To start DNA replication, the Origin Recognition Complex (ORC) and Cdc6 load a Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase loading intermediate. Here we report a 3.9Å structure of the OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHD) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an α-helix in Orc4 and positively charged loops in Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by a Mcm5 loop that embraces Mcm2, but the N-tier ring Mcm2-Mcm5 interface remains open. This structure suggests loading mechanics of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.

The RNA binding proteome ( RBP ome) was previously investigated using UV crosslinking and purification of poly(A)‐associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA ‐associated protein purification ( TRAPP ) based on 254 nm UV crosslinking and purification of all RNA –protein complexes using silica beads. In a variant approach ( PAR ‐ TRAPP ), RNA s were labelled with 4‐thiouracil prior to 350 nm crosslinking. PAR ‐ TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBP s. In comparison, TRAPP identified many more proteins not expected to bind RNA , and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNA s. Exploiting PAR ‐ TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA –peptide conjugates, using iTRAPP , providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBP ome characterization.

BackgroundTRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized.ResultsHere, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25’s endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP.ConclusionsOur results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-017-0444-9) contains supplementary material, which is available to authorized users.