Dynamics of RNA-protein interactions in the HIV-1 Rev-RRE complex visualized by 6-thioguanosine-mediated photocrosslinking.
Expression of the structural proteins of human immunodeficiency virus type 1 (HIV-1) requires the direct interaction of multiple copies of the viral protein Rev with its target RNA, the Rev response element (RRE). RRE is a complex 351-nt RNA that is highly structured and located within the viral env gene. During initial Rev-RRE recognition, Rev binds with high affinity to a bubble structure located within the RRE RNA stem-loop II. We have used a site-specific photocrosslinking method based on 6-thioguanosine (6-thioG) photochemistry to probe the conformation of the high-affinity binding site of RRE RNA and its interactions with Rev protein under physiological conditions. A minimal duplex RNA containing the bubble region of RRE and 12 flanking base pairs was synthesized chemically. Two different RRE constructs with a single photoactive nucleoside (6-thio-dG or 6-thioG) at position 47 or 48 were synthesized. Upon UV irradiation, 6-thioG at both positions formed interstrand covalent crosslinks in RRE RNA. Mapping of crosslink sites by RNA sequencing revealed that 6-thioG at position 47 or 48 crosslinked to A73. In the presence of Rev, both RNA-RNA and RNA-protein crosslinks were observed, however, the RNA-RNA crosslink site was unchanged. Our results provide direct evidence that, during RNA-protein recognition, Rev is in close proximity to O6 of G47 and G48 in the major groove of RRE RNA. Our results also show that the bubble region of RRE RNA has a biologically relevant structure where G47 and G48 are in close proximity to A73 and this RNA structure is not changed significantly upon Rev binding. We propose that Rev protein recognizes and binds to specific structural elements of RRE RNA containing non-Watson-Crick base pairs and such structures could be a determinant for recognition by other RNA-binding proteins. Our site-specific crosslinking methods provide a general approach to capture dynamic states of biologically relevant RNA structures that are otherwise missed by NMR and X-ray crystallographic studies.
Ping
Y H
YH
Department of Pharmacology, Robert Wood Johnson Medical School-UMDNJ, and Microbiology & Molecular Genetics Graduate Program at Rutgers, The State University of New Jersey, Piscataway, 08854, USA.
Liu
Y
Y
Wang
X
X
Neenhold
H R
HR
Rana
T M
TM
D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015694:Gene Products, rev / Q000737:chemistry / Q000378:metabolism*; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry; D015497:HIV-1 / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017421:Sequence Analysis; D013379:Substrate Specificity; D013872:Thionucleosides / Q000737:chemistry; D014466:Ultraviolet Rays; D054321:rev Gene Products, Human Immunodeficiency Virus
D003432:Cross-Linking Reagents; D015694:Gene Products, rev; D013872:Thionucleosides; D054321:rev Gene Products, Human Immunodeficiency Virus; D006151:Guanosine; D012313:RNA; C005783:6-thioguanosine