Analysis of the separation between the ends of mRNA molecules from different organisms by using computational algorithms and smFRET

Abstract

Considering that the innate proximity of RNA ends might have important unknown biological implications, we aimed to determine whether the close proximity of the ends of mRNA molecules is a conserved feature across organisms and gain further insights into the functional effects of the proximity of RNA ends. We present two projects in this thesis; the first one comprises the study of the secondary structure of 274 full native mRNA molecules from 17 different organisms to calculate the contour length (CL) of the external loop as an index of their end-to-end separation. Our computational predictions show bigger variations than previously reported and also than those observed in random sequences. From this project, we found that our results suggest that separations larger than 18.5 nm are not favored, whereas short separations could be related to phenotypical stability. Overall, the results obtained implies the existence of a biological mechanism responsible for the increase in the observed variability, suggesting that the CL features of the exterior loop could be relevant for the initiation of translation, and that a short CL could contribute to the stability of phenotypes. The second one, comprise the single molecule Fluorescence Resonance Energy Transfer (smFRET) system design and calibration to perform the experimental in vitro measurements of the distance between mRNAs ends from 4 organisms from the Eukarya domain. From the second project, we obtained only preliminary results, and some experiments are pending to be performed.