Background: RtcB is a new RNA ligase implicated in tRNA splicing and RNA repair. Results: RtcB has 2Ј,3Ј-cyclic phosphodiesterase and 3Ј-phosphate/5Ј-hydroxyl ligase activities. Conclusion: RtcB seals broken RNAs via a two-step reaction pathway. Significance: The structure, active site, and chemical mechanism of RtcB are unique.

RNA-binding proteins (RBPs) control the fate of nearly every transcript in a cell. However, no existing approach for studying these posttranscriptional gene regulators combines transcriptomewide throughput and biophysical precision. Here, we describe an assay that accomplishes this. Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the Saccharomyces cerevisiae genome. The scale and precision of these measurements revealed many previously unknown features of this well-studied RBP. Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in de novo gene "birth." TGA provided single-nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding. Changes in transcript levels in vts1Δ cells established the regulatory function of these binding sites. The impact of Vts1 on transcript abundance was largely independent of where it bound within an mRNA, challenging prevailing assumptions about how this RBP drives RNA degradation. TGA thus enables a quantitative description of the relationship between variant RNA structures, affinity, and in vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will provide similarly comprehensive and quantitative insights into the function of virtually any RBP.RNA | next-generation sequencing | systems biochemistry | RNA binding proteins | Vts1 R NA-binding proteins (RBPs) constitute 5-10% of the eukaryotic proteome (1-3) and collectively govern the localization, translation, and decay of virtually every transcript (4-6). Despite the ubiquity of RBPs and their central importance in gene regulation, decoding the links between RNA primary sequence and its cadre of regulators remains a major unresolved challenge (7). Current approaches for characterizing RBP function generally involve trade-offs between throughput, comprehensiveness, and quantitative precision. Biophysical measurements can be made with targeted biochemical approaches such as electrophoretic mobility shift assays (EMSAs) or fluorescence polarization (FP) (8, 9), but these methods can only interrogate known RNA-protein interactions and are inherently lowthroughput. Selection-based approaches [e.g., in vitro selection, high-throughput sequencing of RNA, and sequence-specificity landscapes (SEQRS)/RNA bind-n-seq (RBNS)] achieve higher throughput, but these techniques remove binding sites from their natural sequence context and identify "winners" based on more than simple affinity (10). Transcriptome-wide methods, which often use cross-linking and immunoprecipi...

A cherished tenet of nucleic acid enzymology holds that synthesis of polynucleotide 3′-5′ phosphodiesters proceeds via the attack of a 3′-OH on a high-energy 5′ phosphoanhydride: either a nucleoside 5′-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A(5′)pp(5′)N-in the case of polynucleotide ligases. RtcB exemplifies a family of RNA ligases implicated in tRNA splicing and repair. Unlike classic ligases, RtcB seals broken RNAs with 3′-phosphate and 5′-OH ends. Here we show that RtcB executes a three-step ligation pathway entailing (i) reaction of His337 of the enzyme with GTP to form a covalent RtcB-(histidinyl-N)-GMP intermediate; (ii) transfer of guanylate to a polynucleotide 3′-phosphate to form a polynucleotide-(3′)pp(5′)G intermediate; and (iii) attack of a 5′-OH on the -N(3′)pp(5′)G end to form the splice junction. RtcB is structurally sui generis, and its chemical mechanism is unique. The wide distribution of RtcB proteins in bacteria, archaea, and metazoa raises the prospect of an alternative enzymology based on covalently activated 3′ ends. RNA or A(5′)pp(5′)DNA; and (iii) ligase catalyzes attack by a polynucleotide 3′-OH on A(5′)pp(5′)RNA/DNA to form a 3′-5′ phosphodiester bond and release AMP (1). The salient principle of classic ligase catalysis is that the high energy of the ATP phosphoanhydride bond is transferred via the enzyme to the polynucleotide 5′ end, thereby activating the 5′ end for phosphodiester synthesis, with AMP as a favorable leaving group. The shared chemical mechanism of classic RNA and DNA ligases is reflected in their conserved core tertiary structures and active sites (1).Classic ATP-dependent RNA ligases are encoded by diverse taxa in all three phylogenetic domains. In bacteria, fungi, and plants they function as components of multienzyme RNA repair pathways that heal and seal broken RNAs with 2′,3′ cyclic phosphate and 5′-OH ends (2-5). In the healing phase, the 2′,3′ cyclic phosphate end is hydrolyzed by a phosphoesterase enzyme to a 3′-OH, and the 5′-OH end is phosphorylated by a polynucleotide kinase enzyme to yield a 5′-monophosphate. The resulting 3′-OH and 5′-phosphate ends are then suitable for ATP-dependent sealing by RNA ligase. This "healing-and-sealing" pathway is responsible for tRNA splicing in fungi and plants (6, 7), for mRNA splicing in the fungal unfolded protein response (8), and for tRNA restriction repair during bacteriophage infection of Escherichia coli (9).An alternative "direct ligation" pathway for joining 2′,3′ cyclic phosphate and 5′-OH ends during mammalian tRNA splicing was discovered nearly 30 y ago (10-12), when it was shown that the 2′,3′ cyclic phosphate of the cleaved pre-tRNA is incorporated at the splice junction in the mature tRNA. (The junction phosphate in yeast tRNA splicing derives from the γ-phosphate of the NTP substrate for the 5′ kinase step.) Direct ligation languished until 2011, when three laboratories identified bacterial, archaeal, and mammalian RtcB proteins as RNA ligase enzymes capable of seali...

Highlights d The Vts1 IDR promotes its condensation into the non-amyloid prion [SMAUG + ] d [SMAUG + ] hyperactivates Vts1 function d [SMAUG + ] rewires post-transcriptional gene regulation to promote proliferation d Self-assembly is conserved in the human Vts1 homolog hSmaug1

There are many biological contexts in which DNA damage generates "dirty" breaks with 3′-PO 4 (or cyclic-PO 4 ) and 5′-OH ends that cannot be sealed by DNA ligases. Here we show that the Escherichia coli RNA ligase RtcB can splice these dirty DNA ends via a unique chemical mechanism. RtcB transfers GMP from a covalent RtcB-GMP intermediate to a DNA 3′-PO 4 to form a "capped" 3′ end structure, DNA 3′ pp 5′ G. When a suitable DNA 5′-OH end is available, RtcB catalyzes attack of the 5′-OH on DNA 3′ pp 5′ G to form a 3′-5′ phosphodiester splice junction. Our findings unveil an enzymatic capacity for DNA 3′ capping and the sealing of DNA breaks with 3′-PO 4 and 5′-OH termini, with implications for DNA repair and DNA rearrangements.T he Escherichia coli RtcB is a founding member of a recently discovered family of RNA repair/splicing enzymes that join RNA 2′,3′-cyclic-PO 4 or 3′-PO 4 ends to RNA 5′-OH ends (1-4). RtcB executes a four-step pathway that requires GTP as an energy source and Mn 2+ as a cofactor (5-7). RtcB first reacts with GTP to form a covalent RtcB-(histidinyl 337 -N)-GMP intermediate. It then hydrolyzes the RNA 2′,3′-cyclic-PO 4 end to a 3′-PO 4 and transfers guanylate from His337 to the RNA 3′-PO 4 to form an RNA 3′ pp 5′ G intermediate. Finally, RtcB catalyzes the attack of an RNA 5′-OH on the RNA 3′ pp 5′ G end to form the 3′-5′ phosphodiester splice junction and liberate GMP.The unique chemical mechanism of RtcB overturned a longstanding tenet of nucleic acid enzymology, which held that synthesis of polynucleotide 3′-5′ phosphodiesters proceeds via the attack of a 3′-OH on a high-energy 5′-phosphoanhydride: either a nucleoside 5′-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A 5′ pp 5′ N, in the case of classic RNA/DNA ligases. In light of the wide distribution of RtcB proteins in bacteria, archaea, and metazoa, we raised the prospect of an alternative enzymology based on covalently activated 3′-PO 4 ends (6).In principle, the chemistry of RNA 3′-PO 4 /5′-OH end joining by RtcB might be portable to DNA transactions and pertinent to DNA repair. A variety of hydrolytic nucleases incise the DNA phosphodiester backbone to yield 3′-PO 4 and 5′-OH termini that cannot be joined by DNA ligases. Nonligatable 3′-PO 4 ends are also generated during base excision repair catalyzed by DNA glycosylase/lyase enzymes, during the repair of trapped covalent topoisomerase IB-DNA adducts by tyrosyl-DNA phosphodiesterase 1, and during DNA damage inflicted by ionizing radiation. One way nature solves this "dirty end" problem is by deploying a variety of "end healing" enzymes (8-14). These include 3′-phosphoesterases that convert a 3′-PO 4 to a 3′-OH and 5′-kinases that transform a 5′-OH to a 5′-PO 4 , thereby enabling break sealing by the classic ligase pathway. Given what we now know about RtcB, would it not make sense for nature to also endow a pathway for direct joining of DNA 3′-PO 4 and 5′-OH ends, be it via RtcB or another ligase yet to be discovered?We can extend this thought to DN...