRNA were detected. These findings suggest the existence of a complex of U1 RNP particles and hnRNP particles containing beta-globin pre-mRNA.     

Involvement of p38 kinase in hydroxyurea-induced differentiation of K562 cells.

Hydroxyurea is a differentiation-inducing agent of human erythroleukemia K562 cells. However, the cellular mechanisms by which hydroxyurea exerts its effects on tumor cells, leading to the inhibition of cell growth and the induction of differentiation markers, are largely unknown. This study examined the role of different mitogen-activated protein kinase signal transduction pathways in hydroxyurea-induced erythroid differentiation of K562 cells. Using a panel of anti-extracellular signal-related kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 phosphospecific antibodies, we demonstrated that phosphorylation of ERK and JNK is decreased after the treatment of cells with hydroxyurea, whereas phosphorylation of p38 is increased. Moreover, inhibition of ERK activity by PD98059 induced erythroid differentiation, and it acted synergistically with hydroxyurea on hemoglobin synthesis, whereas inhibition of p38 activity by SB203580 inhibited induction of hemoglobin production by hydroxyurea. These findings suggest that the activation of p38 kinase may play important roles in the signal transduction mechanisms of hydroxyurea leading to erythroid differentiation.     

Identification of carbohydrate binding protein 35 in heterogeneous nuclear ribonucleoprotein complex

In previous studies, a lectin designated as carbohydrate binding protein 35 (CBP35) was identified in the nucleus and cytoplasm of cultured mouse 3T3 fibroblasts. In the present study, we observed that treatment of Triton X-100 permeabilized 3T3 cells with ribonuclease A released CBP35 from the nuclei, while parallel treatment with deoxyribonuclease I failed to do so. This conclusion was based on (a) immunofluorescence analysis of the nuclear residue after detergent and enzymatic treatments and (b) immunoblotting analysis of the supernatant fraction produced by these treatments. These results indicate that CBP35 may be associated with the ribonucleoprotein elements of the 3T3 cell nuclei. In corroboration with this conclusion, fractionation of the nucleoplasm derived from 3T3 cells on a cesium sulfate gradient (1.25-1.75 g/mL) localized CBP35 in fractions with densities of 1.30-1.32 g/mL, corresponding to the range of densities reported for heterogeneous nuclear ribonucleoprotein complex (hnRNP). Conversely, when nucleoplasm was fractionated on an affinity column of Sepharose derivatized with N-(epsilon-aminocaproyl)-D-galactosamine, the bound and eluted fraction contained RNA, as well as a set of polypeptides whose molecular weights matched those reported for the core particle of hnRNP. One of these polypeptides was identified as CBP35. These results suggest that CBP35 is a component of hnRNP.     

Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein.

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.