Overlapping and distinct functions of CstF64 and CstF64τ in mammalian mRNA 3′ processing

mRNA 3′ processing is dynamically regulated spatially and temporally. However, the underlying mechanisms remain poorly understood. CstF64τ is a paralog of the general mRNA 3′ processing factor, CstF64, and has been implicated in mediating testis-specific mRNA alternative polyadenylation (APA). However, the functions of CstF64τ in mRNA 3′ processing have not been systematically investigated. We carried out a comprehensive characterization of CstF64τ and compared its properties to those of CstF64. In contrast to previous reports, we found that both CstF64 and CstF64τ are widely expressed in mammalian tissues, and their protein levels display tissue-specific variations. We further demonstrated that CstF64 and CstF64τ have highly similar RNA-binding specificities both in vitro and in vivo. CstF64 and CstF64τ modulate one another''s expression and play overlapping as well as distinct roles in regulating global APA profiles. Interestingly, protein interactome analyses revealed key differences between CstF64 and CstF64τ, including their interactions with another mRNA 3′ processing factor, symplekin. Together, our study of CstF64 and CstF64τ revealed both functional overlap and specificity of these two important mRNA 3′ processing factors and provided new insights into the regulatory mechanisms of mRNA 3′ processing.     

Structure and mechanism of E. coli RNA 2′,3′-cyclic phosphodiesterase

2H (two-histidine) phosphoesterase enzymes are distributed widely in all domains of life and are implicated in diverse RNA and nucleotide transactions, including the transesterification and hydrolysis of cyclic phosphates. Here we report a biochemical and structural characterization of the Escherichia coli 2H protein YapD, which was identified originally as a reversible transesterifying “nuclease/ligase” at RNA 2′,5′-phosphodiesters. We find that YapD is an “end healing” cyclic phosphodiesterase (CPDase) enzyme that hydrolyzes an _HORNA>p substrate with a 2′,3′-cyclic phosphodiester to a _HORNAp product with a 2′-phosphomonoester terminus, without concomitant end joining. Thus we rename this enzyme ThpR (two-histidine 2′,3′-cyclic phosphodiesterase acting on RNA). The 2.0 Å crystal structure of ThpR in a product complex with 2′-AMP highlights the roles of extended histidine-containing motifs ⁴³HxTxxF⁴⁸ and ¹²⁵HxTxxR¹³⁰ in the CPDase reaction. His43-Nε makes a hydrogen bond with the ribose O3′ leaving group, thereby implicating His43 as a general acid catalyst. His125-Nε coordinates the O1P oxygen of the AMP 2′-phosphate (inferred from geometry to derive from the attacking water nucleophile), pointing to His125 as a general base catalyst. Arg130 makes bidentate contact with the AMP 2′-phosphate, suggesting a role in transition-state stabilization. Consistent with these inferences, changing His43, His125, or Arg130 to alanine effaced the CPDase activity of ThpR. Phe48 makes a π–π stack on the adenine nucleobase. Mutating Phe28 to alanine slowed the CPDase by an order of magnitude. The tertiary structure and extended active site motifs of ThpR are conserved in a subfamily of bacterial and archaeal 2H enzymes.     

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes, including 15 putative RNA ligases, one of which, NgrRnl, is a eukaryal homolog of Deinococcus radiodurans RNA ligase, DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3′-OH/5′-PO₄ duplexes in which the 3′-OH strand is RNA. It does so via the “classic” ligase pathway, entailing reaction with ATP to form a covalent NgrRnl–AMP intermediate, transfer of AMP to the nick 5′-PO₄, and attack of the RNA 3′-OH on the adenylylated nick to form a 3′–5′ phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases, NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead, it contains a defining amino-terminal domain that we show is important for 3′-OH/5′-PO₄ nick-sealing and ligase adenylylation, but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl, DraRnl, and their homologs from diverse bacteria, viruses, and unicellular eukarya comprise a new “Rnl5 family” of nick-sealing ligases with a signature domain organization.