Partial RNase Digestion Research Articles

Genome-Wide Profiling of RNA-Protein Interactions Using CLIP-Seq.

UV crosslinking immunoprecipitation (CLIP) is an increasingly popular technique to study protein-RNA interactions in tissues and cells. Whole cells or tissues are ultraviolet irradiated to generate a covalent bond between RNA and proteins that are in close contact. After partial RNase digestion, antibodies specific to an RNA binding protein (RBP) or a protein-epitope tag is then used to immunoprecipitate the protein-RNA complexes. After stringent washing and gel separation the RBP-RNA complex is excised. The RBP is protease digested to allow purification of the bound RNA. Reverse transcription of the RNA followed by high-throughput sequencing of the cDNA library is now often used to identify protein bound RNA on a genome-wide scale. UV irradiation can result in cDNA truncations and/or mutations at the crosslink sites, which complicates the alignment of the sequencing library to the reference genome and the identification of the crosslinking sites. Meanwhile, one or more amino acids of a crosslinked RBP can remain attached to its bound RNA due to incomplete digestion of the protein. As a result, reverse transcriptase may not read through the crosslink sites, and produce cDNA ending at the crosslinked nucleotide. This is harnessed by one variant of CLIP methods to identify crosslinking sites at a nucleotide resolution. This method, individual nucleotide resolution CLIP (iCLIP) circularizes cDNA to capture the truncated cDNA and also increases the efficiency of ligating sequencing adapters to the library. Here, we describe the detailed procedure of iCLIP.

Conformational-dependent and independent RNA binding to the fragile x mental retardation protein.

The interaction between the fragile X mental retardation protein (FMRP) and BC1 RNA has been the subject of controversy. We probed the parameters of RNA binding to FMRP in several ways. Nondenaturing agarose gel analysis showed that BC1 RNA transcripts produced by in vitro transcription contain a population of conformers, which can be modulated by preannealing. Accordingly, FMRP differentially binds to the annealed and unannealed conformer populations. Using partial RNase digestion, we demonstrate that annealed BC1 RNA contains a unique conformer that FMRP likely binds. We further demonstrate that this interaction is 100-fold weaker than that the binding of eEF-1A mRNA and FMRP, and that preannealing is not a general requirement for FMRP's interaction with RNA. In addition, binding does not require the N-terminal 204 amino acids of FMRP, methylated arginine residues and can be recapitulated by both fragile X paralogs. Altogether, our data continue to support a model in which BC1 RNA functions independently of FMRP.

Structural Characterization of the Rous Sarcoma Virus RNA Stability Element

In eukaryotic cells, an mRNA bearing a premature termination codon (PTC) or an abnormally long 3' untranslated region (UTR) is often degraded by the nonsense-mediated mRNA decay (NMD) pathway. Despite the presence of a 5- to 7-kb 3' UTR, unspliced retroviral RNA escapes this degradation. We previously identified the Rous sarcoma virus (RSV) stability element (RSE), an RNA element downstream of the gag natural translation termination codon that prevents degradation of the unspliced viral RNA. Insertion of this element downstream of a PTC in the RSV gag gene also inhibits NMD. Using partial RNase digestion and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, we determined the secondary structure of this element. Incorporating RNase and SHAPE data into structural prediction programs definitively shows that the RSE contains an AU-rich stretch of about 30 single-stranded nucleotides near the 5' end and two substantial stem-loop structures. The overall secondary structure of the RSE appears to be conserved among 20 different avian retroviruses. The structural aspects of this element will serve as a tool in the future design of cis mutants in addressing the mechanism of stabilization.

Photochemical cross-linking of tRNA1Arg to the 30S ribosomal subunit using aryl azide reagents attached to the anticodon loop.

The 2-thiocytidine residue at position 32 of tRNA1Arg from Escherichia coli was modified specifically with three photoaffinity reagents of different lengths, and the corresponding N-acetylarginyl-tRNA1Arg derivatives were cross-linked to the P site of E. coli 70S ribosomes by irradiation. Covalent attachment was dependent upon the presence of a polynucleotide template and exposure to light of the appropriate wavelength. From 4% to 6% of the noncovalently bound tRNA became cross-linked to the ribosome as a result of photolysis, and attachment to the P site was confirmed by the reactivity of arginine in the covalent complexes toward puromycin. Analysis of the irradiated ribosomes by sucrose-gradient sedimentation at low Mg2+ concentration revealed that the tRNA was associated exclusively with the 30S subunit in all cases. Two of the N-acetylarginyl-tRNA1Arg derivatives were attached primarily to ribosomal proteins whereas the third was cross-linked mainly to 16S RNA. Partial RNase digestion of the latter complex demonstrated that the tRNA had become attached to the 3' third of the rRNA molecule. In addition, the tRNA-rRNA bond was shown to be susceptible to cleavage by hydroxylamine and mercaptoethanol.

Probing the structure of 16 S ribosomal RNA from Bacillus brevis.

A majority (approximately 89%) of the nucleotide sequence of Bacillus brevis 16 S rRNA has been determined by a combination of RNA sequencing methods. Several experimental approaches have been used to probe its structure, including (a) partial RNase digestion of 30 S ribosomal subunits, followed by two-dimensional native/denatured gel electrophoresis, in which base-paired fragments were directly identified; (b) identification of positions susceptible to cleavage by RNase A and RNase T1 in 30 S subunits; (c) sites of attack by cobra venom RNase on naked 16 S rRNA; and (d) nucleotides susceptible to attack by bisulfite in 16 S rRNA. These data are discussed with respect to a secondary structure model for B. brevis 16 S rRNA derived by comparative sequence analysis.

The nucleotide sequence of a methionine tRNA which functions in protein elongation in mouse myeloma cells.

The major form of methionine tRNA operational in the elongation of protein synthesis in mouse myeloma cells was purufied from these cells after they had been cultured in the presence of [32P]-phosphate. This [32P]tRNA4-Met species was then digested with T1 RNase or pancreatic RNase so as to obtain both complete and partial RNase digestion products. The nucleotide sequences of these fragments were analysed to enable the derivation of the complete primary structure of this tRNA. tRNA4-Met of mouse myeloma cells is 76 nucleotides in length and contains 15 modified nucleotides. It is the only tRNA yet sequenced which has been found to possess the minor nucleoside 2-methylguanosine (m2G) within the amino acid (a) stem, and also to have an anticodon (c) stem of only 4 and not 5 base-pairs. The loop IV sequence of eukaryotic initiator methionine tRNA (tRNAf-Met) species, -A-U-C-G-m1A-A-A-, IS NOT FOUND IN TRNA4-Met and is therefore absent from at least one of the methionine tRNAs functioning in polypeptide elongation in mammalian cells. This is consistent with the suggested importance of this loop structure in the initiator function of tRNAf-Met in eukaryotic organisms. Three distinct regions of the tRNA cloverleaf, the (b) stem, the anticodon loop (loop II), and loop III, are substantially conserved in structure between tRNAf-Met and tRNA4-Met of mouse myeloma cells. These regions of the structures of mammalian methionine tRNAs probably do not determine whether a certain tRNA-Met will function in the initiation or elongation of protein synthesis, although they might be important in tRNA-Met recognition if the different cytoplasmic tRNA-Met species of mammalian cells are aminoacylated by a single activating enzyme.

Conformational analysis of RNA molecules by partial RNAse digestion and two dimensional acrylamide gel electrophoresis: Application to E. coli 5S RNA

Summary The conformation of sequenced RNA molecules can be analysed by partial RNAse digestion and two-dimensional acrylamide gel electrophoresis: in the first dimension (neutral acrylamide gel) fragments which are not held together by hydrogen bonds dissociate; thanks to the second dimension (8M urea-acrylamide gel) hidden breaks present in the fragments are revealed. Each spot of the resulting two-dimensional map can then be characterized by elution and fingerprinting. The method yields information both on the existence of particularly exposed regions in the molecule and on the pairing state of some parts of the sequence. New results obtained by the application of this method to the analysis of T1 RNAse partial digests of E. coli 5S RNA are described.