CyloFold: secondary structure prediction including pseudoknots

Computational RNA secondary structure prediction approaches differ by the way RNA pseudoknot interactions are handled. For reasons of computational efficiency, most approaches only allow a limited class of pseudoknot interactions or are not considering them at all. Here we present a computational method for RNA secondary structure prediction that is not restricted in terms of pseudoknot complexity. The approach is based on simulating a folding process in a coarse-grained manner by choosing helices based on established energy rules. The steric feasibility of the chosen set of helices is checked during the folding process using a highly coarse-grained 3D model of the RNA structures. Using two data sets of 26 and 241 RNA sequences we find that this approach is competitive compared to the existing RNA secondary structure prediction programs pknotsRG, HotKnots and UnaFold. The key advantages of the new method are that there is no algorithmic restriction in terms of pseudoknot complexity and a test is made for steric feasibility. Availability: The program is available as web server at the site: http://cylofold.abcc.ncifcrf.gov

Structural polymorphism of the HIV-1 leader region explored by computational methods

Experimental studies revealed that the elements of the human immunodeficiency virus type 1 (HIV-1) 5′-untranslated leader region (5′-UTR) can fold in vitro into two alternative conformations, branched (BMH) and ‘linearized’ (LDI) and switch between them to achieve different functionality. In this study we computationally explored in detail, with our massively parallel genetic algorithm (MPGAfold), the propensity of 13 HIV-1 5′-UTRs to fold into the BMH and the LDI conformation types. Besides the BMH conformations these results predict the existence of two functionally equivalent types of LDI conformations. One is similar to what has been shown in vitro to exist in HIV-1 LAI, the other is a novel conformation exemplified by HIV-1 MAL long-distance interactions. These novel MPGAfold results are further corroborated by a consensus probability matrix algorithm applied to a set of 155 HIV-1 sequences. We also have determined in detail the impact of various strain mutations, domain sizes and folds of elongating sequences simulating folding during transcription on HIV-1 RNA secondary structure folding dynamics

DIFFERENT REPLICATION REQUIREMENTS IN THE HOMOLOGOUS 3' ENDS OF A POSITIVE STRAND RNA VIRUS AND ITS SUBVIRAL RNA

SatC is a noncoding subviral RNA associated with Turnip Crinkle Virus (TCV), a small (4054 nt) single-stranded (+)-strand RNA virus belonging to the Carmovirus genus. Because of its small size (356 nt) and TCV-derived 3' end, satC has been successfully used as a model to elucidate sequence and structural requirements for TCV RNA replication. Although satC is considered a model to identify cis-acting elements required for TCV replication, recent findings indicate distinct differences in structures and functions of these related sequences. RNA2D3D predicts that part of the TCV 3' end (H5, H4a, H4b and two pseudoknots) folds into an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits and is required for translation. SatC contains a similar 3' end with 6 nt differences in the 100 nt TSS region. RNA2D3D did not predict a similar structure for satC TSS region, and satC did not bind yeast ribosomes. satC nucleotides were changed into TCV TSS bases to determine which base differences are responsible for the loss of the TSS in satC. Changing these bases all increased ribosome binding but surprisingly none of them had an effect on satC accumulation in protoplasts and plants. Therefore satC may need these and other 3' end base differences for its required conformational switch for efficient replication, and not to inhibit ribosome binding. In vivo genetic selection (SELEX) of the linker sequence between H5 and the Pr showed the conservation of UCC, which led to the discovery of Ø2. Ø2 is required for both viral and satC accumulation in protoplasts. H5-Pr linker had no significant structural change after RdRp binding in satC, which is different with TCV H5-Pr linker. TCV H5-Pr linker had a major structural change upon RdRp binding, and is proposed to be involved in a conformational switch. Replacement of satC H4a with randomized sequence and scoring for fitness in plants by SELEX resulted in winning sequences that contain an H4a-like stem-loop. SELEX of H4a/H4b in satC generated two different structures: wt H4a/H4b-like structure and a single hairpin structure. Two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts. With the protection of CP, satC can form higher amount of dimers that have additional nucleotides at the junction sites in the absence of TCV. The extra nucleotides are not necessarily associated with an active TCV RdRp