The second group comprises multiple structures of u1a rrm in complex with the. The application of crystallography to rna is illustrated with the structure determination of the cug repeat that is linked to type i myotonic dystrophy. Embl, gene expression program, 1 meyerhofstrasse, heidelberg, d69117. Attempts to crystallize the wild type u1a protein and its cognate rna hairpin result in crystals diffracting no. Use of the rnabinding protein u1a as a crystal doudna lab. The design of rna interference rnai reagents is an essential step for performing lossoffunction studies in many experimental systems. For smaller rnas rna purification system has been developed that is capable of producing sufficient rna for crystallography screening batey and kieft, 2007. Such adjustment seems possible, and it is observed in some homologs with known structures.
A software package that integrates three rna analysis tools based on the abstract shapes approach. Molecular biologyrna protocols protocol online your lab. The crystallographic refinement permitted the identification of 91 solvent molecules, with a final agreement factor of %. The role of rna structure in the interaction of u1a protein with u1. This system coexpresses u1apgfp with the rna of interest tagged with u1a aptamers, and has been proven to effectively track rna in vivo.
Use of the u1a protein to facilitate crystallization and structure. Carine crystallography is a crystallographic software which has been developed since 1989 and which is used by thousands of persons around the world for teaching, research and edition is now available in a totally rethought version. Rnastructure is a complete package for rna and dna secondary structure prediction and analysis. Expanding crystallization tools for nucleic acid complexes using. The rna 6000 nano system has a broad dynamic range and can quantitate between 25 500 ngml of rna with a covariance of 10%. The software can also design interacting rna molecules using rnacofold of the viennarna package.
In this chapter, we describe in detail the u1a based tagged rna system. The instrument software automatically compares the peak areas from unknown rna samples to the combined area of the six rna 6000 ladder rna peaks to determine the concentration of the unknown samples. However, rna binding as, for example, in the spliceosomal u1a oubridge et al. These socalled crystallization modules include the u1a rna binding protein and fab bl36, which bind to their cognate hairpins, and the l7ae family of proteins that bind to the kink turn motifs 15,17,18. Raman crystallography is the application of raman spectroscopy to single crystals. The development release allows access to the latest features for feedback. The nmr structure of the 38 kda u1a protein pie rna. Theyincludewildtypeu1arna complex, free wildtype u1a in closed and open conformations, and free sl2 rna, as well as those of three u1a mutants, f56a, f56l and f56w. Affinity maturation of a portable fabrna module for. Collection of tissue for rna analysis laboratory of experimental pathology how to collect and store tissue sample for rna. Anaelu software package anaelu analytical emulator laue utility has been created for aiding the interpretation of twodimensional xray diffraction patterns produced by textured bulk and nanostructured samples.
A utility for collecting statistics from aligned and structurally annotated rna sequences. This technique has been applied to a variety of protein molecules where it has provided unique information about biopolymer folding, substrate binding, and catalysis. The rnaifold software provides two algorithms to solve the inverse folding problem. Although xray diffraction played a role in its solution by james d. List of rna structure prediction software wikipedia. Crystal structure reveals details of nonstandard rna. Crystallographic software university of california. A general strategy to solve the phase problem in rna. Sederivatized rnas for xray crystallography springerlink. This windows software provides an integrated high quality system for building and modelling molecules mopac and iconc are included and the system drives pc gamess and tinker. Notably, the u1 rna hairpin sequence is conserved in viridiplantae and animalia.
It includes algorithms for secondary structure prediction, including facility to predict base pairing probabilities. Md simulations of u1arna systems the md trajectories computed for this study are summarizedintable1. This position is critical for rna binding as the phenylalanine stacks to a certain nucleotide in the complex formed by u1a and rna shiels et al. These socalled crystallization modules include the u1a rna binding protein. Anaelu is composed of three interconnected applications, corresponding to three crystallographic tasks.
Cryoem structure of saccharomyces cerevisiae u1 snrnp offers. Molecular biologyrna protocols protocol online your. Our results provide a new tool set for the crystallization of rna and rna. This indicates that the mutation f56w should not prohibit rna binding to u1a rbd law, 2005, shiels et al.
As a member of the wwpdb, the rcsb pdb curates and annotates pdb data according to agreed upon standards. Proteinassisted rna fragment docking rnax for modeling rna. Polya tail regulation requires cooperative binding of two human u1a proteins to an rna regulatory region called the polyadenylation inhibition element pie. If the rna is universally abundant, homologs may sometimes be purified directly from a thermophile e. Together with foldx protein design tool suite we were able to engineer. When bound to pie rna, u1a proteins also bind to the enzyme responsible for formation of the mature 3. As in protein crystallography, crystal quality may be improved by using nucleic acids with higher thermal stability. The function of many biologically important rnas is conferred by their threedimensional folds, and xray crystallography remains the most powerful tool to determine their structure. The role of rna in biology is varied and rich, with rna playing diverse roles in both healthy and diseased cells. The crystallographic structure of the synthetic oligoribonucleotide, uua6a, has been solved at 2. Structural basis of the rnabinding specificity of human u1a protein.
Oct 19, 2017 d sequence alignment between human and yeast u1a rrm1 demonstrates differences between residues in rnp1 and rnp2 that are known to bind rna in human u1a red triangles and the long loop 2 in. Crystallographic studies of dna and rna sciencedirect. Protein engineering as a tool for crystallography sciencedirect. Md simulations were performed with explicit water and counterions. The structure determination of complex rna molecules such as ribozymes, riboswitches and aptamers by xray crystallography hinges on the preparation of wellordered crystals. When bound to pie rna, u1a proteins also bind to the enzyme responsible for. Pairfold predicts the minimum free energy secondary structure formed by two input dna or rna molecules. The selfcleaving ribozyme of the hepatitis delta virus hdv is the only catalytic rna known to be required for the viability of a human pathogen. The rna was cocrystallized with a mixture of archeoglobus fulgidus l7ae and human u1a. Preparation of rnaprotein complexes for xray crystallography and nmr.
Crystal structure of a hepatitis delta virus ribozyme nature. Crystal structures of transcription factor nusg in light of. Macromolecular crystallographic software links ccp4 cns phenix eden crystallography coot o macros for o uppsala software factory pymol home page sharp mosflm other useful crystallography links crystallography on os x xray absorption edges the protein data bank pdb molecular movies data base nucleic acid databank international. Pairfold can be used, for example, to predict interactions between a probe and target rna molecule, or between pairs of strands in biomolecular nanostructures. The tagged rna system has recently emerged as a feasible noninvasive solution for such purpose. The modules have yielded promising results for rna crystallization and structure determination. Oscail is a windows based software for molecular modelling and crystallography. To solve the crystal structure of fabspinach1 rna complex, we. This series of talks is about rna structure, function, and recognition.
The human u1a rna binding domain rbd1 adopts one of the most common protein folds, the rna recognition motif, and is a paradigm for understanding rna protein interactions. This work was supported by the intramural research program of the nih. Use of the spliceosomal protein u1a to facilitate crystallization and structure determination of complex rnas. In this method, the rna of interest is attached to the 5.
The pdb archive contains information about experimentallydetermined structures of proteins, nucleic acids, and complex assemblies. Aug 22, 2017 highresolution crystal structure reveals a new pathway for rna during a nontraditional form of transcriptionthe process by which rna is produced from a dna template. The role of rna sequence versus structure in rnaprotein interactions remains a. Athena, artemis, hephaestus this software package is based on the ifeffit library of numerical and xas algorithms.
Thermophiles are natural sources of such molecules. The human u1a rnabinding domain rbd1 adopts one of the most common protein folds, the rnarecognition motif, and is a paradigm for understanding rnaprotein interactions. Rna molecules of interest can be constructed to contain a motif that binds to its cognate rna binding protein and the complex is advanced to crystallization trials. These socalled crystallization modules include the u1a rna binding protein and fab bl36 16, which bind to their cognate hairpins. Structure of a rare nonstandard sequence kturn bound by. Cryoem structure of saccharomyces cerevisiae u1 snrnp. Here, we describe the application of raman crystallography to functional rna molecules. A design and visualization tool for rna secondary structure. Crystal structure of human acinus rna recognition motif. Affinity and specificity of protein u1arna complex. If acrrm bound rna in the way observed in related structures, the cterminal.
Xray diffraction from fibrous dna tipped off watson, crick to double helix it is one of the most famous molecular structures ever determined, the now iconic dna double helix. The rna used in crystallization experiments comprised an 11bp duplex in the center of which was the t. Methods in molecular biology methods and protocols, vol 941. Expanding crystallization tools for nucleic acid complexes. Pdf determining structures of rna aptamers and riboswitches. Even mrna, a relatively linear molecule, can contain distinctive three dimensional structures.
The first structure of an rna m5c methyltransferase, fmu. Rna interference reagent design software tools omictools. Highresolution crystal structure reveals a new pathway for rna during a nontraditional form of transcriptionthe process by which rna is produced. Tertiary structure detection software tools rna data. Center for molecular biology of rna at university of california, santa cruz.
U1 spliceosomal rna is the small nuclear rna snrna component of u1 snrnp small nuclear ribonucleoprotein, an rna protein complex that combines with other snrnps, unmodified premrna, and various other proteins to assemble a spliceosome, a large rna protein molecular complex upon which splicing of premrna occurs. Crystallographic studies of dna and rna molecules share many considerations that we outline here. Of particular importance are approaches toward rna crystallography that include exploring. The rna recognition motif rrm is a small approximately 90. The structure of the nterminal rrm of u1a residues 2117 has been determined by nmr spectroscopy32 and xray crystallography residues 392,33 and the complex of the nterminal rrm of u1a residues 297 with sl2 rna 21 bases u1arna has been determined by xray crystallography,34 albeit with two surface mutations. Fmu contains three subdomains which share structural homology to dna m5c methyltransferases and two rna. The availability of sequenced and annotated genomes greatly facilitates rnai experiments in an increasing number of organisms that were previously not genetically tractable. If you want to visualize tertiary structures, you will need to install the software ucsf chimera and to link it to assemble2. Affinity maturation of a portable fabrna module for chaperone. C the u1a model was subjected to rna docking, taking into account the. Crystallization, crystallizability, spliceosomal protein, rna biding domain.