Elucidating the Kinetic Mechanism of Human METTL16.
Elucidating the Kinetic Mechanism of Human METTL16.
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DOI：
10.1021/acs.biochem.2c00601
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发表时间：
2023-01-17
期刊：
影响因子：
2.9
通讯作者：
Brown, Jessica A.
中科院分区：
文献类型：
--
作者：
Breger, Kurtis;Brown, Jessica A.
甲基转移酶样蛋白16(METTL16)是人类体内四种具有催化活性的依赖S腺苷蛋氨酸的m6ARNA甲基转移酶之一。众所周知，METTL16的甲基化靶点是U6小核RNA(U6 SnRNA)和MAT2AmRNA发夹；然而，METTL16可与其他RNA结合，包括转移相关肺腺癌转录本1(MALAT1)的3‘三螺旋。在此，我们对METTL16的动力学机制和生化性质进行了研究。METTL16是一种单体，与MALAT1三螺旋或U6单链RNA形成复合体，与这些RNA结合的解离常数分别为31 nM和18 nM，而与甲基化的U6单链RNA产物的结合为1.1μM。利用U6单链RNA研究甲基化步骤、预孵育和同位素分配试验表明，METTL16在SAM之前与U6单链RNA结合是有序的顺序机制。MetTL16·U6SnRNA·SAM三元复合体的表观解离常数为126min-M。稳态动力学测定的kcat为0.07min-1，单周转率测定为0.56min-1。此外，MetTL16甲基化U6SnRNA的甲基转移酶结构域的表观解离常数为736min-1，kch为0.42min-1，表明缺失的脊椎动物保守区削弱了三元复合体，但不会引起U6SnRNA的限速构象重排。这项研究有助于我们从生物学功能的角度更好地理解METTL16的催化活性。
Methyltransferase-like protein 16 (METTL16) is one of four catalytically active, S-adenosylmethionine (SAM)-dependent m6A RNA methyltransferases in humans. Well-known methylation targets of METTL16 are U6 small nuclear RNA (U6 snRNA) and the MAT2A mRNA hairpins; however, METTL16 binds to other RNAs, including the 3′ triple helix of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Herein, we investigated the kinetic mechanism and biochemical properties of METTL16. METTL16 is a monomer in complex with either the MALAT1 triple helix or U6 snRNA and binds to these RNAs with respective dissociation constants of 31 nM and 18 nM, whereas binding to the methylated U6 snRNA product is 1.1 μM. The MALAT1 triple helix, on the other hand, is not methylated by METTL16 under in vitro conditions. Using the U6 snRNA to study methylation steps, preincubation and isotope partitioning assays indicated an ordered-sequential mechanism, whereby METTL16 binds U6 snRNA before SAM. The apparent dissociation constant for the METTL16·U6 snRNA·SAM ternary complex is 126 μM. Steady-state kinetic assays established a kcat of 0.07 min–1, and single-turnover assays established a kchem of 0.56 min–1. Furthermore, the methyltransferase domain of METTL16 methylated U6 snRNA with an apparent dissociation constant of 736 μM and a kchem of 0.42 min–1, suggesting that the missing vertebrate conserved regions weaken the ternary complex but do not induce any rate-limiting conformational rearrangements of the U6 snRNA. This study helps us to better understand the catalytic activity of METTL16 in the context of its biological functions.
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影响因子：
48
作者：
Linder, Bastian;Grozhik, Anya V.;Olarerin-George, Anthony O.;Meydan, Cem;Mason, Christopher E.;Jaffrey, Samie R.
通讯作者：
Jaffrey, Samie R.
影响因子：
4.8
作者：
Kim, Youngchang;Babnigg, Gyorgy;Jedrzejczak, Robert;Eschenfeldt, William H.;Li, Hui;Maltseva, Natalia;Hatzos-Skintges, Catherine;Gu, Minyi;Makowska-Grzyska, Magdalena;Wu, Ruiying;An, Hao;Chhor, Gekleng;Joachimiak, Andrzej
通讯作者：
Joachimiak, Andrzej
影响因子：
14.9
作者：
Clancy, MJ;Shambaugh, ME;Bokar, JA
通讯作者：
Bokar, JA
影响因子：
4.1
作者：
LITTLEFIELD, JW;DUNN, DB
通讯作者：
DUNN, DB
DOI：
10.1007/978-1-62703-113-4_19
发表时间：
2012
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Kershaw, Christopher J;O'Keefe, Raymond T
通讯作者：
O'Keefe, Raymond T