Structural basis for single-stranded RNA recognition and cleavage by C3PO

Abstract

Translin and translin-associated factor-x are highly conserved in eukaroytes; they can form heteromeric complexes (known as C3POs) and participate in various nucleic acid metabolism pathways. In humans and Drosophila, C3POs cleave the fragmented siRNA passenger strands and facilitate the activation of RNA-induced silencing complex, the effector complex of RNA interference (RNAi). Here, we report three crystal structures of Nanoarchaeum equitans (Ne) C3PO. The apo-NeC3PO structure adopts an open form and unravels a potential substrates entryway for the first time. The NeC3PO:ssRNA and NeC3PO:ssDNA complexes fold like closed football with the substrates captured at the inner cavities. The NeC3PO:ssRNA structure represents the only catalytic form C3PO complex available to date; with mutagenesis and in vitro cleavage assays, the structure provides critical insights into the substrate binding and the two-cation-assisted catalytic mechanisms that are shared by eukaryotic C3POs. The work presented here further advances our understanding on the RNAi pathway.

Structural insights into Drosophila-C3PO complex assembly and ‘Dynamic Side Port’ model in substrate entry and release

“…In addition, negative-stained EM structure of full-length Drosophila C3PO at ∼15 Å resolution and cryo-EM structure of human-C3PO at ∼15 Å resolution were also reported ( 23 ). Notably, all the full-length C3POs adopt closed football-shaped structures, which are very similar to previous reported Translin homo-octamer structures ( 8 , 21 , 22 , 24 ). Strikingly, in vitro mass-spectrometric analysis of Drosophila C3PO revealed a mixture of ratios of 2:6 and 3:5 (TRAX:Translin) in C3PO, which was partially supported by the reported N. equitans C3PO ( Ne- C3PO) structure ( 21 , 23 ).…”

“…Notably, all the full-length C3POs adopt closed football-shaped structures, which are very similar to previous reported Translin homo-octamer structures ( 8 , 21 , 22 , 24 ). Strikingly, in vitro mass-spectrometric analysis of Drosophila C3PO revealed a mixture of ratios of 2:6 and 3:5 (TRAX:Translin) in C3PO, which was partially supported by the reported N. equitans C3PO ( Ne- C3PO) structure ( 21 , 23 ). In addition, human-C3PO structures showed that the two catalytic TRAX subunits were packed side-by-side in a head-to-tail mode, with the active residues facing into the concaved interior cavity ( 21 ).…”

“…Strikingly, in vitro mass-spectrometric analysis of Drosophila C3PO revealed a mixture of ratios of 2:6 and 3:5 (TRAX:Translin) in C3PO, which was partially supported by the reported N. equitans C3PO ( Ne- C3PO) structure ( 21 , 23 ). In addition, human-C3PO structures showed that the two catalytic TRAX subunits were packed side-by-side in a head-to-tail mode, with the active residues facing into the concaved interior cavity ( 21 ). By contrast, Archaeoglobus fulgidus C3PO ( Af- C3PO) in complex with 14 bp dsRNA revealed a symmetric fold with the bound RNA duplex encapsulated within the inner cavity of Af- C3PO ( 24 ).…”

“…In prokaryotes, C3PO-like molecules with unknown biological functions were discovered based on sequence similarities and in vitro cleavage activities ( 18–20 ). The crystal structures of truncated Drosophila -C3PO at 3.4 Å resolution, human-C3PO at 2.9 Å resolution, Archaeoglobus fulgidus C3PO in complex with 14 bp dsRNA at 2.9 Å resolution and Nanoarchaeum equitans C3PO in complex with ssRNA at 2.1 Å resolution were determined ( 8 , 21–23 ). In addition, negative-stained EM structure of full-length Drosophila C3PO at ∼15 Å resolution and cryo-EM structure of human-C3PO at ∼15 Å resolution were also reported ( 23 ).…”

Prosapip1-Dependent Synaptic Adaptations in the Nucleus Accumbens Drive Alcohol Intake, Seeking, and Reward

“…Interestingly, three candidates are related to the microRNA (miRNA) gene-silencing machinery. Specifically, we found that Cnot4, a member of the Ccr4-Not complex that associates with RISC (Collart and Panasenko, 2012), the trinucleotide repeat containing 6a (Tnrc6a, also called GW182), a binding partner of Argonaute (Pfaff et al, 2013), and the trans-lin-associated factor X (Tsnax), which facilitates miRNA loading on RISC (Zhang et al, 2016), were enriched in the NAc polysomal fraction of alcohol-drinking mice but not in alcohol-consuming mice pre-treated with rapamycin. As mTORC1 has recently been shown to play a role in the regulation of miRNA production (Jewell et al, 2015), it would be of interest to assess the consequences of the increased translation of these targets by alcohol.…”