Protein-RNA crosslinking in the subunits of the cytoplasmic ribosome of Tetrahymena thermophila

Use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to introduce RNA-protein crosslinks in the 40S and 60S subunits of the cytoplasmic ribosome of Tetrahymena thermophila is described, and proteins linked covalently to 17S and 26S ribosomal RNAs are identified. RNA-protein crosslinking is accompanied by extensive dimerization and aggregation of ribosomal subunits probably due to formation of interparticle protein-protein crosslinks.     

Crosslinking of ribosomal protein S18 to 16 S RNA in E.coli ribosomal 30 S subunits by the use of a reversible crosslinking agent: trans-diamminedichloroplatinum(II)

We have previously developed [(1987) Biochemistry 26, 5200-5208] the use of trans-diamminedichloroplatinum(II) to induce reversible RNA-protein crosslinks in the ribosomal 30 S subunit. Protein S18 and, to a lesser extent, proteins S13/S14, S11, S4 and S3 could be crosslinked to the 16 S rRNA. The aim of the present work was to identify the crosslinking sites of protein S18. Three sites could be detected: a major one located in region 825-858, and two others located in regions 434-500 and 233-297. This result is discussed in the light of current knowledge of the topographical localization of S18 in the 30 S subunit and of its relation with function.     

Dynamics of RNA-protein interactions in the HIV-1 Rev-RRE complex visualized by 6-thioguanosine-mediated photocrosslinking.

Expression of the structural proteins of human immunodeficiency virus type 1 (HIV-1) requires the direct interaction of multiple copies of the viral protein Rev with its target RNA, the Rev response element (RRE). RRE is a complex 351-nt RNA that is highly structured and located within the viral env gene. During initial Rev-RRE recognition, Rev binds with high affinity to a bubble structure located within the RRE RNA stem-loop II. We have used a site-specific photocrosslinking method based on 6-thioguanosine (6-thioG) photochemistry to probe the conformation of the high-affinity binding site of RRE RNA and its interactions with Rev protein under physiological conditions. A minimal duplex RNA containing the bubble region of RRE and 12 flanking base pairs was synthesized chemically. Two different RRE constructs with a single photoactive nucleoside (6-thio-dG or 6-thioG) at position 47 or 48 were synthesized. Upon UV irradiation, 6-thioG at both positions formed interstrand covalent crosslinks in RRE RNA. Mapping of crosslink sites by RNA sequencing revealed that 6-thioG at position 47 or 48 crosslinked to A73. In the presence of Rev, both RNA-RNA and RNA-protein crosslinks were observed, however, the RNA-RNA crosslink site was unchanged. Our results provide direct evidence that, during RNA-protein recognition, Rev is in close proximity to O6 of G47 and G48 in the major groove of RRE RNA. Our results also show that the bubble region of RRE RNA has a biologically relevant structure where G47 and G48 are in close proximity to A73 and this RNA structure is not changed significantly upon Rev binding. We propose that Rev protein recognizes and binds to specific structural elements of RRE RNA containing non-Watson-Crick base pairs and such structures could be a determinant for recognition by other RNA-binding proteins. Our site-specific crosslinking methods provide a general approach to capture dynamic states of biologically relevant RNA structures that are otherwise missed by NMR and X-ray crystallographic studies.     

Residue selective crosslinking of proteins through photoactivatable or proximity-enabled reactivity

Photo- and chemical crosslinking of proteins have offered various avenues for studying protein structure and protein interactions with biomolecules. Conventional photoactivatable groups generally lack reaction selectivity toward amino acid residues. New photoactivatable groups reacting with selected residues have emerged recently, increasing crosslinking efficiency and facilitating crosslink identification. Traditional chemical crosslinking usually employs highly reactive functional groups, while recent advance has developed latent reactive groups with reactivity triggered by proximity, which reduce spurious crosslinks and improve biocompatibility. The employment of these residue selective chemical functional groups, activated by light or proximity, in small molecule crosslinkers and in genetically encoded unnatural amino acids is summarized. Together with new software development in identifying protein crosslinks, residue selective crosslinking has enhanced the research of elusive protein-protein interactions in vitro, in cell lysate, and in live cells. Residue selective crosslinking is expected to expand to other methods for the investigation of various protein–biomolecule interactions.     

Genome-wide Mapping of Cellular Protein–RNA Interactions Enabled by Chemical Crosslinking

RNA–protein interactions influence many biological processes. Identifying the binding sites of RNA-binding proteins（RBPs） remains one of the most fundamental and important challenges to the studies of such interactions. Capturing RNA and RBPs via chemical crosslinking allows stringent purification procedures that significantly remove the non-specific RNA and protein interactions. Two major types of chemical crosslinking strategies have been developed to date, i.e., UV-enabled crosslinking and enzymatic mechanism-based covalent capture. In this review, we compare such strategies and their current applications, with an emphasis on the technologies themselves rather than the biology that has been revealed. We hope such methods could benefit broader audience and also urge for the development of new methods to study RNA RBP interactions.     

Actin‐induced dimerization of palladin promotes actin‐bundling

A subset of actin binding proteins is able to form crosslinks between two or more actin filaments, thus producing structures of parallel or networked bundles. These actin crosslinking proteins interact with actin through either bivalent binding or dimerization. We recently identified two binding sites within the actin binding domain of palladin, an actin crosslinking protein that plays an important role in normal cell adhesion and motility during wound healing and embryonic development. In this study, we show that actin induces dimerization of palladin. Furthermore, the extent of dimerization reflects earlier comparisons of actin binding and bundling between different domains of palladin. On the basis of these results we hypothesized that actin binding may promote a conformational change that results in dimerization of palladin, which in turn may drive the crosslinking of actin filaments. The proximal distance between two actin binding sites on crosslinking proteins determines the ultrastructural properties of the filament network, therefore we also explored interdomain interactions using a combination of chemical crosslinking experiments and actin cosedimentation assays. Limited proteolysis data reveals that palladin is less susceptible to enzyme digestion after actin binding. Our results suggest that domain movements in palladin are necessary for interactions with actin and are induced by interactions with actin filaments. Accordingly, we put forth a model linking the structural changes to functional dynamics.     

Genome-wide identification of protein binding sites on RNAs in mammalian cells

RNA-binding proteins (RBPs) are proteins that bind to the RNA and participate in forming ribonucleoprotein complexes. They have crucial roles in various biological processes such as RNA splicing, editing, transport, maintenance, degradation, intracellular localization and translation. The RBPs bind RNA with different RNA-sequence specificities and affinities, thus, identification of protein binding sites on RNAs (R-PBSs) will deeper our understanding of RNA-protein interactions. Currently, high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP, also known as CLIP-Seq) is one of the most powerful methods to map RNA-protein binding sites or RNA modification sites. However, this method is only used for identification of single known RBPs and antibodies for RBPs are required. Here we developed a novel method, called capture of protein binding sites on RNAs (RPBS-Cap) to identify genome-wide protein binding sites on RNAs without using antibodies. Double click strategy is used for the RPBS-Cap assay. Proteins and RNAs are UV-crosslinked in vivo first, then the proteins are crosslinked to the magnetic beads. The RNA elements associated with proteins are captured, reverse transcribed and sequenced. Our approach has potential applications for studying genome-wide RNA-protein interactions.     

Analysis of DNA-protein crosslinking activity of malondialdehyde in vitro

In an attempt to identify endogenous chemicals producing DNA-protein crosslinks, we have studied in vitro crosslinking potential of malondialdehyde, a bifunctional chemical that is ubiquitously formed as a product of lipid peroxidation of polyunsaturated fatty acids. We have found that malondialdehyde readily forms crosslinks between DNA and histones under physiological ionic and pH conditions. Formation of DNA-protein crosslinks was limited to proteins that were able to bind to DNA. Malondialdehyde failed to form DNA-protein crosslinks when histone binding to DNA was prevented by elevated ionic strength or when bovine serum albumin was used in the reaction mixture. Malondialdehyde-produced DNA-histone crosslinks were relatively stable at 37 degrees C with t1/2=13.4 days. Crosslinking of histones to DNA proceeds through the initial formation of protein adduct followed by reaction with DNA. Modification of DNA by malondialdehyde does not lead to a subsequent crosslinking of proteins. Significant formation of DNA-protein crosslinks was also registered in isolated kidney and liver nuclei treated with malondialdehyde. Based on its reactivity and stability of the resulting crosslinks, it is suggested that malondialdehyde could be one of the significant sources of endogenous DNA-protein crosslinks.     

A detailed model of the three-dimensional structure of Escherichia coli 16 S ribosomal RNA in situ in the 30 S subunit

A large body of intra-RNA and RNA-protein crosslinking data, obtained in this laboratory, was used to fold the phylogenetically and experimentally established secondary structure of Escherichia coli 16 S RNA into a three-dimensional model. All the crosslinks were induced in intact 30 S subunits (or in some cases in growing E. coli cells), and the sites of crosslinking were precisely localized on the RNA by oligonucleotide analysis. The RNA-protein crosslinking data (including 28 sites, and involving 13 of the 21 30S ribosomal were used to relate the RNA structure to the distribution of the proteins as determined by neutron scattering. The three-dimensional model of the 16 S RNA has overall dimensions of 220 A x 140 A x 90 A, in good agreement with electron microscopic estimates for the 30 S subunit. The shape of the model is also recognizably the same as that seen in electron micrographs, and the positions in the model of bases localized on the 30 S subunit by immunoelectron microscopy (the 5' and 3' termini, the m7G and m6(2)A residues, and C-1400) correspond closely to their experimentally observed positions. The distances between the RNA-protein crosslink sites in the model correlate well with the distances between protein centres of mass obtained by neutron scattering, only two out of 66 distances falling outside the expected tolerance limits. These two distances both involve protein S13, a protein noted for its anomalous behaviour. A comparison with other experimental information not specifically used in deriving the model shows that it fits well with published data on RNA-protein binding sites, mutation sites on the RNA causing resistance to antibiotics, tertiary interactions in the RNA, and a potential secondary structural "switch". Of the sites on 16 S RNA that have been found to be accessible to chemical modification in the 30 S subunit, 87% are at obviously exposed positions in the model. In contrast, 70% of the sites corresponding to positions that have ribose 2'-O-methylations in the eukaryotic 18 S RNA from Xenopus laevis are at non-exposed (i.e. internal) positions in the model. All nine of the modified bases in the E. coli 16 S RNA itself show a remarkable distribution, in that they form a "necklace" in one plane around the "throat" of the subunit. Insertions in eukaryotic 18 S RNA, and corresponding deletions in chloroplast or mammalian mitochondrial ribosomal RNA relative to E. coli 16 S RNA represent distinct sub-domains in the structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crosslinks between intramolecular pairs of ferritin subunits: effects on both H and L subunits and on immunoreactivity of sheep spleen ferritin

Ferritin is a multisubunit protein, controlling iron storage, with a protein coat composed of 24 subunits (up to three distinct types) in different proportions depending on cell type. Little is known about the subunit interactions in ferritin protein coats composed of heterologous subunits, despite the relevance to ferritin structure and ferritin function (iron uptake and release). Synthetic crosslinking is a convenient way to probe subunit contacts. Crosslinks between subunit pairs in ferritin protein coats are also a natural post-translational modification which coincides with different iron content in ferritin from sheep spleen; ferritin from sheep spleen also contains H and L subunits. Crosslinks synthesized by the reaction of ferritin low in natural crosslinks with difluorodinitrobenzene (F2DNB) reproduced the effects of the natural crosslinks on iron uptake and release. We now extend our observations on the structural effects of natural and synthetic crosslinks to include immunoreactivity of the assembled protein, with monoclonal antibodies as a probe. We also demonstrate, for the first time, ferritin peptides involved in an apparent H- and L-subunit contact: two peptides decreased 4X in cyanogen bromide peptide maps after F2DNB crosslinking were residues L-96-138 and H-66-96; the major DNP-dipeptide was Lys-DNP-Lys. Using the structure of an all L-subunit ferritin as a model, the most likely site for the H-L DNP crosslink is L-Lys 104 (C helix) and H-Lys 67 (B helix). The B helix forms the internal subunit dimer interface, a putative site of iron core nucleation. Alteration by crosslinks of the B helix could, therefore, explain the effect of crosslinks on ferritin iron uptake, release, and iron content.     

A microbead-based system for identifying and characterizing RNA-protein interactions by flow cytometry

We present a high throughput, versatile approach to identify RNA-protein interactions and to determine nucleotides important for specific protein binding. In this approach, oligonucleotides are coupled to microbeads and hybridized to RNA-protein complexes. The presence or absence of RNA and/or protein fluorescence indicates the formation of an oligo-RNA-protein complex on each bead. The observed fluorescence is specific for both the hybridization and the RNA-protein interaction. We find that the method can discriminate noncomplementary and mismatch sequences. The observed fluorescence reflects the affinity and specificity of the RNA-protein interaction. In addition, the fluorescence patterns footprint the protein recognition site to determine nucleotides important for protein binding. The system was developed with the human protein U1A binding to RNAs derived from U1 snRNA but can also detect RNA-protein interactions in total RNA backgrounds. We propose that this strategy, in combination with emerging coded bead systems, can identify RNAs and RNA sequences important for interacting with RNA-binding proteins on genomic scales.     

Kinetic studies of RNA-protein interactions using surface plasmon resonance

Although structural, biochemical, and genetic studies have provided much insight into the determinants of specificity and affinity of proteins for RNA, little is currently known about the kinetics that underlie RNA-protein interactions. Protein-RNA complexes are dynamic, and the kinetics of binding and release could influence many processes, such as the ability of RNA-binding proteins to compete for binding sites, the sequential assembly of ribonucleoprotein complexes, and the ability of bound RNA to move between cellular compartments. Therefore, to attain a complete and biologically relevant understanding of RNA-protein interactions, complex formation must be studied not only in equilibrated reactions, but also as a dynamic process. BIACORE, a surface plasmon resonance-based biosensor technology, allows intermolecular interactions to be measured in real time, and can provide both equilibrium and kinetic information about complex formation. This technology is a powerful tool with which to study the dynamics of RNA-protein interactions. We have used BIACORE extensively to obtain detailed insight into the interaction between RNA and proteins carrying RNA recognition motif domains. Here we discuss the physical principles on which BIACORE is based, and the required instrumentation. We describe how to design well-controlled RNA-protein interaction experiments aimed at yielding high-quality data, and outline the steps required for data analysis. In addition, we present examples to illustrate how kinetic studies have provided us with unique insights into the interaction of the spliceosomal U1A protein and the neuronal HuD protein with their respective RNA targets.     

Analysis of EDTA-chelatable proteins from DNA-protein crosslinks induced by a carcinogenic chromium(VI) in cultured intact human cells

DNA-protein crosslinks (DPCs) were induced in intact human leukemic T-lymphocyte MOLT4 cells or isolated nuclei by treatment with potassium chromate, chromium(III) chloride hexahydrate or x-rays. The proteins complexed to DNA were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE). A group of identical non-histone proteins was crosslinked to DNA by any of the three treatments, except that a 51 kDa basic protein was additionally complexed to DNA when either potassium chromate or chromium(III) chloride hexahydrate was the crosslinking agent. Treatment of chromate-induced DNA-protein crosslinks with EDTA or thiourea followed by ultracentifugation dissociated the major proteins from the complex indicating that these proteins were crosslinked to DNA by direct participation of a EDTA-chelatable form of chromium such as Cr(III) through sulfur containing amino acid residues. The 51 kDa protein was not seen in the post-EDTA pellet but was present in the post-thiourea pellet, indicating that it was also crosslinked to DNA by Cr(III) through non-sulfur-containing amino acids. Digestion of x-rays-induced DPCs by DNase I also revealed this protein on two-dimensional gels indicating that the same protein was also crosslinked by oxidative mechanisms. The involvement of oxidative mechanisms in the crosslinking process was indicated as the majority of the proteins in chromate-induced DPCs were resistant to EDTA and thiourea treatment, and were found to crosslink to DNA when x-rays were used as the crosslinking agent. These results suggest that the chromate-induced DPCs are formed by the generation of reactive oxygen species during the intracellular chromate reduction as well as by the biologically generated Cr(III). About 19% of DNA-protein crosslinks actually involve Cr(III) crosslinking DNA to proteins, about 14% involve Cr(III) crosslinking DNA to proteins through non-sulfhydryl containing moieties and about 5% involve Cr(III) crosslinking DNA to sulfhydryl groups on proteins. The remaining 81% of DNA-protein crosslinks appear to be oxidatively crosslinked out of which about 45% appear to be through sulfhydryl groups and another 36% appear to be through non-sulfhydryl groups.     

Molecular basis of double-stranded RNA-protein interactions: structure of a dsRNA-binding domain complexed with dsRNA.

Protein interactions with double-stranded RNA (dsRNA) are critical for many cell processes; however, in contrast to protein-dsDNA interactions, surprisingly little is known about the molecular basis of protein-dsRNA interactions. A large and diverse class of proteins that bind dsRNA do so by utilizing an approximately 70 amino acid motif referred to as the dsRNA-binding domain (dsRBD). We have determined a 1.9 A resolution crystal structure of the second dsRBD of Xenopus laevis RNA-binding protein A complexed with dsRNA. The structure shows that the protein spans 16 bp of dsRNA, interacting with two successive minor grooves and across the intervening major groove on one face of a primarily A-form RNA helix. The nature of these interactions explains dsRBD specificity for dsRNA (over ssRNA or dsDNA) and the apparent lack of sequence specificity. Interestingly, the dsRBD fold resembles a portion of the conserved core structure of a family of polynucleotidyl transferases that includes RuvC, MuA transposase, retroviral integrase and RNase H. Structural comparisons of the dsRBD-dsRNA complex and models proposed for polynucleotidyl transferase-nucleic acid complexes suggest that similarities in nucleic acid binding also exist between these families of proteins.     

Identification of proteins crosslinked to RNA in 40S ribosomal subunits of Saccharomyces cerevisiae

RNA-protein crosslinks were introduced into the 40S ribosomal subunits from Saccharomyces cerevisiae by mild UV treatment. Proteins crosslinked to the 18S rRNA molecule were separated from free proteins by repeated extraction of the treated subunits and centrifugation in glycerol gradients. After digestion with RNase to remove the RNA molecules, proteins were radio-labeled with ¹²⁵I and identified by electrophoresis on two-dimensional polyacrylamide gels with carrier total 40S ribosomal proteins and autoradiography. Proteins S2, S7, S13, S14, S17/22/27, and S18 were linked to the 18S rRNA. A shorter period of irradiation resulted in crosslinking of S2 and S17/22/27 only. Several of these proteins were previously demonstrated to be present in ribosomal core particles or early assembled proteins.     

The role of RNA sequence and structure in RNA--protein interactions

We investigate the sequence and structural properties of RNA-protein interaction sites in 211 RNA-protein chain pairs, the largest set of RNA-protein complexes analyzed to date. Statistical analysis confirms and extends earlier analyses made on smaller data sets. There are 24.6% of hydrogen bonds between RNA and protein that are nucleobase specific, indicating the importance of both nucleobase-specific and -nonspecific interactions. While there is no significant difference between RNA base frequencies in protein-binding and non-binding regions, distinct preferences for RNA bases, RNA structural states, protein residues, and protein secondary structure emerge when nucleobase-specific and -nonspecific interactions are considered separately. Guanine nucleobase and unpaired RNA structural states are significantly preferred in nucleobase-specific interactions; however, nonspecific interactions disfavor guanine, while still favoring unpaired RNA structural states. The opposite preferences of nucleobase-specific and -nonspecific interactions for guanine may explain discrepancies between earlier studies with regard to base preferences in RNA-protein interaction regions. Preferences for amino acid residues differ significantly between nucleobase-specific and -nonspecific interactions, with nonspecific interactions showing the expected bias towards positively charged residues. Irregular protein structures are strongly favored in interactions with the protein backbone, whereas there is little preference for specific protein secondary structure in either nucleobase-specific interaction or -nonspecific interaction. Overall, this study shows strong preferences for both RNA bases and RNA structural states in protein-RNA interactions, indicating their mutual importance in protein recognition.