RNA structural motif recognition based on least-squares distance

RNA structural motifs are recurrent structural elements occurring in RNA molecules. RNA structural motif recognition aims to find RNA substructures that are similar to a query motif, and it is important for RNA structure analysis and RNA function prediction. In view of this, we propose a new method known as RNA Structural Motif Recognition based on Least-Squares distance (LS-RSMR) to effectively recognize RNA structural motifs. A test set consisting of five types of RNA structural motifs occurring in Escherichia coli ribosomal RNA is compiled by us. Experiments are conducted for recognizing these five types of motifs. The experimental results fully reveal the superiority of the proposed LS-RSMR compared with four other state-of-the-art methods.     

RNA模式分析进展

研究表明,R N A模式在基因表达调控方面起着重要作用.由于RN A模式不仅与初级序列有关,更多的表现为高级结构(一般为二级结构)的保守性,所以R N A模式的识别比D N A模式的识别要复杂的多.近十几年里,对R N A模式分析作了大量的计算方面的研究,包括:R N A结构的预测、识别和已知的类型相似的R N A模式、在一组功能或进化相关的基因中找出共同的R N A模式.这里对上述3个方面的计算方法的发展和研究进行了综述.     

Automated classification of RNA 3D motifs and the RNA 3D Motif Atlas

The analysis of atomic-resolution RNA three-dimensional (3D) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-Watson–Crick base pairs. Structurally similar loops define RNA 3D motifs that are conserved in homologous RNA molecules, but can also occur at nonhomologous sites in diverse RNAs, and which often vary in sequence. To further our understanding of RNA motif structure and sequence variability and to provide a useful resource for structure modeling and prediction, we present a new method for automated classification of internal and hairpin loop RNA 3D motifs and a new online database called the RNA 3D Motif Atlas. To classify the motif instances, a representative set of internal and hairpin loops is automatically extracted from a nonredundant list of RNA-containing PDB files. Their structures are compared geometrically, all-against-all, using the FR3D program suite. The loops are clustered into motif groups, taking into account geometric similarity and structural annotations and making allowance for a variable number of bulged bases. The automated procedure that we have implemented identifies all hairpin and internal loop motifs previously described in the literature. All motif instances and motif groups are assigned unique and stable identifiers and are made available in the RNA 3D Motif Atlas (http://rna.bgsu.edu/motifs), which is automatically updated every four weeks. The RNA 3D Motif Atlas provides an interactive user interface for exploring motif diversity and tools for programmatic data access.     

S6:S18 ribosomal protein complex interacts with a structural motif present in its own mRNA

Prokaryotic ribosomal protein genes are typically grouped within highly conserved operons. In many cases, one or more of the encoded proteins not only bind to a specific site in the ribosomal RNA, but also to a motif localized within their own mRNA, and thereby regulate expression of the operon. In this study, we computationally predicted an RNA motif present in many bacterial phyla within the 5′ untranslated region of operons encoding ribosomal proteins S6 and S18. We demonstrated that the S6:S18 complex binds to this motif, which we hereafter refer to as the S6:S18 complex-binding motif (S6S18CBM). This motif is a conserved CCG sequence presented in a bulge flanked by a stem and a hairpin structure. A similar structure containing a CCG trinucleotide forms the S6:S18 complex binding site in 16S ribosomal RNA. We have constructed a 3D structural model of a S6:S18 complex with S6S18CBM, which suggests that the CCG trinucleotide in a specific structural context may be specifically recognized by the S18 protein. This prediction was supported by site-directed mutagenesis of both RNA and protein components. These results provide a molecular basis for understanding protein-RNA recognition and suggest that the S6S18CBM is involved in an auto-regulatory mechanism.     

RNAMotifScanX: a graph alignment approach for RNA structural motif identification

RNA structural motifs are recurrent three-dimensional (3D) components found in the RNA architecture. These RNA structural motifs play important structural or functional roles and usually exhibit highly conserved 3D geometries and base-interaction patterns. Analysis of the RNA 3D structures and elucidation of their molecular functions heavily rely on efficient and accurate identification of these motifs. However, efficient RNA structural motif search tools are lacking due to the high complexity of these motifs. In this work, we present RNAMotifScanX, a motif search tool based on a base-interaction graph alignment algorithm. This novel algorithm enables automatic identification of both partially and fully matched motif instances. RNAMotifScanX considers noncanonical base-pairing interactions, base-stacking interactions, and sequence conservation of the motifs, which leads to significantly improved sensitivity and specificity as compared with other state-of-the-art search tools. RNAMotifScanX also adopts a carefully designed branch-and-bound technique, which enables ultra-fast search of large kink-turn motifs against a 23S rRNA. The software package RNAMotifScanX is implemented using GNU C++, and is freely available from http://genome.ucf.edu/RNAMotifScanX.     

Annotation of tertiary interactions in RNA structures reveals variations and correlations

RNA tertiary motifs play an important role in RNA folding and biochemical functions. To help interpret the complex organization of RNA tertiary interactions, we comprehensively analyze a data set of 54 high-resolution RNA crystal structures for motif occurrence and correlations. Specifically, we search seven recognized categories of RNA tertiary motifs (coaxial helix, A-minor, ribose zipper, pseudoknot, kissing hairpin, tRNA D-loop/T-loop, and tetraloop-tetraloop receptor) by various computer programs. For the nonredundant RNA data set, we find 613 RNA tertiary interactions, most of which occur in the 16S and 23S rRNAs. An analysis of these motifs reveals the diversity and variety of A-minor motif interactions and the various possible loop-loop receptor interactions that expand upon the tetraloop-tetraloop receptor. Correlations between motifs, such as pseudoknot or coaxial helix with A-minor, reveal higher-order patterns. These findings may ultimately help define tertiary structure restraints for RNA tertiary structure prediction. A complete annotation of the RNA diagrams for our data set is available at http://www.biomath.nyu.edu/motifs/.     

RNA Ligation and the Origin of tRNA

A straightforward origin of transfer RNA,(tRNA), is difficult to envision because of the apparentlycomplex idiosyncratic interaction between the D-loop and T-loop. Recently, multiple examples of the T-loop structuralmotif have been identified in ribosomal RNA. These examplesshow that the long-range interactions between the T-loop andD-loops seen in tRNA are not an essential part of the motifbut rather are facilitated by it. Thus, the core T-loopstructure could already have existed in a small RNA prior tothe emergence of the tRNA. The tRNA might then have arisenby expansion of an RNA that carried the motif. With thisidea in mind, Di Giulio's earlier hypothesis that tRNAevolved by a simple duplication or ligation of a minihelixRNA was re-examined. It is shown that an essentially moderntRNA structure can in fact be generated by the ligation oftwo 38-nucleotide RNA minihelices of appropriate sequence.Although rare, such sequences occur with sufficientfrequency, (1 in 3 × 10⁷), that they could be found in astandard in vitro RNA selection experiment. Theresults demonstrate that a series of RNA duplications, aspreviously proposed, can in principal account for the originof tRNA. More generally, the results point out that RNAligation can be a powerful driving force for increasedcomplexity in the RNA World.     

G-ribo motif favors the formation of pseudoknots in ribosomal RNA

Analysis of the pseudoknots existing in the ribosomal RNA showed that four of them are formed with the help of G-ribo, a recently identified RNA recurrent motif. The analysis of these pseudoknots revealed two major aspects in the G-ribo motif structure, which together provide the structural context favoring the formation of two different types of pseudoknots. The first aspect pertains to a particular side-by-side juxtaposition of two double helices that facilitates switches of the polynucleotide chain between different strands. The second aspect deals with the presence of an adenosine at a specific place where it can stabilize a particular arrangement of two quasicoaxial helices required for the pseudoknot formation. Additional analysis shows that the latter aspect is also present in other pseudoknots not related to the G-ribo motif or the ribosome, and thus represents a general structural element favoring the formation of pseudoknots.     

Sequence and structural conservation in RNA ribose zippers

The "ribose zipper", an important element of RNA tertiary structure, is characterized by consecutive hydrogen-bonding interactions between ribose 2'-hydroxyls from different regions of an RNA chain or between RNA chains. These tertiary contacts have previously been observed to also involve base-backbone and base-base interactions (A-minor type). We searched for ribose zipper tertiary interactions in the crystal structures of the large ribosomal subunit RNAs of Haloarcula marismortui and Deinococcus radiodurans, and the small ribosomal subunit RNA of Thermus thermophilus and identified a total of 97 ribose zippers. Of these, 20 were found in T. thermophilus 16 S rRNA, 44 in H. marismortui 23 S rRNA (plus 2 bridging 5 S and 23 S rRNAs) and 30 in D. radiodurans 23 S rRNA (plus 1 bridging 5 S and 23 S rRNAs). These were analyzed in terms of sequence conservation, structural conservation and stability, location in secondary structure, and phylogenetic conservation. Eleven types of ribose zippers were defined based on ribose-base interactions. Of these 11, seven were observed in the ribosomal RNAs. The most common of these is the canonical ribose zipper, originally observed in the P4-P6 group I intron fragment. All ribose zippers were formed by antiparallel chain interactions and only a single example extended beyond two residues, forming an overlapping ribose zipper of three consecutive residues near the small subunit A-site. Almost all ribose zippers link stem (Watson-Crick duplex) or stem-like (base-paired), with loop (external, internal, or junction) chain segments. About two-thirds of the observed ribose zippers interact with ribosomal proteins. Most of these ribosomal proteins bridge the ribose zipper chain segments with basic amino acid residues hydrogen bonding to the RNA backbone. Proteins involved in crucial ribosome function and in early stages of ribosomal assembly also stabilize ribose zipper interactions. All ribose zippers show strong sequence conservation both within these three ribosomal RNA structures and in a large database of aligned prokaryotic sequences. The physical basis of the sequence conservation is stacked base triples formed between consecutive base-pairs on the stem or stem-like segment with bases (often adenines) from the loop-side segment. These triples have previously been characterized as Type I and Type II A-minor motifs and are stabilized by base-base and base-ribose hydrogen bonds. The sequence and structure conservation of ribose zippers can be directly used in tertiary structure prediction and may have applications in molecular modeling and design.     

Recurrent RNA motifs as probes for studying RNA-protein interactions in the ribosome

To understand how the nucleotide sequence of ribosomal RNA determines its tertiary structure, we developed a new approach for identification of those features of rRNA sequence that are responsible for formation of different short- and long-range interactions. The approach is based on the co-analysis of several examples of a particular recurrent RNA motif. For different cases of the motif, we design combinatorial gene libraries in which equivalent nucleotide positions are randomized. Through in vivo expression of the designed libraries we select those variants that provide for functional ribosomes. Then, analysis of the nucleotide sequences of the selected clones would allow us to determine the sequence constraints imposed on each case of the motif. The constraints shared by all cases are interpreted as providing for the integrity of the motif, while those ones specific for individual cases would enable the motif to fit into the particular structural context. Here we demonstrate the validity of this approach for three examples of the so-called along-groove packing motif found in different parts of ribosomal RNA.     

A structural database for k-turn motifs in RNA

The kink-turn (k-turn) is a common structural motif in RNA that introduces a tight kink into the helical axis. k-turns play an important architectural role in RNA structures and serve as binding sites for a number of proteins. We have created a database of known and postulated k-turn sequences and three-dimensional (3D) structures, available via the internet. This site provides (1) a database of sequence and structure, as a resource for the RNA community, and (2) a tool to enable the manipulation and comparison of 3D structures where known.     

The UAA/GAN internal loop motif: a new RNA structural element that forms a cross-strand AAA stack and long-range tertiary interactions

Analysis of aligned RNA sequences and high-resolution crystal structures has revealed a new RNA structural element, termed the UAA/GAN motif. Found in internal loops of the 23 S rRNA, as well as in RNase P RNA and group I and II introns, this six-nucleotide motif adopts a distinctive local structure that includes two base-pairs with non-canonical conformations and three conserved adenine bases, which form a cross-strand AAA stack in the minor groove. Most importantly, the motif invariably forms long-range tertiary contacts, as the AAA stack typically forms A-minor interactions and the flipped-out N nucleotide forms additional contacts that are specific to the structural context of each loop. The widespread presence of this motif and its propensity to form long-range contacts suggest that it plays a critical role in defining the architectures of structured RNAs.     

Diversity of base-pair conformations and their occurrence in rRNA structure and RNA structural motifs

In addition to the canonical base-pairs comprising the standard Watson-Crick (C:G and U:A) and wobble U:G conformations, an analysis of the base-pair types and conformations in the rRNAs in the high-resolution crystal structures of the Thermus thermophilus 30S and Haloarcula marismortui 50S ribosomal subunits has identified a wide variety of non-canonical base-pair types and conformations. However, the existing nomenclatures do not describe all of the observed non-canonical conformations or describe them with some ambiguity. Thus, a standardized system is required to classify all of these non-canonical conformations appropriately. Here, we propose a new, simple and systematic nomenclature that unambiguously classifies base-pair conformations occurring in base-pairs, base-triples and base-quadruples that are associated with secondary and tertiary interactions. This system is based on the topological arrangement of the two bases and glycosidic bonds in a given base-pair. Base-pairs in the internal positions of regular secondary structure helices usually form with canonical base-pair groups (C:G, U:A, and U:G) and canonical conformations (C:G WC, U:A WC, and U:G Wb). In contrast, non-helical base-pairs outside of regular structure helices usually have non-canonical base-pair groups and conformations. In addition, many non-helical base-pairs are involved in RNA motifs that form a defined set of non-canonical conformations. Thus, each rare non-canonical conformation may be functionally and structurally important. Finally, the topology-based isostericity of base-pair conformations can rationalize base-pair exchanges in the evolution of RNA molecules.     

NMR structure of a 4 x 4 nucleotide RNA internal loop from an R2 retrotransposon: identification of a three purine-purine sheared pair motif and comparison to MC-SYM predictions

The NMR solution structure is reported of a duplex, 5'GUGAAGCCCGU/3'UCACAGGAGGC, containing a 4 × 4 nucleotide internal loop from an R2 retrotransposon RNA. The loop contains three sheared purine-purine pairs and reveals a structural element found in other RNAs, which we refer to as the 3RRs motif. Optical melting measurements of the thermodynamics of the duplex indicate that the internal loop is 1.6 kcal/mol more stable at 37°C than predicted. The results identify the 3RRs motif as a common structural element that can facilitate prediction of 3D structure. Known examples include internal loops having the pairings: 5'GAA/3'AGG, 5'GAG/3'AGG, 5'GAA/3'AAG, and 5'AAG/3'AGG. The structural information is compared with predictions made with the MC-Sym program.     

The Thermus thermophilus DEAD box helicase Hera contains a modified RNA recognition motif domain loosely connected to the helicase core

DEAD box family helicases consist of a helicase core that is formed by two flexibly linked RecA-like domains. The helicase activity can be regulated by N- or C-terminal extensions flanking the core. Thermus thermophilus heat resistant RNA-dependent ATPase (Hera) is the first DEAD box helicase that forms a dimer using a unique dimerization domain. In addition to the dimerization domain, Hera contains a C-terminal RNA binding domain (RBD) that shares sequence homology only to uncharacterized proteins of the Deinococcus/Thermus group. The crystal structure of Hera_RBD reveals the fold of an altered RNA recognition motif (RRM) with limited structural homology to the RBD of the DEAD box helicase YxiN from Bacillus subtilis. Comparison with RRM/RNA complexes shows that a RNA binding mode different than that suggested for YxiN, but similar to U1A, can be inferred for Hera. The orientation of the RBD relative to the helicase core was defined in a second crystal structure of a Hera fragment including the C-terminal RecA domain, the dimerization domain, and the RBD. The structures allow construction of a model for the entire Hera helicase dimer. A likely binding surface for large RNA substrates that spans both RecA-like domains and the RBD is identified.     

Structure of the RNA Binding Domain of a DEAD-Box Helicase Bound to Its Ribosomal RNA Target Reveals a Novel Mode of Recognition by an RNA Recognition Motif

DEAD-box RNA helicases of the bacterial DbpA subfamily are localized to their biological substrate when a carboxy-terminal RNA recognition motif domain binds tightly and specifically to a segment of 23S ribosomal RNA (rRNA) that includes hairpin 92 of the peptidyl transferase center. A complex between a fragment of 23S rRNA and the RNA binding domain (RBD) of the Bacillus subtilis DbpA protein YxiN was crystallized and its structure was determined to 2.9 Å resolution, revealing an RNA recognition mode that differs from those observed with other RNA recognition motifs. The RBD is bound between two RNA strands at a three-way junction. Multiple phosphates of the RNA backbone interact with an electropositive band generated by lysines of the RBD. Nucleotides of the single-stranded loop of hairpin 92 interact with the RBD, including the guanosine base of G2553, which forms three hydrogen bonds with the peptide backbone. A G2553U mutation reduces the RNA binding affinity by 2 orders of magnitude, confirming that G2553 is a sequence specificity determinant in RNA binding. Binding of the RBD to 23S rRNA in the late stages of ribosome subunit maturation would position the ATP-binding duplex destabilization fragment of the protein for interaction with rRNA in the peptidyl transferase cleft of the subunit, allowing it to “melt out” unstable secondary structures and allow proper folding.     

Structure of the pyrimidine-rich internal loop in the poliovirus 3'-UTR: the importance of maintaining pseudo-2-fold symmetry in RNA helices containing two adjacent non-canonical base-pairs

Formation of non-canonical base-pairs in RNA often plays a very important functional role. In addition they frequently serve as factors in stabilizing the secondary structure elements that provide the frame of large compact RNA structures. Here we describe the structure of an internal loop containing a 5'CU3'/5'UU3' non-canonical tandem base-pair motif, which is conserved within the 3'-UTR of poliovirus-like enteroviruses. Structural details reveal striking regularities of the local helix geometry, resulting from alternating geometrical adjustments, which are important for understanding and predicting stabilities and configurations of tandem non-canonical base-pairs. The C-U and U-U base-pairs severely contract the minor groove of the sugar-phosphate backbone, which might be important for protein recognition or binding to other RNA elements.     

Zinc Finger-like Motif Conserved in a Family of RNA Binding Proteins

NP220s compose a family of RNA binding proteins together with matrin 3, one of major proteins of the nuclear matrix. They have repeats of RNA recognition motif (RRM; MH2) homologous to RRM in heterogeneous nuclear RNPs I/L in addition to MH1 and MH3 with unknown function. In search of additional homologous sequences, we found the reported sequence of rat matrin 3 is partially incorrect. Correction of this sequence showed that the NP220 family has a fourth homologous motif with the characteristics of a Cys₂-His₂ zinc finger-like motif. The sequence of this motif is perfectly conserved in human and mouse NP220s despite their 75% overall sequence homology.     

一种结合SELEX与二代测序技术筛选RNA结合蛋白特异识别序列新方法的建立

RNA结合蛋白通过特异识别RNA底物发挥重要的生物学作用。指数富集的配体系统进化(Systematic evolution of ligands by exponential enrichment,SELEX)技术是一种体外筛选核酸底物的基本方法,SELEX技术通过重复多轮筛选从随机核酸序列库中筛选出特异性与靶物质高度亲和的核酸底物,本研究将利用该技术与二代高通量测序(NGS)相结合,体外合成含有20个随机碱基的RNA文库,将所要研究的蛋白构建到带有可被链亲和酶素磁珠捕获的SBP标记的载体上去,显著提高筛选效率,仅需1轮筛选即可获得所需RNA底物motif。通过该方法获得了人的hn RNP A1的UP1结构域特异识别AGG和AG二种RNA序列,并通过EMSA实验证实其可以与获得的RNA motif结合。这一方法的建立对于研究RNA结合蛋白识别底物的序列特异性,并进一步了解其在生物体内的调控机制有重要意义。