In-vivo secondary structure analysis of the small nuclear RNA U1 using psoralen cross-linking.
Intramolecular base-pairing interactions have been probed in the small nuclear RNA U1 in vivo. HeLa cells were treated with the psoralen derivative aminomethyltrioxsalen, and cross-linking was carried out by irradiating the intact cells with light of 365 nm wavelength. Cross-linking resulted in a discrete shift in electrophoretic mobility of approximately 65 to 70% of the U1. This intramolecularly cross-linked U1 RNA, termed XU1, was purified and shown to co-migrate with uncross-linked U1 upon photo-reversal of psoralen cross-links with light of 254 nm wavelength. XU1 was also generated by the in-vitro cross-linking of deproteinized U1, suggesting that the secondary structure of U1 RNA in solution is similar to that of U1 ribonucleoprotein in the cell. A sequencing analysis was developed, based on partial enzymatic and alkaline cleavage of psoralen-treated RNA, to identify the position of psoralen cross-links and to distinguish between psoralen monoadducts and diadducts (cross-links). Sequencing of 3' and 5' end-labeled XU1 provided direct evidence for the presence of a unique intramolecular cross-link in XU1, located on uridine 116 (U116). This result is consistent with several secondary-structure models for U1 in which U116 is located in a base-paired stem. The proximity of uridine 96 (U96) to U116 on the opposite side of the base-paired stem suggested that U116 was cross-linked to U96. An additional U1 species having an electrophoretic mobility between those of U1 and XU1 was also generated by psoralen treatment. Analysis of this U1 species, termed U1M, revealed a psoralen monoadduct on U96. Further longwave (365 nm) irradiation of purified U1M resulted in its conversion to XU1 by completion of the U96-U116 cross-link. This suggested that cross-linking at the U96-U116 site occurred as a two-step process in which the psoralen first reacted with U96 and then with U116. Sequencing analysis also identified a psoralen monoadduct on uridine 45 (U45) of XU1. Efficient psoralen-adduct formation, which resulted in cross-linking at the U96-U116 site and monoaddition on U45, suggests that these regions are relatively accessible in the native U1 small nuclear ribonucleoprotein particle in vivo.
Calvet
J P
JP
Department of Biochemistry, University of Kansas Medical Center, Kansas City 66103.
Myers
J A
JA
D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D012342:RNA, Small Nuclear / Q000187:drug effects; D014307:Trioxsalen / Q000031:analogs & derivatives / Q000494:pharmacology; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D012342:RNA, Small Nuclear; C018822:aminomethyltrioxsalen; D014307:Trioxsalen