Molecular Mechanism Of RNA Structures Remodeling By The DExH/D-box RNA Helicase RHA

RNA helicases are present in almost all living organisms,including animals,plants,humans,and so on,widely involved in various biological processes relate to RNA metabolism,such as activation of transcription and promotion of translation,etc.RNA helicases can be divided into multiple superfamilies,and the most common is the superfamily2（SF2）.The human RNA helicase A（RHA）investigated in this paper belonged to SF2,was a member of DEx H/D-box family,also named as NDH II or DHX9.RHA was mainly found in the nucleus,and it was a protein that shuttled between the nucleus and the cytoplasm.RHA has two double-stranded RNA binding domains ds RBD1 and ds RBD2 at the amino terminal,DEIH and HELICc at the helicase core region,HA2,OB-fold and RGG at the carboxyl terminal.The current researches on the mechanism of RHA were not in-depth,and the research methods are mainly biochemical methods at the overall level,lack of research methods at the single molecular level.Aiming at these problems,eukaryotic and prokaryotic expression systems were used to express RHA firstly in this paper,and obtained full-length RHA protein with good activity.Then,biochemical methods combined with single molecule fluorescence resonance energy transfer technique were used to investigate the molecular mechanism of RHA remodeling RNA structures,and RHA-assisted RNA structure conversion was observed at the single molecule level in real time.The main experimental works in this paper were as follows:1 Expression and purification of RNA helicase RHARHA performs various functions in organisms,and the acquisition of RHA protein is the premise to study the mechanism of action of RHA in vitro.The baculovirus system with adherent Sf9 as host cells and the mammalian cell system with suspended 293F as host cells were used for the expression of full-length RHA in this paper.Sf9 cells were infected by baculovirus,which integrated full-length RHA gene,and obvious lesions of Sf9 cells could be observed after 72 h,and then harvested cells.And 293F cells were harvested after cells were transfected with the recombinant plasmid for 48 h.After the cells were broken by ultrasound,Ni²⁺-NTA-agarose and Capto DEAE Sepharose were used for two-step purification of RHA.The results showed that the expression of full-length RHA in Sf9 cells was significant higher than that in 293F cells,and the RHA expressed in two kinds of cells all had good activity.In addition,E.coli prokaryotic expression system was also used to express the deletion mutated RHA,but the protein was difficult to be expressed.2 Investigation of the molecular mechanism of different types of ds RNA unwinding by RHAThe various biological processes that RHA involved are closely related to its unwinding activity,and the study of the unwinding mechanism is particularly important to understand how RHA participates in these biological processes.RNA helicases have two common unwinding fashions,local strand separation and directional translocation,RHA is a typical translocation helicase,and the process of unwinding is directed from 3’to 5’ends,ds RNA with different terminals were designed in this paper,and the experiments verified that the substrate ds RNA of RHA needed to have a single strand extension at the 3’end（3’-overhang）,that is,ds RNA with 5’-overhang or blunt end were not be unwound by RHA.In order to explore the minimum length of 3’-overhang of ds RNA that enables RHA to perform its unwinding activity,substrates with 19 bp double strand were designed in this paper,and the lengths of 3’-overhang of the different substrates were 5 nt,6 nt,7 nt and 10 nt,respectively.The results of unwinding experiments indicated that the ds RNA unwound by RHA needed a3’-overhang with not less than 6 nt.In addition,three ds RNA substrates with double strand length of 19 bp were uesd in this paper,and the 3’-overhang length of these ds RNA were 10 nt,16 nt and 22 nt,respectively.These substrates were used in the unwinding experiments to obtain the kinetic data,such as the unwinding rates and the final unwound amplitudes,then these data were fitted.The results indicated that the length of 3’-overhang is helpful for the unwinding reaction,and the unwinding rates and final unwound amplitudes were increased with the length of 3’-overhang;The high concentrations of RHA also had the activity to promote the reannealing of the unwinding products ss RNA to form ds RNA;and RHA participated in the unwinding reaction as a polymer.Furthermore,three nonhydrolyzabel ATP analogs,adenylyl imidodiphosphonate（ADPNP）,ADP-beryllium fluoride（ADP-Be F_X）and ADP-aluminum fluoride（ADP-Al F₄）,were used to replace the ATP in the unwinding reaction of RHA,the results showed that RHA could not perform the helicase activity with the participation of non-hydrolyzed ATP analogs,that is,RHA indeed required the energy from ATP hydrolysis to unwind ds RNA.3 Investigation of the molecular mechanism of RNA structure conversion regulated by RHA at the single molecule levelRHA not only has the helicase activity to unwind ds RNA,but also has the annealing activity to promote ss RNA to form ds RNA.On this basis,experiments were carried out to explore whether RHA regulated RNA structure conversion against thermodynamic equilibrium,that is,whether RHA promoted the coversion from stable to unstable structure.Three RNA strands used in experiments were named X,Y,and Z,and the RNA sequences were from the internal ribosome entry site（IRES）of foot-and-mouth disease virus.The 5’end of X was labeled with Cy5,the 5’end and 3’end of Y were labeled with biotin and Cy3,respectively,the Z was not labeled.The bases of Y and Z were complementary paired to form ds RNA YZ without loops structure,while the X and Y formed ds RNA XY with two loops structure.In theory,ds RNA YZ was more stable than ds RNA XY.Here,determination of melting temperature（Tm）confirmed that ds RNA YZ was structurally more stable than XY,and then observed RHA promoted ds RNA YZ and ss RNA X convert into ds RNA XY in presence of ATP by non-denaturing PAGE and fluorescence intensity measurement experiments,it was first found that RHA regulated the RNA structure conversion against thermodynamic equilibrium in vitro.In order to obtain more dynamic data of structure conversion,the single molecule fluorescent resenonse energy transfer technology was also used in this paper,realized the synchronization of sampling and acquiring images by the self-designed manual real-time sampling device,and observed RHA-assisted RNA structure conversion at the single molecule level in real time.The fluorescent molecular traces obtained at different concentrations of ATP were analyzed,and the results shown that that intermediate structure generated in the RHA-assisted conversion.Moreover,the results of the biochemical experiments and single molecule experiments indicated that RHA-assisted RNA structure conversion against thermodynamic equilibrium in an ATP-dependent manner.The higher concentration of ATP increased the rates of RNA structure conversion and producted more intermediate structure and final structure ds RNA XY.RHA has a variety of biological functions,not only promotes the replication of various viruses,but also participates in the antiviral immunity of the organisms.Thus,research on the mechanism of action of RHA is particularly important to understand how it performs various biological functions,especially its functional localization in promoting viral replication and antiviral immunity.