Regulation of the thiamine pyrophosphate (TPP)‐sensing riboswitch in NMT1 mRNA from Neurospora crassa

Abstract

The expression of Neurospora crassa NMT1 involved in thiamine pyrophosphate (TPP) metabolism is regulated at the level of mRNA splicing by a TPP‐sensing riboswitch within the precursor NMT1 mRNA. Here, using the systematic helix‐based computational method, we investigated the regulation of this riboswitch. We find that the function of the riboswitch does not depend on the transcription process. Whether TPP is present or not, the riboswitch predominately folds into the ON state, while the OFF state aptamer structure does not appear during transcription. Since the transition from the ON state to the aptamer structure is extremely slow, TPP may interact with the RNA before full formation of the aptamer structure, promoting the switch flipping. The potential to fully form helix P0 of the ON state is necessary to restore ligand‐dependent gene control by the riboswitch.

Intrinsic Regulatory Role of RNA Structural Arrangement in Alternative Splicing Control

“…In consequence of TPP binding, the large structural rearrangement is believed to enable the selection of alternative 5′ss by the spliceosome with concomitant repression of a branch site, resulting in reduction of nmt1 expression. Intriguingly, Gong and others provided even deeper elucidation of the kinetics of TPP-riboswitch structural response by exploiting a systematic helix-based computational method [ 260 ]. The authors proposed a co-transcriptional mode of TPP binding before the riboswitch folds into thermodynamically optimal “ON” state in order to facilitate the otherwise extremely slow and energetically excessive structure transition.…”

Regulation of the ThiM riboswitch is facilitated by the trapped structure formed during transcription of the wild‐type sequence

“…As a kind of RNA‐based gene devices, riboswitches regulate downstream gene expression by forming one of the two alternative functional states (unbound/bound) in response to specific signals. Like the NMT1 TPP and metF riboswitches [35,76], the ThiM riboswitch has a well‐known bound functional state but an unclear ligand‐free functional state, which is difficult to refine by crystallography or NMR spectroscopy. According to the populational kinetics of states formed during the transcription, the helix‐based RNA folding theory successfully reveals the TPP‐induced switch mechanism and its functional structures.…”

“…If the switchpoint locates near the end of transcription, such as that in the NMT1 TPP (Fig. 7B) and SAM‐III riboswitches [35,76], the above factors will not have a big impact on their performances. For these riboswitches, ligand binding could perturb the equilibrium toward the bound functional state at the decision point.…”

“…To improve the computational efficiency, the transition node approximation is employed to decrease the calculated conformational space when the nascent RNA chain is longer than 120 nt [35,37,38]. Assuming that min E is the highest free energy of the states with big initial populations (a cutoff population of 10%) at one step, the newly formed states with energies lower than (min E + 3) kcal·mol −1 possibly have accumulated populations.…”

“…By incorporating native secondary structural information, the kinetic Monte Carlo (MC) simulation gives access to investigate co‐transcriptional folding on the secondary structure level via comparison with experimental results to get the relationship between real time and MC steps [29]. Based on the similar parameters as kinetic MC, the helix‐based RNA folding theory has been proven to be a reliable approach to study mechanisms of riboswitches and functions of other mRNAs [35–40]. Compared to the above methods, it is quite suitable to feature the incorporation of competing conformations and ligand‐binding kinetics for riboswitches [36,38,41].…”