6th Dysferlin Conference, 3-6 April 2013, Arlington, Virginia, USA.

The 2013 Dysferlin Conference, sponsored and organized by the Jain Foundation, was held from April 3-6, 2013 in Arlington, VA. Participants included 34 researcher speakers, 5 dysferlinopathy patients and all 8 members of the Jain Foundation team. Dysferlinopathy is a rare disease that typically robs patients of mobility during their second or third decade of life. The goals of these Dysferlin Conferences are to bring experts in the field together so that they will collaborate with one another, to quicken the pace of understanding the biology of the disease and to build effective platforms to ameliorate disease. This is important because the function of dysferlin and how to compensate for its absence is still not well understood, in spite of the fact that the dysferlin gene was identified more than a decade ago. The objective of this conference, therefore, was to share and discuss the newest unpublished research defining the role of dysferlin in skeletal muscle, why its absence causes muscular dystrophy and possible therapies for dysferlin-deficient muscular dystrophy patients.

An allosteric self-splicing ribozyme triggered by a bacterial second messenger.

Group I self-splicing ribozymes commonly function as components of selfish mobile genetic elements. We identified an allosteric group I ribozyme, wherein self-splicing is regulated by a distinct riboswitch class that senses the bacterial second messenger c-di-GMP. The tandem RNA sensory system resides in the 5' untranslated region of the messenger RNA for a putative virulence gene in the pathogenic bacterium Clostridium difficile. c-di-GMP binding by the riboswitch induces folding changes at atypical splice site junctions to modulate alternative RNA processing. Our findings indicate that some self-splicing ribozymes are not selfish elements but are harnessed by cells as metabolite sensors and genetic regulators.

Roseoflavin is a natural antibacterial compound that binds to FMN riboswitches and regulates gene expression.

Riboswitches in messenger RNAs carry receptor domains called aptamers that can bind to metabolites and control expression of associated genes. The Gram-positive bacterium Bacillus subtilis has two representatives of a class of riboswitches that bind flavin mononucleotide (FMN). These riboswitches control genes responsible for the biosynthesis and transport of riboflavin, a precursor of FMN. We found that roseoflavin, a chemical analog of FMN and riboflavin that has antimicrobial activity, can directly bind to FMN riboswitch aptamers and downregulate the expression of an FMN riboswitch-lacZ reporter gene in B. subtilis. A role for the riboswitch in the antimicrobial mechanism of roseoflavin is supported by our observation that some previously identified roseoflavin-resistant bacteria have mutations within an FMN aptamer. Riboswitch mutations in these resistant bacteria disrupt ligand binding and derepress reporter gene expression in the presence of either riboflavin or roseoflavin. If FMN riboswitches are a major target for roseoflavin antimicrobial action, then future efforts to develop compounds that trigger FMN riboswitch function could lead to the identification of new antimicrobial drugs.

Riboswitches that sense S-adenosylhomocysteine and activate genes involved in coenzyme recycling.

We have identified a highly conserved RNA motif that occurs upstream of genes involved in S-adenosyl-L-methionine (SAM) recycling in many Gram-positive and Gram-negative species of bacteria. The phylogenetic distribution and the conserved structural features of representatives of this motif are indicative of riboswitch function. Riboswitches are widespread metabolite-sensing gene control elements that are typically found in the 5' untranslated regions (UTRs) of bacterial mRNAs. We experimentally verified that examples of this RNA motif specifically recognize S-adenosylhomocysteine (SAH) in protein-free in vitro assays, and confirmed that these RNAs strongly discriminate against SAM and other closely related analogs. A representative SAH motif was found to activate expression of a downstream gene in vivo when the metabolite is bound. These observations confirm that SAH motif RNAs are distinct ligand-binding aptamers for a riboswitch class that selectively binds SAH and controls genes essential for recycling expended SAM coenzymes.

Identification of 22 candidate structured RNAs in bacteria using the CMfinder comparative genomics pipeline.

We applied a computational pipeline based on comparative genomics to bacteria, and identified 22 novel candidate RNA motifs. We predicted six to be riboswitches, which are mRNA elements that regulate gene expression on binding a specific metabolite. In separate studies, we confirmed that two of these are novel riboswitches. Three other riboswitch candidates are upstream of either a putative transporter gene in the order Lactobacillales, citric acid cycle genes in Burkholderiales or molybdenum cofactor biosynthesis genes in several phyla. The remaining riboswitch candidate, the widespread Genes for the Environment, for Membranes and for Motility (GEMM) motif, is associated with genes important for natural competence in Vibrio cholerae and the use of metal ions as electron acceptors in Geobacter sulfurreducens. Among the other motifs, one has a genetic distribution similar to a previously published candidate riboswitch, ykkC/yxkD, but has a different structure. We identified possible non-coding RNAs in five phyla, and several additional cis-regulatory RNAs, including one in epsilon-proteobacteria (upstream of purD, involved in purine biosynthesis), and one in Cyanobacteria (within an ATP synthase operon). These candidate RNAs add to the growing list of RNA motifs involved in multiple cellular processes, and suggest that many additional RNAs remain to be discovered.