Over coffee we were flipping through the May edition of the Howard Hughes Medical Institute's Newsletter when we saw an article on riboswitches, RNA aptamers and the RNA World. The piece features Ron Breaker from Yale, who is most known for describing riboswitches. These RNA elements usually sit on the 5' end of an mRNA and can change conformation when they bind to various small molecules, such as metabolites. These structural changes can affect whether the downstream message on the transcript is translated into protein. So riboswitches are sophisticated versions of aptamers which are RNA elements that simply bind to small molecules.
The article covers the key discoveries that have led to our current understanding of riboswitches and possible implications such as the feasibility of the RNA world hypothesis.
(I remember a conversation I had with Ron where he mentioned "RNA World" about once every two sentences. You gotta love that!)
RNA ... it's a crazy thing to get involved with. Yesterday I visited Jim Deshler's lab at BU and we were discussing how hard it is to study RNA and RNA-protein interactions. You see when proteins bind to eachother they interact using distinct surfaces - everything is well defined. RNA protein interactions are a mess. According to many researchers once RNA binding proteins are bound to their target sequences they multimerize and spread along the length of the RNA polymer. Thus all RNA binding proteins not only bind to certain RNA motifs with high affinity but can also bind any RNA sequence with low affinity. Isolating specific RNA element binding proteins can be a horrible experience. This new CLIP technique developed by the Darnell lab may help identify specific RNA-protein interactions. Then RNA binding proteins can be very weird. Apparently a good chunk of the prion like proteins in the yeast genome are actually RNA binding proteins. I bet that this ratio is similar in higher eukaryotes. Scarry.
Then there is RNA folding - a real mess. There are good programs (such as MFold) but often it is hard to model how any species of RNA folds within the context of a cell with all of these RNA binding proteins hanging around. In addition RNA elements can probably fold in multiple ways as is the case with riboswitches.
OK enough! Time for happy hour ...
Mfold is great for testing a sequence that you plan on using for in vitro transcription, but as we all know it's relegated to the thermodynamically stable species. I've found that it useful to perform consequetive runs adding bases onto the 3' end to simulate co-transcriptional folding (lock in an upstream stable structure and see what happens as the 3' end grows). It was successful in helping a friend change/troubleshoot their design of a transfection construct to overexpress an RNA with a specific structure.
RNA folding is a b%#@&h of a problem. I think that I've stumbled onto a general RNA element found in many transcripts but MFold and other programs don't recognize anything special in these putative elements. I've also stumbled onto an interesting fold (as predicted by MFold) that might disrupt this element in a particular transcript. We should sitdown and talk ...