An RNA-protein contact determined by 5-bromouridine substitution, photocrosslinking and sequencing.

An analogue of the replicase translational operator of bacteriophage R17, that contains a 5-bromouridine at position -5 (RNA 1), complexes with a dimer of the coat protein and photocrosslinks to the coat protein in high yield upon excitation at 308 nm with a xenon chloride excimer laser. Tryptic digestion of the crosslinked nucleoprotein complex followed by Edman degradation of the tryptic fragment bearing the RNA indicates crosslinking to tyrosine 85 of the coat protein. A control experiment with a Tyr 85 to Ser 85 variant coat protein showed binding but no photocrosslinking at saturating protein concentration. This is consistent with the observation from model compound studies of preferential photocrosslinking of BrU to the electron rich aromatic amino acids tryptophan, tyrosine, and histidine with 308 nm excitation.     

Nucleoside and nucleotide inactivation of R17 coat protein: evidence for a transient covalent RNA-protein bond

R17 coat protein forms a specific complex with a 21-nucleotide RNA hairpin containing the initiation site for the phage replicase gene. The RNA binding activity of the protein is inhibited by prior incubation with 5-bromouridine (BrU). The inactivation occurs with pseudo-first-order kinetics, and the inactive protein is stable to dilution. RNA binding activity of the BrU-inactivated protein is restored upon incubation with dithiothreitol. Inactivation of coat protein by N-ethylmaleimide or p-(chloromercuri)-benzenesulfonate indicates that a cysteine residue is located near the RNA binding site. Since 5-bromodeoxyuridine does not inactivate coat protein, a specific binding event appears to be required before inactivation can occur. Surprisingly, unmodified cytidine nucleotides also inactivate coat protein, with a specificity similar to the modified analogues. These results are discussed with regard to the formation of a transient covalent RNA-protein bond.     

Direct cross-linking of snRNP proteins F and 70K to snRNAs by ultra-violet radiation in situ.

Protein-RNA interactions in small nuclear ribonucleoproteins (UsnRNPs) from HeLa cells were investigated by irradiation of purified nucleoplasmic snRNPs U1 to U6 with UV light at 254 nm. The cross-linked proteins were analyzed on one- and two-dimensional gel electrophoresis systems, and the existence of a stable cross-linkage was demonstrated by isolating protein-oligonucleotide complexes from snRNPs containing 32P-labelled snRNAs after exhaustive digestion with a mixture of RNases of different specificities. The primary target of the UV-light induced cross-linking reaction between protein and RNA was protein F. It was also found to be cross-linked to U1 snRNA in purified U1 snRNPs. Protein F is known to be one of the common snRNP proteins, which together with D, E and G protect a 15-25 nucleotide long stretch of snRNAs U1, U2, U4 and U5, the so-called domain A or Sm binding site against nuclease digestion (Liautard et al., 1982). It is therefore likely that the core-protein may bind directly and specifically to the common snRNA domain A, or else to a sub-region of this. The second protein which was demonstrated to be cross-linked to snRNA was the U1 specific protein 70K. Since it has been shown that binding of protein 70K to U1 RNP requires the presence of the 5' stem and loop of U1 RNA (Hamm et al., 1987) it is likely that the 70K protein directly interacts with a sub-region of the first stem loop structure.     

紫外交联免疫沉淀技术原理及其应用

紫外交联免疫沉淀（UV cross-linking immunoprecipitation,CLIP）技术最初建立于2003年。通过紫外交联、免疫沉淀、逆转录及后续的高通量测序等步骤,可在全转录组范围鉴定特定RNA结合蛋白（RNA-binding proteins,RBP）的靶标RNA序列和结合位点。在近20年的应用过程中,该技术被不断改进和完善,可操作性、实验结果的准确性都有所提升,技术的应用范围也有所拓展。本文对CLIP技术的基本原理、实验方法、实际应用进行介绍,着重比较几种主流CLIP技术的异同,并对如何选择具体的技术路线提出建议。     

Probing Tat peptide-TAR RNA interactions by psoralen photo-cross-linking

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. We have used a site-specific cross-linking method based on psoralen photochemistry to determine the effect of core residues from the Tat sequence on the protein orientation in the Tat-TAR complex and on the specificity of Tat-TAR binding. We synthesized two Tat fragments, Tat(42-72) and Tat(37-72), and incorporated a psoralen-modified amino acid at position 41 during solid-phase assembly of the peptides. We used these psoralen-Tat conjugates to form specific complexes with TAR RNA. Upon near-ultraviolet irradiation (360 nm), psoralen-Asp41-Tat(37-72) cross-linked to a single site in the TAR RNA sequence. The RNA-protein complex was purified and the cross-link site on TAR RNA was determined by primer extension analysis, which revealed that Asp41 of Tat is close to U42 of the lower stem region of TAR RNA. Specificity of the RNA-peptide cross-linking reactions was determined by competition experiments. Our results show that the addition of only four residues (Cys37-Thr40) from the Tat core region significantly enhanced the specificity of the Tat peptide-TAR interactions without altering the site or chemical nature of the cross-link. These studies provide new insights into RNA-protein recognition that could be useful in designing peptidomimetics for RNA targeting. Such psoralen-peptide conjugates provide a new class of probes for sequence-specific protein-nucleic acid interactions and could be used to selectively control gene expression or to induce site-directed mutations.     

Specific interaction of HeLa cell proteins with coxsackievirus B3 3'UTR: La autoantigen binds the 3' and 5'UTR independently of the poly(A) tail

Coxsackievirus B3 (CVB3) is a positive, single-stranded RNA virus. The secondary structure of the 3' untranslated region (3'UTR) of CVB3 RNA consists of three stem-loops and is followed by a poly(A) tail sequence. These stem-loop structures have been suggested to participate in the regulation of viral replication through interaction with cellular proteins that are yet to be identified. In this study, by competitive UV cross-linking using mutated 3'UTR probes we have demonstrated that the poly(A) tail is essential for promoting HeLa cell protein interactions with the 3'UTR because deletion of this sequence abolished most of the protein interactions. Unexpectedly, mutations that disrupted the tertiary loop-loop interactions without affecting the stem-loops did not apparently affect these protein interactions, indicating that secondary structure rather than the high-order structure may play a major role in recruiting these RNA binding proteins. Among the observed 3'UTR RNA binding proteins, we have confirmed a 52 kDa protein as the human La autoantigen by using purified recombinant protein and a polyclonal La antibody. This protein can interact with both the 3' and 5'UTRs independently of the poly(A) tail. Further analysis by two-stage UV cross-linking, we found that the 3' and 5'UTR sequences may share the same binding site on the La protein.     

Identification of domains in brome mosaic virus RNA-1 and coat protein necessary for specific interaction and encapsidation.

Even though many single-stranded RNAs are present in the cytoplasm of infected cells, encapsidation by brome mosaic virus (BMV) coat protein is specific for BMV RNA. Although the highly conserved 3' region of each of the three BMV genomic RNAs is an attractive candidate for the site of recognition by the coat protein, band shift and UV cross-linking assays in the presence of specific and nonspecific competitors revealed only nonspecific interactions. However, BMV RNA-1 formed a retarded complex (complex I) with the coat protein in the absence of competitors, and two domains of RNA-1 that specifically bound coat protein in a small complex (complex II), presumably early in the encapsidation process, were identified. Strong nonspecific, cooperative binding was observed in the presence of high concentrations of coat protein, suggesting that this provides the mechanism leading to rapid encapsidation seen in vivo. In contrast, no binding to a coat protein mutant lacking the N-terminal 25 amino acids that has been shown to be incapable of encapsidation in vivo (R. Sacher and P. Ahlquist, J. Virol. 63:4545-4552, 1989) was detected in vitro. The use of deletion mutants of RNA-1 revealed the presence of domains within the coding region of protein 1a that formed complexes with purified coat protein. One deletion mutant (B1SX) lacking these domains was only slightly more effective in dissociating RNA-1-coat protein complexes than were nonspecific competitors, further suggesting that regions other than the 3' end can participate in the selective encapsidation of BMV RNAs.     

Selection of high affinity RNA ligands to the bacteriophage R17 coat protein.

RNA ligands with high affinity for the bacteriophage R17 coat protein were isolated from a pool of random RNA molecules using SELEX. Of the 38 ligands isolated, 36 were found to contain a hairpin very similar to the naturally occurring coat protein binding site in the R17 genome. The common features of these 36 sequences provide a consensus binding site and predict components of a hairpin that promote favorable interaction with the coat protein. These include a tetraloop of primary sequence AUCA and a variable-length stem with a bulged adenosine residue at a specific stem position. The predicted consensus agrees well with the highest-affinity RNA binding site of the R17 coat protein, identified through classical but laborious techniques. These results demonstrate the value of SELEX as a tool for isolating high affinity RNA ligands to a specific target protein, and the further value of those ligands to point the researcher toward natural sequences for that target protein.     

A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: application to iron regulatory protein.

We describe a new procedure to identify RNA or DNA binding sites in proteins, based on a combination of UV cross-linking and single-hit chemical peptide cleavage. Site-directed mutagenesis is used to create a series of mutants with single Asn-Gly sequences in the protein to be analysed. Recombinant mutant proteins are incubated with their radiolabelled target sequence and UV irradiated. Covalently linked RNA- or DNA-protein complexes are digested with hydroxylamine and labelled peptides identified by SDS-PAGE and autoradiography. The analysis requires only small amounts of protein and is achieved within a relatively short time. Using this method we mapped the site at which human iron regulatory protein (IRP) is UV cross-linked to iron responsive element RNA to amino acid residues 116-151.     

Characterization of Escherichia coli SecA protein binding to a site on its mRNA involved in autoregulation.

In order to understand further the autogenous regulation of Escherichia coli secA translation, we have set up a purified system to study the binding of SecA protein to portions of its mRNA. Specific SecA protein-RNA binding was demonstrated by UV cross-linking, filter binding, and gel shift assays. Use of the filter binding assay allowed optimization of binding, which was influenced by Mg2+ and ATP concentrations, and a measurement of the affinity of this interaction. A nested series of RNAs lacking either 5' or 3' portions of geneX-secA sequences were used to localize the SecA protein binding site to sequences around the geneX-secA intergenic region. These studies imply that SecA protein directly regulates its own translation by a specific RNA binding activity that presumably blocks translational initiation.     

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.     

Ribosomal protein S5 interacts with the internal ribosomal entry site of hepatitis C virus

Translational initiation of hepatitis C virus (HCV) genome RNA occurs via its highly structured 5' noncoding region called the internal ribosome entry site (IRES). Recent studies indicate that HCV IRES and 40 S ribosomal subunit form a stable binary complex that is believed to be important for the subsequent assembly of the 48 S initiation complex. Ribosomal protein (rp) S9 has been suggested as the prime candidate protein for binding of the HCV IRES to the 40 S subunit. RpS9 has a molecular mass of approximately 25 kDa in UV cross-linking experiments. In the present study, we examined the approximately 25-kDa proteins of the 40 S ribosome that form complexes with the HCV IRES upon UV cross-linking. Immunoprecipitation with specific antibodies against two 25-kDa 40 S proteins, rpS5 and rpS9, clearly identified rpS5 as the protein bound to the IRES. Thus, our results support rpS5 as the critical element in positioning the HCV RNA on the 40 S ribosomal subunit during translation initiation.     

Detection of a Novel ATP-Dependent Cross-Linked Protein at the 5′ Splice Site-U1 Small Nuclear RNA Duplex by Methylene Blue-Mediated Photo-Cross-Linking

Assembly of spliceosomes involves a number of sequential steps in which small nuclear ribonucleoprotein particles (snRNPs) and some non-snRNP proteins recognize the splice site sequences and undergo various conformational rearrangements. A number of important intermolecular RNA-RNA duplexes are formed transiently during the process of splice site recognition. Various steps in the assembly pathway are dependent upon ATP hydrolysis, either for protein phosphorylation or for the activity of helicases, which may modulate the RNA structures. Major efforts have been made to identify proteins that interact with specific regions of the pre-mRNA during the stages of spliceosome assembly and catalysis by site-specific UV cross-linking. However, UV cross-linking is often inefficient for the detection of proteins that interact with base-paired RNA. Here we have used the complementary approach of methylene blue-mediated photo-cross-linking to detect specifically proteins that interact with the duplexes formed between pre-mRNA and small nuclear RNA (snRNA). We have detected a novel cross-link between a 65-kDa protein (p65) and the 5′ splice site. A range of data suggest that p65 cross-links to the transient duplex formed by U1 snRNA and the 5′ splice site. Moreover, although p65 cross-linking requires only a 5′ splice site within the pre-mRNA, it also requires ATP hydrolysis, suggesting that its detection reflects a very early ATP-dependent event during splicing.     

Analysis of multiple forms of nuclear factor I in human and murine cell lines.

Nuclear factor I (NFI) is a group of related site-specific DNA-binding proteins that function in adenovirus DNA replication and cellular RNA metabolism. We have measured both the levels and forms of NFI that interact with a well-characterized 26-base-pair NFI-binding site. Five different NFI-DNA complexes were seen in HeLa nuclear extracts by using a gel mobility shift (GMS) assay. In addition, at least six forms of NFI were shown to cross-link directly to DNA by using a UV cross-linking assay. The distinct GMS complexes detected were composed of different subspecies of NFI polypeptides as assayed by UV cross-linking. Different murine cell lines possessed varying levels and forms of NFI binding activity, as judged by nitrocellulose filter binding and GMS assays. The growth state of NIH 3T3 cells affected both the types of NFI-DNA complexes seen in a GMS assay and the forms of the protein detected by UV cross-linking.     

Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site

The interaction between phage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA--protein interaction. Nuclease protection and selection experiments define the binding site to about 20 contiguous nucleotides which form a hairpin. A nitrocellulose filter retention assay is used to show that the binding between the coat protein and a synthetic 21-nucleotide RNA fragment conforms to a simple bimolecular reaction. Unit stoichiometry and a Kd of about 1 nM are obtained at 2 degrees C in buffer containing 0.19 M salt. The interaction is highly sequence specific since a variety of RNAs failed to compete with the 21-nucleotide fragment for coat protein binding.     

Specific RNA binding by Q beta coat protein

The interaction between the bacteriophage Q beta coat protein and its specific binding site on Q beta genomic RNA was characterized by using a nitrocellulose filter binding assay. Q beta coat protein bound to a synthetic 29-nucleotide RNA hairpin with an association constant of 400 microM-1 at 4 degrees C, 0.2 M ionic strength, pH 6.0. Complex formation had a broad pH optimum centered around pH 6.0 and was favored by both enthalpy and entropy. The salt dependence of Ka revealed that four to five ion pairs may be formed in the complex although approximately 80% of the free energy of complex formation is contributed by nonelectrostatic interactions. Truncation experiments revealed that coat protein binding required only the presence of a hairpin with an eight base pair stem and a three-base loop. Analysis of the binding properties of hairpin variants showed that the sequence of the stem was not important for coat protein recognition and only one of the three loop residues was essential. A bulged adenosine present in the coat protein binding site was not required for coat protein binding. Q beta coat protein binding specific is therefore primarily achieved by the structure and not by the sequence of the operator.     

Cooperative binding of R17 coat protein to RNA.

The binding of the R17 coat protein to synthetic RNAs containing one or two coat protein binding sites was characterized by using nitrocellulose filter and gel-retention assays. RNAs with two available sites bound coat protein in a cooperative manner, resulting in a higher affinity and reduced sensitivity to pH, ionic strength, and temperature when compared with RNAs containing only a single site. The cooperativity can contribute up to -5 kcal/mol to the overall binding affinity with the greatest cooperativity found at low pH, high ionic strength, and high temperatures. Similar solution properties for the encapsidation of the related fr and f2 phage suggest that the cooperativity is due to favorable interactions between the two coat proteins bound to the RNA. This system therefore resembles an intermediate state of phage assembly. No cooperative binding was observed for RNAs containing a single site and a 5' or 3' extension of nonspecific sequence, indicating that R17 coat protein has a very low nonspecific binding affinity. Unexpectedly weak binding was observed for several RNAs due to the presence of alternative conformational states of the RNA.