A viral ADP-ribosyltransferase attaches RNA chains to host proteins.

The mechanisms by which viruses hijack the genetic machinery of the cells they infect are of current interest. When bacteriophage T4 infects Escherichia coli, it uses three different adenosine diphosphate (ADP)-ribosyltransferases (ARTs) to reprogram the transcriptional and translational apparatus of the host by ADP-ribosylation using nicotinamide adenine dinucleotide (NAD) as a substrate1,2. NAD has previously been identified as a 5' modification of cellular RNAs3-5. Here we report that the T4 ART ModB accepts not only NAD but also NAD-capped RNA (NAD-RNA) as a substrate and attaches entire RNA chains to acceptor proteins in an 'RNAylation' reaction. ModB specifically RNAylates the ribosomal proteins rS1 and rL2 at defined Arg residues, and selected E. coli and T4 phage RNAs are linked to rS1 in vivo. T4 phages that express an inactive mutant of ModB have a decreased burst size and slowed lysis of E. coli. Our findings reveal a distinct biological role for NAD-RNA, namely the activation of the RNA for enzymatic transfer to proteins. The attachment of specific RNAs to ribosomal proteins might provide a strategy for the phage to modulate the host's translation machinery. This work reveals a direct connection between RNA modification and post-translational protein modification. ARTs have important roles far beyond viral infections6, so RNAylation may have far-reaching implications.

APC couples neuronal mRNAs to multiple kinesins, EB1, and shrinking microtubule ends for bidirectional mRNA motility.

Understanding where in the cytoplasm mRNAs are translated is increasingly recognized as being as important as knowing the timing and level of protein expression. mRNAs are localized via active motor-driven transport along microtubules (MTs) but the underlying essential factors and dynamic interactions are largely unknown. Using biochemical in vitro reconstitutions with purified mammalian proteins, multicolor TIRF-microscopy, and interaction kinetics measurements, we show that adenomatous polyposis coli (APC) enables kinesin-1- and kinesin-2-based mRNA transport, and that APC is an ideal adaptor for long-range mRNA transport as it forms highly stable complexes with 3'UTR fragments of several neuronal mRNAs (APC-RNPs). The kinesin-1 KIF5A binds and transports several neuronal mRNP components such as FMRP, PURα and mRNA fragments weakly, whereas the transport frequency of the mRNA fragments is significantly increased by APC. APC-RNP-motor complexes can assemble on MTs, generating highly processive mRNA transport events. We further find that end-binding protein 1 (EB1) recruits APC-RNPs to dynamically growing MT ends and APC-RNPs track shrinking MTs, producing MT minus-end-directed RNA motility due to the high dwell times of APC on MTs. Our findings establish APC as a versatile mRNA-kinesin adaptor and a key factor for the assembly and bidirectional movement of neuronal transport mRNPs.

B-to-A transition in target DNA during retroviral integration.

Integration into host target DNA (tDNA), a hallmark of retroviral replication, is mediated by the intasome, a multimer of integrase (IN) assembled on viral DNA (vDNA) ends. To ascertain aspects of tDNA recognition during integration, we have solved the 3.5 Å resolution cryo-EM structure of the mouse mammary tumor virus (MMTV) strand transfer complex (STC) intasome. The tDNA adopts an A-like conformation in the region encompassing the sites of vDNA joining, which exposes the sugar-phosphate backbone for IN-mediated strand transfer. Examination of existing retroviral STC structures revealed conservation of A-form tDNA in the analogous regions of these complexes. Furthermore, analyses of sequence preferences in genomic integration sites selectively targeted by six different retroviruses highlighted consistent propensity for A-philic sequences at the sites of vDNA joining. Our structure additionally revealed several novel MMTV IN-DNA interactions, as well as contacts seen in prior STC structures, including conserved Pro125 and Tyr149 residues interacting with tDNA. In infected cells, Pro125 substitutions impacted the global pattern of MMTV integration without significantly altering local base sequence preferences at vDNA insertion sites. Collectively, these data advance our understanding of retroviral intasome structure and function, as well as factors that influence patterns of vDNA integration in genomic DNA.

In Vitro Reconstitution of Kinesin-Based, Axonal mRNA Transport.

Motor protein-driven transport of mRNAs on microtubules and their local translation underlie important neuronal functions such as development, growth cone steering, and synaptic plasticity. While there is abundant data on how membrane-bound cargoes such as vesicles, endosomes, or mitochondria are coupled to motor proteins, surprisingly little is known on the direct interactions of RNA-protein complexes and kinesins or dynein. Provided the potential building blocks are identified, in vitro reconstitutions coupled to Total Internal Reflection Microscopy (TIRF-M) are a powerful and highly sensitive tool to understand how single molecules dynamically interact to assemble into functional complexes. Here we describe how we assemble TIRF-M imaging chambers suitable for the imaging of single protein-RNA complexes. We give advice on optimal sample preparation procedures and explain how a minimal axonal mRNA transport complex can be assembled in vitro. As these assays work at picomolar-range concentrations of proteins and RNAs, they allow the investigation of molecules that cannot be obtained at high concentrations, such as many large or disordered proteins. This now opens the possibility to study how RNA-binding proteins (RBPs), RNAs, and microtubule-associated proteins act together in real-time at single-molecule sensitivity to create cytoplasmic mRNA distributions.

Mammalian Neuronal mRNA Transport Complexes: The Few Knowns and the Many Unknowns.

Hundreds of messenger RNAs (mRNAs) are transported into neurites to provide templates for the assembly of local protein networks. These networks enable a neuron to configure different cellular domains for specialized functions. According to current evidence, mRNAs are mostly transported in rather small packages of one to three copies, rarely containing different transcripts. This opens up fascinating logistic problems: how are hundreds of different mRNA cargoes sorted into distinct packages and how are they coupled to and released from motor proteins to produce the observed mRNA distributions? Are all mRNAs transported by the same transport machinery, or are there different adaptors or motors for different transcripts or classes of mRNAs? A variety of often indirect evidence exists for the involvement of proteins in mRNA localization, but relatively little is known about the essential activities required for the actual transport process. Here, we summarize the different types of available evidence for interactions that connect mammalian mRNAs to motor proteins to highlight at which point further research is needed to uncover critical missing links. We further argue that a combination of discovery approaches reporting direct interactions, in vitro reconstitution, and fast perturbations in cells is an ideal future strategy to unravel essential interactions and specific functions of proteins in mRNA transport processes.

A Novel NAD-RNA Decapping Pathway Discovered by Synthetic Light-Up NAD-RNAs.

The complexity of the transcriptome is governed by the intricate interplay of transcription, RNA processing, translocation, and decay. In eukaryotes, the removal of the 5'-RNA cap is essential for the initiation of RNA degradation. In addition to the canonical 5'-N7-methyl guanosine cap in eukaryotes, the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD) was identified as a new 5'-RNA cap structure in prokaryotic and eukaryotic organisms. So far, two classes of NAD-RNA decapping enzymes have been identified, namely Nudix enzymes that liberate nicotinamide mononucleotide (NMN) and DXO-enzymes that remove the entire NAD cap. Herein, we introduce 8-(furan-2-yl)-substituted NAD-capped-RNA (FurNAD-RNA) as a new research tool for the identification and characterization of novel NAD-RNA decapping enzymes. These compounds are found to be suitable for various enzymatic reactions that result in the release of a fluorescence quencher, either nicotinamide (NAM) or nicotinamide mononucleotide (NMN), from the RNA which causes a fluorescence turn-on. FurNAD-RNAs allow for real-time quantification of decapping activity, parallelization, high-throughput screening and identification of novel decapping enzymes in vitro. Using FurNAD-RNAs, we discovered that the eukaryotic glycohydrolase CD38 processes NAD-capped RNA in vitro into ADP-ribose-modified-RNA and nicotinamide and therefore might act as a decapping enzyme in vivo. The existence of multiple pathways suggests that the decapping of NAD-RNA is an important and regulated process in eukaryotes.

Retroviral integrase protein and intasome nucleoprotein complex structures.

Retroviral replication proceeds through the integration of a DNA copy of the viral RNA genome into the host cellular genome, a process that is mediated by the viral integrase (IN) protein. IN catalyzes two distinct chemical reactions: 3'-processing, whereby the viral DNA is recessed by a di- or trinucleotide at its 3'-ends, and strand transfer, in which the processed viral DNA ends are inserted into host chromosomal DNA. Although IN has been studied as a recombinant protein since the 1980s, detailed structural understanding of its catalytic functions awaited high resolution structures of functional IN-DNA complexes or intasomes, initially obtained in 2010 for the spumavirus prototype foamy virus (PFV). Since then, two additional retroviral intasome structures, from the α-retrovirus Rous sarcoma virus (RSV) and ß-retrovirus mouse mammary tumor virus (MMTV), have emerged. Here, we briefly review the history of IN structural biology prior to the intasome era, and then compare the intasome structures of PFV, MMTV and RSV in detail. Whereas the PFV intasome is characterized by a tetrameric assembly of IN around the viral DNA ends, the newer structures harbor octameric IN assemblies. Although the higher order architectures of MMTV and RSV intasomes differ from that of the PFV intasome, they possess remarkably similar intasomal core structures. Thus, retroviral integration machineries have adapted evolutionarily to utilize disparate IN elements to construct convergent intasome core structures for catalytic function.

Structure and function of the bacterial decapping enzyme NudC.

RNA capping and decapping are thought to be distinctive features of eukaryotes. The redox cofactor NAD was recently discovered to be attached to small regulatory RNAs in bacteria in a cap-like manner, and Nudix hydrolase NudC was found to act as a NAD-decapping enzyme in vitro and in vivo. Here, crystal structures of Escherichia coli NudC in complex with substrate NAD and with cleavage product NMN reveal the catalytic residues lining the binding pocket and principles underlying molecular recognition of substrate and product. Biochemical mutation analysis identifies the conserved Nudix motif as the catalytic center of the enzyme, which needs to be homodimeric, as the catalytic pocket is composed of amino acids from both monomers. NudC is single-strand specific and has a purine preference for the 5'-terminal nucleotide. The enzyme strongly prefers NAD-linked RNA (NAD-RNA) over NAD and binds to a diverse set of cellular RNAs in an unspecific manner.