N-terminal α-methylation of RCC1 is necessary for stable chromatin association and normal mitosis

Abstract

Regulator of chromatin condensation 1 (RCC1) is the only known guanine nucleotide-exchange factor for the Ran GTPase and has pivotal roles in nucleo-cytoplasmic transport, mitosis, and nuclear-envelope assembly 1 . RCC1 associates dynamically with chromatin through binding to histones H2A and/or H2B in a Ran-regulated manner 2,3 . Here, we report that, unexpectedly, the amino-terminal serine or proline residue of RCC1 is uniquely methylated on its α-amino group. Methylation requires removal of the initiating methionine, and the presence of proline and lysine at positions 3 and 4, respectively. Methylation-defective mutants of RCC1 bind less effectively than wild-type protein to chromatin during mitosis, which causes spindle-pole defects. We propose a bimodal attachment mechanism for RCC1 in which the tail promotes stable RCC1 association with chromatin through DNA binding in an α-N-methylation-dependent manner. These data provide the first known function for N-terminal protein methylation.RCC1 has a propeller-like structure, one face of which binds to Ran 4 , whereas the other face binds chromatin. A flexible N-terminal tail contains a nuclear localization signal (NLS) 5,6 . Although the propeller seems rigid, Ran allosterically modulates RCC1 association with chromatin 3 , and the RCC1 D182A mutant, which does not detectably bind Ran, shows reduced chromatin association 7,8 . Removal of the N-terminal tail also leads to exclusion of RCC1 from chromosomes 7 , whereas phosphorylation on Ser 2 and Ser 11 in human RCC1 reportedly permits stable association with mitotic chromosomes 8,9 . These phosphorylations have been proposed to reduce the affinity of the NLS for its nuclear transport receptor, Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ 4 Correspondence should be addressed to I.G.M. (igm9c@virginia.edu).Note: Supplementary Information is available on the Nature Cell Biology website. AUTHOR CONTRIBUTIONST.C. made the RCC1 mutant proteins, identified the methyltransferase activity and its recognition motif, and performed the assays to identify its function. T.L.M. and J.S. performed the mass spectrometry. C.E.S.-T. made the antibodies against methylated RCC1 and performed the experiments with them. D.F.H. directed the mass spectrometry. I.G.M. directed the biochemical and cell biological studies. T.C. and I.G.M. wrote the paper. All authors discussed results and contributed to the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript importin-α3, which, when bound to RCC1, interferes with chromatin attachment 9 . However, Ser 2 is found only in simians and is a proline residue in other mammalian species.The N-terminus of RCC1 resembles histone tails, which also contain NLSs, and which are phosphorylated, acetylated and methylated in complex patterns that comprise a histone code 10,11 . We asked whether ...

Importin-β Is a GDP-to-GTP Exchange Factor of Ran

“…This hypothesis is further supported by the fact that RanBP1 mutants are defective in nuclear import (35). A mechanism involving an in situ formation of Ran-GTP in the transport complex would present the advantage of being independent of RCC1 and Ran-GTP, two versatile proteins that are involved in many vital functions in the cell other than nucleocytoplasmic transport, such as chromatin condensation, mitosis regulation, spindle assembly, and post-mitotic nuclear envelope assembly (2,48). In different physiological stages of the cell, their concentrations may vary significantly.…”

Localized RanGTP Accumulation Promotes Microtubule Nucleation at Kinetochores in Somatic Mammalian Cells

“…Functional spindles also form in cells lacking centrosomes, e.g., in higher plants and some animal oocytes. In Xenopus eggs, spindles assemble through MT nucleation from chromatin in a process requiring the guanosine triphosphatase (GTPase) Ran (Carazo-Salas et al, 1999;Kalab et al, 1999) which, in its GTP-bound form, is generated near chromatin under the localized action of the nucleotide exchange factor RCC1 (Moore et al, 2002;Li et al, 2003;Chen et al, 2007). In the Xenopus system, RanGTP releases spindle assembly factors containing nuclear localization signals (NLSs), such as NuMa and TPX2 (Gruss et al, 2001;Nachury et al, 2001;Wiese et al, 2001), from the inhibitory binding to importin ␣/␤, in the immediate vicinity of chromatin, thus promoting nucleation of MTs therein (reviewed by Weis, 2003;Ciciarello et al, 2007).…”

Structural basis for substrate recognition by the human N-terminal methyltransferase 1

“…α-N-terminal methylation has been hypothesized to regulate protein stability via N-end rule pathways or mediate protein-protein interactions (Tooley and Schaner Tooley 2014). Intriguingly, α-N-terminal methylation was recently found to mediate protein-DNA interactions between chromatin and regulator of chromatin condensation (RCC1) (Chen et al 2007;Hitakomate et al 2010), potentially through the positively charged and exhaustively methylated N terminus of RCC1. RCC1 is the only known guanine nucleotide exchange factor for the Ran GTPase, which plays indispensable roles in nucleocytoplasmic transport, mitosis, and nuclear envelope assembly (Chen et al 2007).…”

“…Intriguingly, α-N-terminal methylation was recently found to mediate protein-DNA interactions between chromatin and regulator of chromatin condensation (RCC1) (Chen et al 2007;Hitakomate et al 2010), potentially through the positively charged and exhaustively methylated N terminus of RCC1. RCC1 is the only known guanine nucleotide exchange factor for the Ran GTPase, which plays indispensable roles in nucleocytoplasmic transport, mitosis, and nuclear envelope assembly (Chen et al 2007). Notably, α-N-terminal methyltransferase 1 (NTMT1/ NRMT1/METTL11A) catalyzes the N-terminal methylation of RCC1, representing a novel regulatory mechanism for the Ran GTPase activity (Tooley et al 2010;Webb et al 2010).…”

“…Notably, α-N-terminal methyltransferase 1 (NTMT1/ NRMT1/METTL11A) catalyzes the N-terminal methylation of RCC1, representing a novel regulatory mechanism for the Ran GTPase activity (Tooley et al 2010;Webb et al 2010). Abrogation of N-terminal methylation of RCC1 by mutations or knockdown of NTMT1 significantly diminishes the binding ability and association of RCC1 to chromatin and causes spindle pole defects and aberrant mitosis, underlining the functional significance of NTMT1-mediated α-N-methylation in mitotic spindle assembly and chromosome segregation (Chen et al 2007). …”