Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA

The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5′ terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT). To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1), a 1-nucleotide interhelical loop and an 8-bp stem (S2) that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging

Pyrvinium pamoate changes alternative splicing of the serotonin receptor 2C by influencing its RNA structure

The serotonin receptor 2C plays a central role in mood and appetite control. It undergoes pre-mRNA editing as well as alternative splicing. The RNA editing suggests that the pre-mRNA forms a stable secondary structure in vivo. To identify substances that promote alternative exons inclusion, we set up a high-throughput screen and identified pyrvinium pamoate as a drug-promoting exon inclusion without editing. Circular dichroism spectroscopy indicates that pyrvinium pamoate binds directly to the pre-mRNA and changes its structure. SHAPE (selective 2\u27-hydroxyl acylation analysed by primer extension) assays show that part of the regulated 5\u27-splice site forms intramolecular base pairs that are removed by this structural change, which likely allows splice site recognition and exon inclusion. Genome-wide analyses show that pyrvinium pamoate regulates \u3e300 alternative exons that form secondary structures enriched in A-U base pairs. Our data demonstrate that alternative splicing of structured pre-mRNAs can be regulated by small molecules that directly bind to the RNA, which is reminiscent to an RNA riboswitch

ProbKnot: Fast prediction of RNA secondary structure including pseudoknots

It is a significant challenge to predict RNA secondary structures including pseudoknots. Here, a new algorithm capable of predicting pseudoknots of any topology, ProbKnot, is reported. ProbKnot assembles maximum expected accuracy structures from computed base-pairing probabilities in O(N2) time, where N is the length of the sequence. The performance of ProbKnot was measured by comparing predicted structures with known structures for a large database of RNA sequences with fewer than 700 nucleotides. The percentage of known pairs correctly predicted was 69.3%. Additionally, the percentage of predicted pairs in the known structure was 61.3%. This performance is the highest of four tested algorithms that are capable of pseudoknot prediction. The program is available for download at: http://rna.urmc.rochester.edu/RNAstructure.html

Improving and applying RNA secondary structure prediction

Thesis (Ph. D.)--University of Rochester. Department of Chemistry, 2014.All the roles of RNA molecules in the cell are not yet known. Over the years many RNA functions have been discovered, such as catalyzing reactions and regulating gene expression, functions originally attributed to proteins. It is often important to know the structure of an RNA in order to understand its function. RNA secondary structure is formed by stems, loops and pseudoknots, structures with nucleotides in the loop forming pairs outside of the loop. Pseudoknot-free secondary structures can be predicted using dynamic programming algorithms. It is, however, a challenge to predict RNA secondary structures including pseudoknots; the prediction of lowest free energy pseudoknotted structures is known to be NP-complete. We developed an algorithm capable of predicting pseudoknots of any topology, ProbKnot. ProbKnot assembles maximum expected accuracy structures from computed base-pairing probabilities that scales with the squares of the length of the sequence. The probabilities are computed using the partition function algorithm based on thermodynamic parameters that do not account for pseudoknotted structures. ProbKnot performs the best of four tested algorithms that are capable of pseudoknot prediction. Base pair probabilities can also be calculated from an ensemble of structures. We developed a Monte Carlo secondary structure sampling method that includes replica exchange. The obtained pair probabilities from the sample match those from the partition function to less than 1% RMSD when pseudoknots are not allowed. This method can be used to predict structures with pseudoknots, thus bypassing the need for a dynamic programming algorithm, which is notoriously slow when pseudoknots are allowed. Artificially designed nucleic acids have been successfully used in nanostructures. The inverse problem of finding a sequence of nucleotides that can assume desired secondary conformation is challenging because there are only four natural RNA bases and many other structures are often possible for a given sequence. To facilitate rapid design, we built a database of RNA helices and loops that demonstrate traits observed in natural RNA structures and have little tendency to cross-hybridize with each other. When using the database as compared to randomly chosen sequences, sequences for biologically-relevant structures are designed approximately 32 times faster

Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA

The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5′ terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT). To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1), a 1-nucleotide interhelical loop and an 8-bp stem (S2) that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging

Transcriptional Regulation on Aneuploid Chromosomes in Diverse Candida albicans Mutants

Abstract Candida albicans is a diploid fungus and a predominant opportunistic human pathogen. Notably, C. albicans employs reversible chromosomal aneuploidies as a means of survival in adverse environments. We previously characterized transcription on the monosomic chromosome 5 (Ch5) that arises with adaptation to growth on the toxic sugar sorbose in the mutant Sor125(55). We now extend this analysis to the trisomic hybrid Ch4/7 within Sor125(55) and a diverse group of three mutants harboring a single Ch5. We find a similar pattern of transcriptional changes on either type of aneuploid chromosome within these mutants wherein expression of many genes follows chromosome ploidy, consistent with a direct mechanism to regulate genes important for adaptation to growth. In contrast, a significant number of genes are expressed at the disomic level, implying distinct mechanisms compensating for gene dose on monosomic or trisomic chromosomes consistent with maintaining cell homeostasis. Finally, we find evidence for an additional mechanism that elevates expression of genes on normal disomic Ch4 and Ch7 in mutants to levels commensurate with that found on the trisomic Ch4/7b in Sor125(55). Several of these genes are similarly differentially regulated among mutants, suggesting they play key functions in either maintaining aneuploidy or adaptation to growth conditions