1. RNA Remodeling by RNA Chaperones Monitored by RNA Structure Probing
Susann Friedrich, Tobias Schmidt, Sven-Erik Behrens Methods Mol Biol. 2020;2106:179-192. doi: 10.1007/978-1-0716-0231-7_11.
RNA structure probing enables the characterization of RNA secondary structures by established procedures such as the enzyme- or chemical-based detection of single- or double-stranded regions. A specific type of application involves the detection of changes of RNA structures and conformations that are induced by proteins with RNA chaperone activity. This chapter outlines a protocol to analyze RNA structures in vitro in the presence of an RNA-binding protein with RNA chaperone activity. For this purpose, we make use of the methylating agents dimethyl sulfate (DMS) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMCT). DMS and CMCT specifically modify nucleotides that are not involved in base-pairing or tertiary structure hydrogen bonding and that are not protected by a ligand such as a protein. Modified bases are identified by primer extension. As an example, we describe how the RNA chaperone activity of an isoform of the RNA-binding protein AUF1 induces the flaviviral RNA switch required for viral genome cyclization and viral replication.This chapter includes comprehensive protocols for in vitro synthesis of RNA, 32P-5'-end labeling of DNA primers, primer extension, as well as the preparation and running of analytical gels. The described methodology should be applicable to any other RNA and protein of interest to identify protein-directed RNA remodeling.
2. Normal parameters for diagnostic transcranial magnetic stimulation using a parabolic coil with biphasic pulse stimulation
Pimthong Jitsakulchaidej, Pakorn Wivatvongvana, Kittipong Kitisak BMC Neurol. 2022 Dec 31;22(1):510. doi: 10.1186/s12883-022-02977-8.
Background: TMS is being used to aid in the diagnosis of central nervous system (CNS) illnesses. It is useful in planning rehabilitation programs and setting appropriate goals for patients. We used a parabolic coil with biphasic pulse stimulation to find normal values for diagnostic TMS parameters. Objectives: 1. To determine the normal motor threshold (MT), motor evoked potentials (MEP), central motor conduction time (CMCT), intracortical facilitation (ICF), short-interval intracortical inhibition (SICI), and silent period (SP) values. 2. To measure the MEP latencies of abductor pollicis brevis (APB) and extensor digitorum brevis (EDB) at various ages, heights, and arm and leg lengths. Study design: Descriptive Study. Setting: Department of Rehabilitation Medicine, Chiang Mai University, Thailand. Subjects: Forty-eight healthy participants volunteered for the study. Methods: All participants received a single diagnostic TMS using a parabolic coil with biphasic pulse stimulation on the left primary motor cortex (M1). All parameters: MT, MEP, CMCT, ICF, SICI, and SP were recorded through surface EMGs at the right APB and EDB. Outcome parameters were reported by the mean and standard deviation (SD) or median and interquartile range (IQR), according to data distribution. MEP latencies of APB and EDB were also measured at various ages, heights, and arm and leg lengths. Results: APB-MEP latencies at 120% and 140% MT were 21.77 ± 1.47 and 21.17 ± 1.44 ms. APB-CMCT at 120% and 140% MT were 7.81 ± 1.32 and 7.19 ± 1.21 ms. APB-MEP amplitudes at 120% and 140% MT were 1.04 (0.80-1.68) and 2.24 (1.47-3.52) mV. EDB-MEP latencies at 120% and 140% MT were 37.14 ± 2.85 and 36.46 ± 2.53 ms. EDB-CMCT at 120% and 140% MT were 14.33 ± 2.50 and 13.63 ± 2.57 ms. EDB-MEP amplitudes at 120% and 140% MT were 0.60 (0.38-0.98) and 0.95 (0.69-1.55) mV. ICF amplitudes of APB and EDB were 2.26 (1.61-3.49) and 1.26 (0.88-1.98) mV. SICI amplitudes of APB and EDB were 0.21 (0.13-0.51) and 0.18 (0.09-0.29) mV. MEP latencies of APB at 120% and 140% MT were different between heights < 160 cm and ≥ 160 cm (p < 0.001 and p < 0.001) and different between arm lengths < 65 and ≥ 65 cm (p = 0.022 and p = 0.002). Conclusion: We established diagnostic TMS measurements using a parabolic coil with a biphasic pulse configuration. EDB has a higher MT than APB. The 140/120 MEP ratio of APB and EDB is two-fold. The optimal MEP recording for APB is 120%, whereas EDB is 140% of MT. CMCT by the F-wave is more convenient and tolerable for patients. ICF provides a twofold increase in MEP amplitude. SICI provides a ¼-fold of MEP amplitude. SP from APB and EDB are 121.58 ± 21.50 and 181.01 ± 40.99 ms, respectively. Height and MEP latencies have a modest relationship, whereas height and arm length share a strong positive correlation.
3. RNA Secondary Structure Study by Chemical Probing Methods Using DMS and CMCT
Fatima Alghoul, Gilbert Eriani, Franck Martin Methods Mol Biol. 2021;2300:241-250. doi: 10.1007/978-1-0716-1386-3_18.
RNA folds into secondary structures that can serve in understanding various RNA functions (Weeks KM. Curr Opin Struct Biol 20(3):295-304, 2010). Chemical probing is a method that enables the characterization of RNA secondary structures using chemical reagents that specifically modify RNA nucleotides that are located in single-stranded areas. In our protocol, we used Dimethyl Sulfate (DMS) and Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-p-Toluene sulfonate (CMCT) that are both base-specific modifying reagents (Behm-Ansmant I, et al. J Nucleic Acids 2011:408053, 2011). These modifications are mapped by primer extension arrests using 5' fluorescently labeled primers. In this protocol, we show a comprehensive method to identify RNA secondary structures in vitro using fluorescently labeled oligos. To demonstrate the efficiency of the method, we give an example of a structure we have designed which corresponds to a part of the 5'-UTR regulatory element called Translation Inhibitory Element (TIE) from Hox a3 mRNA (Xue S, et al. Nature 517(7532):33-38, 2015).
