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.     

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.     

Nucleic acid binding properties of major proteins from the heterogeneous nuclear ribonucleoproteins of wheat.

Glycine-rich core hnRNP proteins purified from wheat bind tightly to single-stranded but not to double-stranded nucleic acids with a preference for natural RNA over single-stranded DNA. Binding results in i) a progressive disruption of the residual secondary structure of the polynucleotide and the formation of an extended nucleoprotein filament until a protein to polynucleotide weight ratio of about 5:1 is attained. As more protein is added, this is followed by ii) the formation of globular structures along the polynucleotide chain with a concomitant reduction in the contour length of the nucleoprotein complex. These two features of the interaction--unwinding and condensation into beads--are analogous to the previously described behavior of the major glycine-rich core hnRNP protein from Artemia salina (Thomas et al. (1981) Proc. Natl. Acad. Sci. USA 78, 2888) and may represent the basic functional properties of this relatively well conserved group of nuclear proteins.     

The detection of messenger ribonucleic acid sequences in heterogeneous nuclear ribonucleic acid fractions of the oestrogen-stimulated rat uterus.

Active xanthine oxidase was labelled specifically with 33S in the cyanide-labile site of the molybdenum centre. The Very Rapid molybdenum (V) e.p.r. signal, generated from this, shows strong coupling of 33S to molybdenum, providing unambiguous evidence that, at least in the signal-giving species, this sulphur atom is a ligand of molybdenum. The structure of the signal-giving species is discussed.     

The behaviour of heterogeneous nuclear ribonucleic acid in partially and completely denaturing conditions

It is shown that the heterogeneous nuclear RNA (HnRNA) synthesized in the presence of actinomycin and at low and high temperatures sediments in low-ionic-strength sucrose gradients between the rRNA components, similar to the unmethylated RNA synthesized under ;step-down' conditions. If the ionic strength is increased then the HnRNA sediments more rapidly than 28S rRNA, with a large proportion about the 45S precursor rRNA position. Initially this was thought to be due to aggregation of the HnRNA; however, centrifugation and electrophoresis in completely denaturing conditions suggest that the molecular weight of this species of RNA is very large The experiments reveal that HnRNA is conformationally unstable relative to the nucleolar RNA and that the slower sedimentation rate of HnRNA in 5mm-EDTA-Tris base-sucrose gradients reflects the greater expansion of the HnRNA relative to the nucleolar RNA. The implications of this finding are discussed.     

The oestrogen-stimulated synthesis of heterogeneous nuclear ribonucleic acid in the uterus of immature rats

An early response to the administration of oestradiol-17beta to immature rats is the synthesis of uterine RNA of very high-molecular-weight. This RNA is shown to be heterogeneous nuclear RNA. Increased precursor incorporation into the heterogeneous nRNA is not confined to entities of precise molecular weight but appears to involve much of the size range of the species. These findings are discussed with respect to the mode of action of oestradiol.