Tuberous sclerosis complex (TSC) integrates upstream stimuli and regulates cell growth by controlling the activity of mTORC1. TSC complex functions as a GTPase-activating protein (GAP) towards small GTPase Rheb and inhibits Rheb-mediated activation of mTORC1. Mutations in TSC genes cause tuberous sclerosis. In this study, the near-atomic resolution structure of human TSC complex reveals an arch-shaped architecture, with a 2:2:1 stoichiometry of TSC1, TSC2, and TBC1D7. This asymmetric complex consists of two interweaved TSC1 coiled-coil and one TBC1D7 that spans over the tail-to-tail TSC2 dimer. The two TSC2 GAP domains are symmetrically cradled within the core module formed by TSC2 dimerization domain and central coiled-coil of TSC1. Structural and biochemical analyses reveal TSC2 GAP-Rheb complimentary interactions and suggest a catalytic mechanism, by which an asparagine thumb (N1643) stabilizes γ-phosphate of GTP and accelerate GTP hydrolysis of Rheb. Our study reveals mechanisms of TSC complex assembly and GAP activity.

5-Methylcytosine is found in both DNA and RNA; although its functions in DNA are well established, the exact role of 5-methylcytidine (m⁵C) in RNA remains poorly defined. Here we identified, by employing a quantitative proteomics method, multiple candidate recognition proteins of m⁵C in RNA, including several YTH domain-containing family (YTHDF) proteins. We showed that YTHDF2 could bind directly to m⁵C in RNA, albeit at a lower affinity than that toward N⁶-methyladenosine (m⁶A) in RNA, and this binding involves Trp⁴³², a conserved residue located in the hydrophobic pocket of YTHDF2 that is also required for m⁶A recognition. RNA bisulfite sequencing results revealed that, after CRISPR-Cas9-mediated knockout of the YTHDF2 gene, the majority of m⁵C sites in rRNA (rRNA) exhibited substantially augmented levels of methylation. Moreover, we found that YTHDF2 is involved in pre-rRNA processing in cells. Together, our data expanded the functions of the YTHDF2 protein in post-transcriptional regulations of RNA and provided novel insights into the functions of m⁵C in RNA biology.