RESUMO
N-methyladenosine (mA), a ubiquitous RNA modification, is installed by METTL3-METTL14 complex. The structure of the heterodimeric complex between the methyltransferase domains (MTDs) of METTL3 and METTL14 has been previously determined. However, the MTDs alone possess no enzymatic activity. Here we present the solution structure for the zinc finger domain (ZFD) of METTL3, the inclusion of which fulfills the methyltransferase activity of METTL3-METTL14. We show that the ZFD specifically binds to an RNA containing 5'-GGACU-3' consensus sequence, but does not to one without. The ZFD thus serves as the target recognition domain, a structural feature previously shown for DNA methyltransferases, and cooperates with the MTDs of METTL3-METTL14 for catalysis. However, the interaction between the ZFD and the specific RNA is extremely weak, with the binding affinity at several hundred micromolar under physiological conditions. The ZFD contains two CCCH-type zinc fingers connected by an anti-parallel β-sheet. Mutational analysis and NMR titrations have mapped the functional interface to a contiguous surface. As a division of labor, the RNA-binding interface comprises basic residues from zinc finger 1 and hydrophobic residues from β-sheet and zinc finger 2. Further we show that the linker between the ZFD and MTD of METTL3 is flexible but partially folded, which may permit the cooperation between the two domains during catalysis. Together, the structural characterization of METTL3 ZFD paves the way to elucidate the atomic details of the entire process of RNA mA modification.
RESUMO
Objective To evaluate the impact of breathing motion on target volume and the factors influencing the set-up errors during tangential whole breast irradiation.Methods From Jan 2003 to Dec 2003,patients with early-stage breast cancer after breast conserving surgery,were selected to be eligible for the study.All patients were immobilized in treatment position by breast beard of Med-Tec 250.The motion of the breast treatment volume was observed on a fluoroscope in different directions under free breathing in 16 patients.The set-up errors in different dimensions during irradiation were measured by weekly portal films (PF) in comparison with digital reconstructed radiographs (DRR) in 11 patients.Results The central lung distance (CLD) variation during free breathing was (2.1?1.2) mm which is greater than the motion to- wards the other directions.By comparing the PF and DRR,the systemic error,random error and overall er- ror in the outer,inner and cranio-caudal directions was 1.9,1.6,2.5 and 2.4,1.7,3.1 and 2.6,2.3, 3.5 mm,respectively.In addition,the discrepancy of the treatment position in cranio-caudal direction and breast volume was most obvious at the beginning 2 weeks with the peak of breast volume at the second week. It decreased gradually during the following 3 weeks.Conclusions This study suggests that the mean value of the motion of the breast target volume during one breathing cycle is less than 2 mm.The set-up errors dur- ing irradiation is the greatest in cranio-caudal direction,suggesting that the fixing precision of the breast board should be further improved.The set-up error during irradiation are most obvious at the beginning two weeks,with the peak of the breast volume in the second week.