Ultraviolet light-induced crosslinking of HnRNA to informofer proteins in vitro

RNA-protein interaction in the 30S subunits of rat liver hnRNP has been studied by crosslinking of informofer proteins to hnRNA induced by UV irradiation.Irradiation of 30S particles with 254 nm UV light in doses of 1×10⁵ erg/mm² leads to the extensive crosslinking hnRNA to informofer proteins. The crosslinked material was analyzed either by resedimentation in a 15–30% sucrose gradient in the presence of 3 M guanidine-HCl and 1 M NaCl or by centrifugation in a Cs₂SO₄ density gradient containing guanidine-HCl and sarkosyl. The crosslinked complexes sedimented at about 25S in the sucrose gradient and proved to be heterogeneous in isopycnic centrifugation experiments. The proteins of the crosslinked complexes were analyzed by polyacrylamide gel electrophoresis. Proteins with M_r values of 70 000, 58 000, 43 000 and 40 000 appeared to be crosslinked with hnRNAs of the 30S particles.In the unirradiated 30S particles after centrifugation in the Cs₂SO₄ density gradient containing guanidine-HCl and sarkosyl two minor proteins were observed with M_r values of 70 000 and 58 000, banded in density zones characteristic for free RNA.     

Ultraviolet light-induced recombination

Stimulation of transduction in $\text{Escherichia coli}$ by ultraviolet irradiation of the transducing phage P1 requires the $\text{uvrA-uvrB}$ nuclease but not the $\text{uvrC}$ product or DNA polymerase I. It is hypothesized that the first step in “normal” recombination can be bypassed by any procedure generating single-stranded ends of DNA (as, for example, by $\text{uvra-uvrB}$ nuclease activity).     

Ultraviolet light-induced changes in ribonuclease conformation

Ultraviolet light-induced inactivation of RNase A is accompanied by a decrease in the circular dichroism (CD) at the extrema observed at 239 nm and 275 nm. As inactivation progresses a new CD band centered near 325 nm also develops. For RNase maintaining up to one half of its original activity an isoelliptic point may be noted near 257 nm. These findings are consistent with initial normalization of a “buried” tyrosyl residue followed by chemical modification of the protein. Further chemical change and extensive conformational reorganization of the protein appear to accompany advanced inactivation.     

Ultraviolet light-induced mutation of diploid human lymphoblasts

Ultraviolet irradiation (254 nm) of immortal diploid human lymphoblasts killed cells, caused mutation at three genetic loci studied, and transiently inhibited ³H-TdR uptake into DNA. A shoulder of about 6 J/m² and a D₀ of 6 J/m² was observed for survival. Mutation rose in a monotonic non-linear fashion through 6 J/m²; above 6 J/m², complex behavior approximating a plateau in induced mutation was observed. Irradiation at 4.4 J/m² caused a transient increase in the number of cells synthesizing DNA and a decrease in the rate of DNA synthesis relative to mock-irradiated controls. The parameter of rate of DNA synthesis per cell in DNA synthetic phase showed a rapid recovery toward control values between 2 and 4 h after irradiation and a slower recovery to control values by 22 h post-irradiation.Fractionated dose schedules were used to measure the effects of allowing a time interval between doses at nontoxic fluences (2.2 j/m²), moderately toxic fluences (8.8 J/m²) and toxic fluences (17.6 J/m²). These measurements indicate that in the non-toxic range of fluences common to human exposure, mutational response is mediated by a post-irradiation process which seems to show to shkow enchanced ability to protect against mutation induced by subsequent irradiation. However, at moderately toxic fluences there was little effect of dose fractionation, and at toxic fluences, a time-dependent increase in mutation fraction was observed at separation times greater than 7 h. We suggest that these latter observations arise primarily from cell cycle heterogeneity with regard to sensitivity to UV killing and mutation.     

Genomic DNA regions whose complementary strands are prone to UV light-induced crosslinking

We prepared an extensive set of DNA restriction fragments, irradiated them with UV light, and detected crosslinked complementary strands by electrophoresis in denaturing agarose gels. These experimental data were quantified by densitometry to determine tetranucleotide contributions to crosslinking. The tetranucleotide contributions were used to predict genomic maps of the crosslinking probability that permitted us to identify two strongly crosslinking genomic regions having 295 and 389 base pairs in length. The two sequences shared the (ATTTTATA).(TATAAAAT) octamer, which is a candidate for the hotspot of UV light-induced crosslinking between the complementary strands of DNA.     

Ultraviolet light-induced inhibition of small nuclear RNA synthesis

Two apparently distinct types of inhibition of the synthesis of U1, U2, U3, U4, and U5 small nuclear RNA, induced by ultraviolet (UV) radiation, have been described before: immediate and delayed. Our present observation can be summarized as follows: a) neither the immediate nor the delayed inhibition appear to be mediated by the formation of cyclobutane pyrimidine dimers, since they were not prevented by photoreactivating light, in ICR 2A frog cells; b) the inhibition of U1 RNA synthesis, monitored in HeLA cells within the first few minutes after irradiation, extrapolated to a substantial suppression at time zero of postirradiation cell incubation, providing further support for the proposal that the immediate inhibition is a reaction separate from the delayed UV light-induced inhibition of U1 RNA synthesis; c) the transition from the pattern of the immediate inhibition to that of the delayed inhibition (disappearance of the UV-resistant fraction of U1 RNA synthesis and increased rate of inhibition) occurred gradually, without an apparent threshold, within the first 2 hr of incubation after irradiation; and d) the incident UV dose that resulted in a 37% level of residual U1 RNA synthesis (D37) during the delayed inhibition was about 7 J/m2, with an apparent UV dose threshold, and was about 60 J/m2 for the immediate inhibition.     

U.V. induced covalent crosslinking of E.coli ribosomal RNA to specific proteins

$\text{E.}\text{coli}$ 70S ribosomes uniformly labeled $\text{in}\text{vivo}$ with ³²PO₄ were subjected to varying doses of u.v. radiation and then to the combined action of the RNases A and T₁. Following these treatments the ribosomal proteins were separated by trichloroacetic acid precipitation from the noncovalently attached RNA degradation fragments. Subsequent two-dimensional gel electrophoresis and autoradiography of these proteins revealed that significant ³²PO₄ was associated with unique ribosomal proteins, L2 was among these.     

Ultraviolet light-induced increase in tumor cell susceptibility to TNF-dependent and TNF-independent natural cell-mediated cytotoxicity

Previously we demonstrated that two consecutive in vitro irradiations of MCA 102 cells with high doses of UVC light (610 and 457 J/m2) resulted in a selection of a permanent line MCA 102UV that manifested high sensitivity to natural cell-mediated cytotoxicity (NCMC). In the present study analysis of the effector cells involved in lysis of these tumor cells was performed by comparing the cytotoxicity of normal spleen cells which mediated both NK and NC cell activity with (a) normal spleen cells in which NC activity was neutralized by anti-TNF Abs (NK+,NC-), (b) NK-depleted or NK-deficient spleen cells (NK-,NC+), and (c) NK-deficient or -depleted spleen cells with NC activity neutralized by anti-TNF Abs (NK-,NC-). Results of these studies indicate that lysis of the original MCA 102 tumor cells was relatively low and was mediated by NC cells. UV irradiation significantly increased MCA 102 tumor cell sensitivity to lysis by both NK and NC cells. Analysis of the mechanisms involved in UV-induced NK sensitivity revealed that UV irradiation increased tumor cell susceptibility to lytic NK-derived granules. NC sensitivity of MCA 102UV tumor cells was associated with their increase in sensitivity to TNF and selection of MCA 102UV cells for resistance to rTNF resulted in a decrease in their susceptibility to NC cells. To determine how fast UV-induced sensitivity to NCMC and rTNF can be established, 51Cr-labeled MCA 102 cells were irradiated in vitro with 38-304 J/m2 of UVC light and their sensitivity to lysis by spleen cells and rTNF was tested immediately in an 18-hr cytotoxicity assay. UV treatment with the same doses was repeated 12 days later. The data obtained showed that tumor cell sensitivity to NCMC and TNF appeared shortly after UV irradiation, was stable, and was further substantially augmented by the second round of UV treatment. Thus, in vitro UV irradiation of tumor cells could be an effective modulator of tumor cell sensitivity to TNF-dependent and TNF-independent cell-mediated cytotoxicity.     

Chemical crosslinking and the stabilization of proteins and enzymes.

The technique of chemical crosslinking has been used to enhance the stability of proteins and enzymes. In this procedure, the molecule is braced with chemical crosslinks either intramolecularly or intermolecularly to another species to reinforce its active structure. Various chemicals have been used for this purpose. The bifunctional reagents are the most prominent. These compounds are derived from group-specific reagents and may be classified into homobifunctional, heterobifunctional, and zero-length crosslinkers. Different physical and chemical characteristics have been incorporated into these chemicals. Their versatility holds great potential in preparing chemically, thermally, and mechanically stable proteins and enzymes for industrial applications.     

Binding of Xenopus oocyte masking proteins to mRNA sequences.

It has been shown previously that maternal mRNA, synthesized and stored in growing oocytes, is stabilized and blocked from translation through various mechanisms including restricted polyadenylation and the binding of proteins to 3' regulatory elements. In addition to binding sequence-specific proteins, the bulk of stored mRNA is packaged with a set of 'masking' proteins, the most abundant of which are the phosphoproteins pp56 and pp60. In this report these proteins are shown to be bound to heterogeneous mRNA sequences and not to the 3' poly(A) tract. Crosslinking studies demonstrate that all of the pp56/60 present makes direct contact with the RNA. In vitro binding studies confirm that pp56/60 interact with single-stranded RNA of heterogeneous sequence, such as occurring in the maternal mRNA encoding cyclin B1. However, binding is equally effective to capped and polyadenylated cyclin mRNA, to truncated mRNA lacking 5' and 3' non-coding regions and even to the antisense sequence. Lengths of 70-80 nucleotides are protected from ribonuclease digestion after protein binding. Although no extended binding motif could be detected, binding does appear to have some specificity in that it is not competed out by 100-fold excess of double-stranded RNA, transfer RNA, poly(A) and various other homopolymers and heteropolymers. The sequence which competes most efficiently is the mixed polypyrimidine, poly(C,U). Crosslinking of RNA-protein complexes, followed by ribonuclease digestion, suggests that the arrangement of proteins on RNA is as dimers. Dimerization appears to be stabilized by phosphorylation of pp56/60. These results are discussed in terms of the known structures of pp56/60.     

Xanthine oxidase-catalyzed crosslinking of cell membrane proteins

Isolated erythrocyte membranes exposed to protease-free xanthine oxidase plus xanthine and ferric iron undergo lipid peroxidation and protein crosslinking (appearance of high molecular weight aggregates on sodium dodecyl sulfate (SDS) gel electrophoresis). Spectrin is more susceptible to crosslinking than the other polypeptides. Thiol-reducible bonds (disulfides) as well as nonreducible bonds are generated, the former type relatively rapidly (detected within 10-20 min) and the latter type more slowly (usually detected after 1 h). Reducible crosslinking is inhibited by catalase, but not by superoxide dismutase, desferrioxamine, butylated hydroxyltoluene, and mannitol; whereas nonreducible crosslinking, like free radical lipid peroxidation, is inhibited by all of these agents except mannitol. Zinc(II) also inhibits lipid peroxidation, but stimulates disulfide bond formation to the virtual exclusion of all other crosslinking. Our results indicate that disulfide formation is dependent on H2O2, but not O2- or iron. However, O2-, H2O2, and iron are all required for lipid peroxidation and nondisulfide crosslinking, suggesting the intermediacy of OH generated via the iron-catalyzed Haber-Weiss reaction. The possible role of malonaldehyde (MDA, a by-product of lipid peroxidation) in the latter type of crosslinking was examined. Solubilized samples of xanthine/xanthine oxidase-treated membranes showed a strong visible fluorescence (emission maximum 450 nm; excitation 390 nm). This resembled the fluorescence of membranes treated with authentic MDA, which forms conjugated imine linkages between amino groups. Fluorescence scanning of SDS gels from MDA-treated membranes showed a strong signal coincident with crosslinked proteins and also one in the low molecular weight, nonprotein region, suggestive of aminolipid conjugates. Similar scanning on xanthine/xanthine oxidase-reacted membranes indicated that all fluorescence is associated with the lipid fraction. Thus, nonreducible protein crosslinks in this system do not appear to be of the MDA-derived, Schiff base type.     

Formation of two-dimensional complexes of F-actin and crosslinking proteins on lipid monolayers: demonstration of unipolar alpha-actinin-F-actin crosslinking.

A method is described for forming two-dimensional (2-D) paracrystalline complexes of F-actin and bundling/gelation proteins on positively charged lipid monolayers. These arrays facilitate detailed structural studies of protein interactions with F-actin by eliminating superposition effects present in 3-D bundles. Bundles of F-actin have been produced using the glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase, the cytoskeletal protein erythrocyte adducin as well as smooth muscle alpha-actinin from chicken gizzard. All of the 2-D bundles formed contain F-actin with a 13/6 helical structure. F-actin-aldolase bundles have an interfilament spacing of 12.6 nm and a superlattice arrangement of actin filaments that can be explained by expression of a local twofold axis in the neighborhood of the aldolase. Well ordered F-actin-alpha-actinin 2-D bundles have an interfilament spacing of 36 nm and contain crosslinks 33 nm in length angled approximately 25-35 degrees to the filament axis. Images and optical diffraction patterns of these bundles suggest that they consist of parallel, unipolar arrays of actin filaments. This observation is consistent with an actin crosslinking function at adhesion plaques where actin filaments are bound to the cell membrane with uniform polarity.     

Ultraviolet light-induced apoptotic death is impaired by the HMG-CoA reductase inhibitor lovastatin

HMG-CoA reductase inhibitors (i.e., statins) attenuate C-terminal isoprenylation of Rho GTPases, thereby inhibiting UV-C-induced activation of c-Jun-N-terminal kinases/stress-activated protein kinases (JNKs/SAPKs). Inhibition of UV-C-triggered JNK/SAPK activation by lovastatin is due to inhibition of Rac-SEK1/MKK4-mediated phosphorylation of JNKs/SAPKs at Thr183/Tyr185. UV-C-stimulated phosphorylation of p38 kinase (Thr180/Tyr182) is also impaired by lovastatin. Cell killing provoked by UV-C irradiation was significantly inhibited by lovastatin. This was paralleled by a reduced frequency of chromosomal aberrations, accelerated recovery from UV-C-induced transient replication blockage, inhibition of Chk1 kinase activation and impaired cyclinB1 expression. Furthermore, UV-C-induced activation of caspases and apoptotic death was largely reduced by lovastatin. Inhibition of JNK/SAPK by transient overexpression of dominant-negative JNK1/SAPK1 also conferred resistance to UV-C light and attenuated activation of caspase 3. Based on the data, we suggest that lovastatin-provoked resistance to UV-C light is due to the inhibition of UV-C-inducible Rac-SEK1/MKK4-JNK/SAPK-dependent signal mechanisms regulating cell cycle progression and activation of caspases and apoptotic death.     

Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA

Macrophages derived from tristetraprolin (TTP)-deficient mice exhibited increased tumor necrosis factor alpha (TNFalpha) release as a consequence of increased stability of TNFalpha mRNA. TTP was then shown to destabilize TNFalpha mRNA after binding directly to the AU-rich region (ARE) of the 3'-untranslated region of the TNFalpha mRNA. In mammals and in Xenopus, TTP is the prototype of a small family of three known zinc finger proteins containing two CCCH zinc fingers spaced 18 amino acids apart; a fourth more distantly related family member has been identified in Xenopus and fish. We show here that representatives of all four family members were able to bind to the TNFalpha ARE in a cell-free system and, in most cases, promote the breakdown of TNFalpha mRNA in intact cells. Because the primary sequences of these CCCH proteins are most closely related in their tandem zinc finger domains, we tested whether various fragments of TTP that contained both zinc fingers resembled the intact protein in these assays. We found that amino- and carboxyl-terminal truncated forms of TTP, as well as a 77 amino acid fragment that contained both zinc fingers, could bind to the TNFalpha ARE in cell-free cross-linking and gel shift assays. In addition, these truncated forms of TTP could also stimulate the apparent deadenylation and/or breakdown of TNFalpha mRNA in intact cells. Alignments of the tandem zinc finger domains from all four groups of homologous proteins have identified invariant residues as well as group-specific signature amino acids that presumably contribute to ARE binding and protein-specific activities, respectively.     

Structure and evolution of the actin crosslinking proteins

The actin crosslinking proteins exhibit marked diversity in size and shape and crosslink actin filaments in different ways. Amino acid sequence analysis of many of these proteins has provided clues to the origin of their diversity. Spectrin, alpha-actinin, ABP-120, ABP-280, fimbrin, and dystrophin share a homologous sequence segment that is implicated as the common actin binding domain. The remainder of each protein consists of repetitive and non-repetitive sequence segments that have been shuffled and multiplied in evolution to produce a variety of proteins that are related in function and in composition, but that differ significantly in structure.     

A colorimetric procedure to monitor the extent of crosslinking of proteins by bisimidoesters

Reaction of proteins with bisimidoesters is one of the popular crosslinking techniques. The extent of the reaction cannot be followed directly using the commonly used free amino group-determining reagents ninhydrin and 2,4,6-trinitrobenzene sulfonic acid since they react with bisimidoesters. However, we found that the extent of amidination of protein could be followed colorimetrically by using 1-fluoro-2,4-dinitrobenzene.