UPA-seq: prediction of functional lncRNAs using differential sensitivity to UV crosslinking.

While a large number of long noncoding RNAs (lncRNAs) are transcribed from the genome of higher eukaryotes, systematic prediction of their functionality has been challenging due to the lack of conserved sequence motifs or structures. Assuming that some lncRNAs function as large ribonucleoprotein complexes and thus are easily crosslinked to proteins upon UV irradiation, we performed RNA-seq analyses of RNAs recovered from the aqueous phase after UV irradiation and phenol-chloroform extraction (UPA-seq). As expected, the numbers of UPA-seq reads mapped to known functional lncRNAs were remarkably reduced upon UV irradiation. Comparison with ENCODE eCLIP data revealed that lncRNAs that exhibited greater decreases upon UV irradiation preferentially associated with proteins containing prion-like domains (PrLDs). Fluorescent in situ hybridization (FISH) analyses revealed the nuclear localization of novel functional lncRNA candidates, including one that accumulated at the site of transcription. We propose that UPA-seq provides a useful tool for the selection of lncRNA candidates to be analyzed in depth in subsequent functional studies.

Generation of novel covalent RNA-protein complexes in cells by ultraviolet B irradiation: implications for autoimmunity.

OBJECTIVE: To determine whether ultraviolet B (UVB) irradiation induces novel modifications in autoantigens targeted during experimental photoinduced epidermal damage. METHODS: To search for novel UVB-induced autoantigen modifications, lysates made from UVB-irradiated human keratinocytes or HeLa cells were immunoblotted using human autoantibodies that recognize ribonucleoprotein autoantigens. Novel autoantigen structures identified were further characterized using nucleases and RNA hybridization. RESULTS: Human sera that recognize U1-70 kd (U1-70K) and La by immunoblotting also recognized multiple novel species when they were used to immunoblot lysates of UVB-irradiated keratinocytes or HeLa cells. These species were not present in control cells and were not observed when apoptosis was induced by Fas ligation or cytotoxic lymphocyte granule contents. Biochemical analysis using multiple assays revealed that these novel UVB-induced molecular species result from the covalent crosslinking between the U1 RNA and the hYRNA molecules with their associated proteins, including U1-70K, La, and likely components of the Sm particle. CONCLUSION: These data demonstrate that UVB irradiation of live cells can directly induce covalent RNA-protein complexes, which are recognized by human autoantibodies. As previously described for other autoantigens, these covalent complexes of RNA and proteins may have important consequences in terms of antigen capture and processing.

Response of a DNA-binding protein to radiation-induced oxidative stress.

The DNA-binding protein MC1 is a chromosomal protein extracted from the archaebacterium Methanosarcina sp. CHTI55. It binds any DNA, and exhibits an enhanced affinity for some short sequences and structures (circles, cruciform DNA). Moreover, the protein bends DNA strongly at the binding site. MC1 was submitted to oxidative stress through gamma-ray irradiation. In our experimental conditions, damage is essentially due to hydroxyl radicals issued from water radiolysis. Upon irradiation, the regular complex between MC1 and DNA disappears, while a new complex appears. In the new complex, the protein loses its ability to recognise preferential sequences and DNA circles, and bends DNA less strongly than in the regular one. The new complex disappears and the protein becomes totally inactivated by high doses.A model has been proposed to explain these experimental results. Two targets, R(1) and R(2), are concomitantly destroyed in the protein, with different kinetics. R(2) oxidation has no effect on the regular binding, whereas R(1) oxidation modifies the functioning of MC1: loss of preferential site and structure recognition, weaker bending. The destruction of both R(1) and R(2) targets leads to a total inactivation of the protein. This model accounts for the data obtained by titrations of DNA with irradiated proteins. When the protein is irradiated in the complex with DNA, bound DNA protects its binding site on the protein very efficiently. The highly oxidisable tryptophan and methionine could be the amino acid residues implicated in the inactivation process.

Fas ligand and Bax gene transcription contributes to Ro60 ribonucleoprotein redistribution in UV-A irradiated human keratinocytes.

OBJECTIVES: Ro ribonucleoproteins are of particular interest because they are serologic markers of photosensitive variants of lupus such as the subacute cutaneous lupus erythematosus (SCLE), in which the polycyclic skin lesions are triggered by exposure to the sun. We study the role of apoptosis in the expression of Ro antigen. METHODS: We used UV-A irradiated keratinocytes. RESULTS: We demonstrate in cultured human UVA-irradiated keratinocytes that the enhanced expression of Ro60 ribonucleoprotein is caused by antigenic redistribution consecutive to Fas-L and Bax gene activation.

Expression of SS-A/Ro and SS-B/La antigens in skin biopsy specimens of patients with photosensitive forms of lupus erythematosus.

CONTEXT: The reason that only some patients with lupus erythematosus (LE) develop autoantibodies to SS-A/Ro and SS-B/La antigens and photosensitivity is unknown. One hypothesis is that both events are related to the level of expression of these antigens in the skin. OBJECTIVE AND DESIGN: To test this hypothesis, we measured the expression of the 52-kd SS-A/Ro, 60-kd SS-A/Ro, and 48-kd SS-B/La antigens in normal sun-protected and sun-exposed skin in 14 patients with LE with photosensitivity, 12 patients with LE without photosensitivity, and 4 normal individuals. The presence of circulating antibodies to these antigens was measured in all patients. SETTING: Outpatient clinic in an academic medical center. RESULTS: We found that the expression of the 52-kd SS-A/Ro, 60-kd SS-A/Ro, and 48-kd SS-B/La antigens in skin biopsy specimens obtained from the same site was 4- to 10-fold higher in patients with LE with photosensitivity than in those patients with LE without photosensitivity (P<.001). Antigen expression was highly correlated with the presence and titer of circulating anti-SS-A/Ro and anti-SS-B/La antibodies (P<.001). CONCLUSIONS: These findings indicate that photosensitivity and the presence and titer of circulating anti-SS-A/Ro and anti-SS-B/La antibodies are both directly correlated with the expression of accessible and immunoreactive SS-A/Ro and SS-B/La antigens in the skin specimens of patients with LE. Thus, the expression of these antigens in keratinocytes may be an important determinant of the development of both SS-A/Ro and SS-B/La autoantibodies and of photosensitive forms of LE.

Ultraviolet B irradiation and cytomegalovirus infection synergize to induce the cell surface expression of 52-kD/Ro antigen.

Cultured human fibroblasts (MRC-5) have been previously demonstrated to express calreticulin, but not Ro autoantigen, on their surface after human cytomegalovirus (CMV) infection. The present study addresses the question of whether other stimuli, alone or in combination with CMV, can induce the surface expression of Ro autoantigens on human fibroblasts. Using a fixed-cell ELISA to detect autoantigen expression, a synergistic effect between ultraviolet B (UVB) exposure and CMV infection on the surface expression of 52-kD/Ro antigen, but not 60-kD/Ro or calreticulin, was observed. The enhanced expression of 52-kD/Ro antigen was significant and specific, compared with untreated cells, cells infected with CMV alone or irradiated with UVB only, and cells subjected to other treatments, such as low pH. Immunofluorescence studies confirmed these findings and indicated that cells expressed 52-kD/Ro protein on their surface at 24 h after a combined UVB and CMV treatment. These studies provide evidence that synergy between UVB irradiation and CMV infection may play a role in the induction of cell surface expression of the human autoantigen, 52-kD/Ro.

Stress-induced cell surface expression and antigenic alteration of the Ro/SSA autoantigen.

Recent studies have shown that Ro/SSA autoantigen is heterogeneous. There are two isoform families; the 60 kD forms (Ro60) and the 52 kD forms (Ro52). Recently we have found that autoantibodies to the Ro/SSA proteins are conformation dependent. Anti-Ro60 antibodies are mainly directed to the native protein and conversely anti-Ro52 antibodies are directed only to the denatured protein. It has been known that UV irradiation to cultured keratinocytes induces cell surface expression of Ro/SSA and this phenomenon has been thought to be related with photosensitivity in patients with anti-Ro/SSA antibodies. We studied the quantitative and qualitative changes of the Ro/SSA protein induced by stress, such as with heat shock and UV irradiation, and found that only Ro52 could be expressed on the cell surface of human peripheral lymphocytes by either heat shock or UV irradiation. Moreover, flow cytometric analysis revealed that HS-treated and UV-treated lymphocytes could be stained with patient sera, and by using a technique which combined immunoprecipitation and Western immunoblotting, it has been confirmed that Ro52 expressed on the cell surface can be recognized by anti-Ro/SSA antibodies in native form while cytoplasmic Ro52 cannot be recognized. These data suggest that Ro52 can be antigenic in vivo when expressed on the cell surface and may explain the mechanism of direct tissue damage by anti-Ro/SSA antibodies.

Human immunodeficiency virus type 1 Rev-responsive element RNA binds to host cell-specific proteins.

RNase protection-gel retention studies show human host cell-specific ribonucleoprotein complexes with human immunodeficiency virus type 1 Rev-responsive element (RRE) RNA. Nuclear proteins from rodent or murine cells appear to lack the ability to form these complexes. Human-mouse somatic cell hybrids retaining a single human chromosome, either 6 or 12, form the RRE-nuclear-protein complexes. One of the complexes requires the entire RRE RNA, while the other needs RRE RNA stem-loops 1 and 2 only. Two major proteins with molecular masses of 120 and 62 kDa specifically bind to RRE RNA. Rodent cells (CHO) either lack or contain small amounts of these RRE-binding proteins.

Impact of ultraviolet radiation on the cellular expression of Ro/SS-A-autoantigenic polypeptides.

Modulation of Ro/SS-A autoantigens in epidermal keratinocytes has been implicated in the pathogenesis of photosensitive forms of Ro/SS-A-antibody-associated cutaneous lupus erythematosus (LE) such as subacute cutaneous LE and neonatal LE. Since Ro/SS-A ribonucleoprotein particles have recently been shown to be very complex molecular structures, we have performed studies to determine whether the expression of three of the Ro/SS-A antigenic polypeptides might be differentially regulated in transformed human epidermal keratinocytes following UVB radiation. Our findings indicate that both total cellular and cell surface levels of calreticulin are upregulated by UVB exposure more so than are either the 52- or 60-kD Ro/SS-A antigens. These results suggest that calreticulin could be a critical component of the Ro/SS-A ribonucleoprotein complex involved in the pathogenesis of Ro/SS-A-antibody-associated LE skin lesions.

Human RNaseP RNA and nucleolar 7-2 RNA share conserved 'To' antigen-binding domains.

RNase P in both prokaryotes and eukaryotes is a ribonucleoprotein that cleaves tRNA precursors to generate the 5' termini of the mature tRNAs. Many patients with autoimmune diseases produce antibodies against a 40 kDa protein (designated To or Th antigen) which is an integral component of eukaryotic RNaseP as well as nucleolar 7-2 RNP which is identical to the mitochondrial RNA processing (MRP) RNP. Interestingly, the To antigen found in human cells and the C5 protein, the only protein component of E. coli RNaseP, are antigenically related. In this study, we show that a 56 nucleotide-long sequence, corresponding to nucleotides 20-75 near the 5' end of human RNaseP RNA, is sufficient to bind the To antigen. We previously showed that the human To antigen binds to a short distinct structural domain near the 5' end of human 7-2/MRP RNA. There is no obvious primary sequence homology between the To antigen binding sites in RNaseP RNA and 7-2/MRP RNA; however, these sequences are capable of assuming a similar secondary structure which corresponds to the recently proposed 'cage' structure for RNaseP RNAs and 7-2/MRP RNA (Forster and Altman (1989) Cell 62: 407-409). These data are supportive of the idea that these two RNAs may have evolved from a common progenitor molecule.

NAB2: a yeast nuclear polyadenylated RNA-binding protein essential for cell viability.

A variety of nuclear ribonucleoproteins are believed to associate directly with nascent RNA polymerase II transcripts and remain associated during subsequent nuclear RNA processing reactions, including pre-mRNA polyadenylation and splicing as well as nucleocytoplasmic mRNA transport. To investigate the functions of these proteins by using a combined biochemical and genetic approach, we have isolated nuclear polyadenylated RNA-binding (NAB) proteins from Saccharomyces cerevisiae. Living yeast cells were irradiated with UV light to covalently cross-link proteins intimately associated with RNA in vivo. Polyadenylated RNAs were then selectively purified, and the covalent RNA-protein complexes were used to elicit antibodies in mice. Both monoclonal and polyclonal antibodies which detect a variety of NAB proteins were prepared. Here we characterize one of these proteins, NAB2. NAB2 is one of the major proteins associated with nuclear polyadenylated RNA in vivo, as detected by UV light-induced cross-linking. Cellular immunofluorescence, using both monoclonal and polyclonal antibodies, demonstrates that the NAB2 protein is localized within the nucleus. The deduced primary structure of NAB2 indicates that it is composed of at least two distinct types of RNA-binding motifs: (i) an RGG box recently described in a variety of heterogeneous nuclear RNA-, pre-rRNA-, mRNA-, and small nucleolar RNA-binding proteins and (ii) CCCH motif repeats related to the zinc-binding motifs of the largest subunit of RNA polymerases I, II, and III. In vitro RNA homopolymer/single-stranded DNA binding studies indicate that although both the RGG box and CCCH motifs bind poly(G), poly(U), and single-stranded DNA, the CCCH motifs also bind to poly(A). NAB2 is located on chromosome VII within a cluster of ribonucleoprotein genes, and its expression is essential for cell growth.

Modification of mRNA-associated proteins during changes in growth conditions.

Following serum stimulation of quiescent 3T6 cells, an elevated in vivo rate of translation was observed. These studies were designed to identify the proteins associated with polysomal mRNA under different growth conditions in an attempt to establish a relationship between translational rate and the mRNA-associated proteins. Ultraviolet cross-linking of proteins to mRNA was employed to ensure that only genuine mRNA-associated proteins were investigated. Our results revealed little change in the population of mRNA-binding proteins, although minor variations in the synthesis of several proteins, most notably a 32 kilodalton species, were observed during growth transitions. These investigations demonstrate further that most of the mRNA-binding proteins were phosphorylated with the degree of phosphorylation of several proteins influenced by growth conditions.

The effects of hormonal and other stimuli on cell-surface Ro/SSA antigen expression by human keratinocytes in vitro: their possible role in the induction of cutaneous lupus lesions.

Ultraviolet B light (UVB) has previously been shown to induce the expression of the extractable nuclear antigens (e.g. Ro/SSA) on the surfaces of human keratinocytes in vitro. This study assessed whether injurious, metabolic, inflammatory, immunological or hormonal stimuli would also induce this expression or modulate that produced by UVB. No stimulus initiated expression alone, but 17-beta oestradiol doubled that found in response to UVB. These findings confirm the potential role of UVB in the initiation and potentiation of cutaneous lupus lesions and may help to explain the female preponderance of the disease.

Ultraviolet radiation (UVR) induces cell-surface Ro/SSA antigen expression by human keratinocytes in vitro: a possible mechanism for the UVR induction of cutaneous lupus lesions.

Antinuclear antibodies are useful markers of connective tissue disease. In this study, UVB but not UVA induced the expression of Ro/SSA antigen on keratinocyte surfaces in vitro. This expression was also found with the extractable nuclear antigens RnP and Sm, but not with single or double-stranded DNA. The expression was prevented by blocking protein synthesis, suggesting that it was an active process. The results suggest that UVB exposure may result in the expression of Ro/SSA antigen on the surfaces of basal keratinocytes in vivo. This antigen could then bind circulating antibody leading to the cutaneous lesions in neonatal and subacute cutaneous lupus erythematosus.

Viral small nuclear ribonucleoproteins bind a protein implicated in messenger RNA destabilization.

Herpesvirus saimiri (HVS) is one of several primate viruses that carry genes for small RNAs. The five H. saimiri-encoded U RNAs (HSURs) are the most abundant viral transcripts expressed in transformed marmoset T lymphocytes. They assemble with host proteins common to spliceosomal small nuclear ribonucleoproteins (snRNPs). HSURs 1, 2, and 5 exhibit sequences at their 5' ends identical to the AUUUA motif, which targets a number of protooncogene, cytokine, and lymphokine mRNAs for rapid degradation. We show that a 32-kDa protein previously demonstrated to bind to the 3' untranslated region of several unstable messages can be UV crosslinked specifically to HSUR 1, 2, and 5 transcripts in vitro, as well as to endogenous HSUR snRNPs. Our results suggest an unusual role for these viral snRNPs: HSURs may function to attenuate the rapid degradation of certain cellular mRNAs, thereby facilitating viral transformation of host T lymphocytes.

A mammalian protein of 220 kDa binds pre-mRNAs in the spliceosome: a potential homologue of the yeast PRP8 protein.

A mammalian protein of approximately 220 kDa (p220) was UV-crosslinked to precursor mRNAs (pre-mRNAs) under splicing conditions. The kinetics and biochemical requirements of the UV-crosslinking of p220 corresponded to the kinetics and biochemical requirements of spliceosome formation. On Western blots, antibodies against the yeast splicing factor PRP8 recognized a doublet of proteins, the faster migrating of which comigrated with p220. Furthermore, UV-crosslinked p220 was immunoprecipitated with anti-PRP8 antisera. These results suggest structural conservation of the splicing factor PRP8 from yeast to mammals and show that this protein is in close proximity to the pre-mRNA in the spliceosome.

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA.

Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region.

Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but did not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed.

U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins.

The U1 small nuclear ribonucleoprotein particle (U1 snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to U1 RNA, using a U1 snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of U1 RNA in reconstituted U1 snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the U1 snRNP, with the 5' end of U1 RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the U1 RNA rather than assembling in the U1 snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to U1 RNA in the absence of bound 70K and Sm core proteins.