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Using global data for around 180 countries and territories and 170 food/feed types primarily derived from FAOSTAT, we have systematically analyzed the changes in greenhouse gas (GHG) emission intensity (GHG i ) (kg CO 2eq per kg protein production) over the past six decades. We found that, with large spatial heterogeneity, emission intensity decreased by nearly two-thirds from 1961 to 2019, predominantly in the earlier years due to agronomic improvement in productivity. However, in the most recent decade, emission intensity has become stagnant, and in a few countries even showed an increase, due to the rapid increase in livestock production and land use changes. The trade of final produced protein between countries has potentially reduced the global GHG i , especially for countries that are net importers with high GHG i , such as many in Africa and South Asia. Overall, a continuous decline of emission intensity in the future relies on countries with higher emission intensity to increase agricultural productivity and minimize land use changes. Countries with lower emission intensity should reduce livestock production and increase the free trade of agricultural products and improve the trade optimality., Competing Interests: Competing interests statement:The authors declare no competing interest.

Crosstalk between metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to disease. Here, we investigated whether maintenance of circadian rhythms depends on specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to signal from a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function across a series of pancreatic adenocarcinoma cell lines. Metabolic profiling of congenic tumor cell clones revealed substantial diversity among these lines that we used to identify clones to generate circadian reporter lines. We observed diverse circadian profiles among these lines that varied with their metabolic phenotype: The most hypometabolic line [exhibiting low levels of oxidative phosphorylation (OxPhos) and glycolysis] had the strongest rhythms, while the most hypermetabolic line had the weakest rhythms. Pharmacological enhancement of OxPhos decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, inhibition of OxPhos enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells., Competing Interests: Competing interests statement:The authors declare no competing interest.

Tpt1 is an essential agent of fungal and plant tRNA splicing that removes an internal RNA 2'-phosphate generated by tRNA ligase. Tpt1 also removes the 2'-phosphouridine mark installed by Ark1 kinase in the V-loop of archaeal tRNAs. Tpt1 performs a two-step reaction in which the 2'-PO 4 attacks NAD + to form an RNA-2'-phospho-(ADP-ribose) intermediate, and transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Here, we present structures of archaeal Tpt1 enzymes, captured as product complexes with ADP-ribose-1″-PO 4 , ADP-ribose-2″-PO 4 , and 2'-OH RNA, and as substrate complexes with 2',5'-ADP and NAD + , that illuminate 2'-PO 4 junction recognition and catalysis. We show that archaeal Tpt1 enzymes can use the 2'-PO 4 -containing metabolites NADP + and NADPH as substrates for 2'-PO 4 transfer to NAD + . A role in 2'-phospho-NADP(H) dynamics provides a rationale for the prevalence of Tpt1 in taxa that lack a capacity for internal RNA 2'-phosphorylation.

The King Baboon spider, Pelinobius muticus , is a burrowing African tarantula. Its impressive size and appealing coloration are tempered by reports describing severe localized pain, swelling, itchiness, and muscle cramping after accidental envenomation. Hyperalgesia is the most prominent symptom after bites from P. muticus , but the molecular basis by which the venom induces pain is unknown. Proteotranscriptomic analysis of P. muticus venom uncovered a cysteine-rich peptide, δ/κ-theraphotoxin-Pm1a (δ/κ-TRTX-Pm1a), that elicited nocifensive behavior when injected into mice. In small dorsal root ganglion neurons, synthetic δ/κ-TRTX-Pm1a (sPm1a) induced hyperexcitability by enhancing tetrodotoxin-resistant sodium currents, impairing repolarization and lowering the threshold of action potential firing, consistent with the severe pain associated with envenomation. The molecular mechanism of nociceptor sensitization by sPm1a involves multimodal actions over several ion channel targets, including Na V 1.8, K V 2.1, and tetrodotoxin-sensitive Na V channels. The promiscuous targeting of peptides like δ/κ-TRTX-Pm1a may be an evolutionary adaptation in pain-inducing defensive venoms., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

The system of long noncoding RNA (lncRNA)-mediated transcriptional interference that represses fission yeast phosphate homoeostasis gene pho1 provides a sensitive readout of genetic influences on cotranscriptional 3'-processing and termination and a tool for discovery of regulators of this phase of the Pol2 transcription cycle. Here, we conducted a genetic screen for relief of transcriptional interference that unveiled a mechanism by which Pol2 termination is enhanced via a gain-of-function mutation, G476S, in the RNA-binding domain of an essential termination factor, Seb1. The genetic and physical evidence for gain-of-function is compelling: 1) seb1-G476S de-represses pho1 and tgp1 , both of which are subject to lncRNA-mediated transcriptional interference; 2) seb1-G476S elicits precocious lncRNA transcription termination in response to lncRNA 5'-proximal poly(A) signals; 3) seb1-G476S derepression of pho1 is effaced by loss-of-function mutations in cleavage and polyadenylation factor (CPF) subunits and termination factor Rhn1; 4) synthetic lethality of seb1-G476S with pho1 derepressive mutants rpb1-CTD-S7A and aps1 ∆ is rescued by CPF/Rhn1 loss-of-function alleles; and 5) seb1-G476S elicits an upstream shift in poly(A) site preference in several messenger RNA genes. A crystal structure of the Seb1-G476S RNA-binding domain indicates potential for gain of contacts from Ser476 to RNA nucleobases. To our knowledge, this is a unique instance of a gain-of-function phenotype in a eukaryal transcription termination protein., Competing Interests: The authors declare no competing interest.

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The N-terminal motor domain of the AdnB subunit hydrolyzes ATP to drive rapid and processive 3' to 5' translocation of AdnAB on the tracking DNA strand. ATP hydrolysis is mechanically productive when oscillating protein domain motions synchronized with the ATPase cycle propel the DNA tracking strand forward by a single-nucleotide step, in what is thought to entail a pawl-and-ratchet-like fashion. By gauging the effects of alanine mutations of the 16 amino acids at the AdnB-DNA interface on DNA-dependent ATP hydrolysis, DNA translocation, and DSB resection in ensemble and single-molecule assays, we gained key insights into which DNA contacts couple ATP hydrolysis to motor activity. The results implicate AdnB Trp325, which intercalates into the tracking strand and stacks on a nucleobase, as the singular essential constituent of the ratchet pawl, without which ATP hydrolysis on ssDNA is mechanically futile. Loss of Thr663 and Thr118 contacts with tracking strand phosphates and of His665 with a nucleobase drastically slows the AdnAB motor during DSB resection. Our findings for AdnAB prompt us to analogize its mechanism to that of an automobile clutch., Competing Interests: The authors declare no competing interest.

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Here we report cryoelectron microscopy (cryo-EM) structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5' single-strand DNA (ssDNA) tail by the AdnA nuclease. The structures reveal the path of the 5' ssDNA through the AdnA nuclease domain and the mechanism of 5' strand cleavage; the path of the 3' tracking strand through the AdnB motor and the DNA contacts that couple ATP hydrolysis to mechanical work; the position of the AdnA iron-sulfur cluster subdomain at the Y junction and its likely role in maintaining the split trajectories of the unwound 5' and 3' strands. Single-molecule DNA curtain analysis of DSB resection reveals that AdnAB is highly processive but prone to spontaneous pausing at random sites on duplex DNA. A striking property of AdnAB is that the velocity of DSB resection slows after the enzyme experiences a spontaneous pause. Our results highlight shared as well as distinctive properties of AdnAB vis-à-vis the RecBCD and AddAB clades of bacterial DSB-resecting motor nucleases., Competing Interests: The authors declare no competing interest.

Long interspersed element-1 (LINE-1 or L1) amplifies via retrotransposition. Active L1s encode 2 proteins (ORF1p and ORF2p) that bind their encoding transcript to promote retrotransposition in cis The L1-encoded proteins also promote the retrotransposition of small-interspersed element RNAs, noncoding RNAs, and messenger RNAs in trans Some L1-mediated retrotransposition events consist of a copy of U6 RNA conjoined to a variably 5'-truncated L1, but how U6/L1 chimeras are formed requires elucidation. Here, we report the following: The RNA ligase RtcB can join U6 RNAs ending in a 2',3'-cyclic phosphate to L1 RNAs containing a 5'-OH in vitro; depletion of endogenous RtcB in HeLa cell extracts reduces U6/L1 RNA ligation efficiency; retrotransposition of U6/L1 RNAs leads to U6/L1 pseudogene formation; and a unique cohort of U6/L1 chimeric RNAs are present in multiple human cell lines. Thus, these data suggest that U6 small nuclear RNA (snRNA) and RtcB participate in the formation of chimeric RNAs and that retrotransposition of chimeric RNA contributes to interindividual genetic variation., Competing Interests: Conflict of interest statement: J.V.M. is an inventor on patent US6150160, is a paid consultant for Gilead Sciences and a privately held company founded by Flagship Pioneering, and is on the American Society of Human Genetics Board of Directors., (Copyright © 2019 the Author(s). Published by PNAS.)

Precise modulation of hepatic glucose metabolism is crucial during the fasting and feeding cycle and is controlled by the actions of circulating insulin and glucagon. The insulin-signaling pathway requires insulin receptor substrate 1 (IRS1) and IRS2, which are found to be dysregulated in diabetes and obesity. The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1A) is a fasting-induced transcriptional coactivator. In nonalcoholic fatty liver disease and in patients with type 2 diabetes, low hepatic PGC1A levels are associated with insulin resistance. However, how PGC1A activity impacts the hepatic insulin-signaling pathway is still unclear. We used gain- and loss-of-function models in mouse primary hepatocytes and measured hepatocyte insulin response by gene and protein expression and ex vivo glucose production. We found that the PGC1A level determines the relative ratio of IRS1 and IRS2 in hepatocytes, impacting insulin receptor signaling via protein kinase B/AKT (AKT). PGC1A drove the expression of IRS2 downstream of glucagon signaling while simultaneously reducing IRS1 expression. We illustrate that glucagon- or PGC1A-induced IRS2 expression was dependent on cAMP Response Element Binding Protein activity and that this was essential for suppression of hepatocyte gluconeogenesis in response to insulin in vitro. We also show that increased hepatic PGC1A improves glucose homeostasis in vivo, revealing a counterregulatory role for PGC1A in repressing uncontrolled glucose production in response to insulin signaling. These data highlight a mechanism by which PGC1A plays dual roles in the control of gluconeogenesis during the fasting-to-fed transition through regulated balance between IRS1 and IRS2 expression., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The carboxy-terminal domain (CTD) code encrypted within the Y 1 S 2 P 3 T 4 S 5 P 6 S 7 heptad repeats of RNA polymerase II (Pol2) is deeply rooted in eukaryal biology. Key steps to deciphering the code are identifying the events in gene expression that are governed by individual "letters" and then defining a vocabulary of multiletter "words" and their meaning. Thr4 and Ser7 exert opposite effects on the fission yeast pho1 gene, expression of which is repressed under phosphate-replete conditions by transcription of an upstream flanking long noncoding RNA (lncRNA). Here we attribute the derepression of pho1 by a CTD -S7A mutation to precocious termination of lncRNA synthesis, an effect that is erased by mutations of cleavage-polyadenylation factor (CPF) subunits Ctf1, Ssu72, Ppn1, Swd22, and Dis2 and termination factor Rhn1. By contrast, a CTD -T4A mutation hyperrepresses pho1 , as do CPF subunit and Rhn1 mutations, implying that T4A reduces lncRNA termination. Moreover, CTD -T4A is synthetically lethal with ppn1 ∆ and swd22 ∆, signifying that Thr4 and the Ppn1•Swd22 module play important, functionally redundant roles in promoting Pol2 termination. We find that Ppn1 and Swd22 become essential for viability when the CTD array is curtailed and that S7A overcomes the need for Ppn1•Swd22 in the short CTD context. Mutational synergies highlight redundant essential functions of ( i ) Ppn1•Swd22 and Rhn1, ( ii ) Ppn1•Swd22 and Ctf1, and ( iii ) Ssu72 and Dis2 phosphatases. CTD alleles Y1F , S2A , and T4A have overlapping synthetic lethalities with ppn1 ∆ and swd22 ∆, suggesting that Tyr1-Ser2-Thr4 form a three-letter CTD word that abets termination, with Rhn1 being a likely "reader" of this word., Competing Interests: The authors declare no conflict of interest.

Mutations in LRRK2 are known to be the most common genetic cause of sporadic and familial Parkinson's disease (PD). Multiple lines of LRRK2 transgenic or knockin mice have been developed, yet none exhibit substantial dopamine (DA)-neuron degeneration. Here we develop human tyrosine hydroxylase (TH) promoter-controlled tetracycline-sensitive LRRK2 G2019S (GS) and LRRK2 G2019S kinase-dead (GS/DA) transgenic mice and show that LRRK2 GS expression leads to an age- and kinase-dependent cell-autonomous neurodegeneration of DA and norepinephrine (NE) neurons. Accompanying the loss of DA neurons are DA-dependent behavioral deficits and α-synuclein pathology that are also LRRK2 GS kinase-dependent. Transmission EM reveals that that there is an LRRK2 GS kinase-dependent significant reduction in synaptic vesicle number and a greater abundance of clathrin-coated vesicles in DA neurons. These transgenic mice indicate that LRRK2-induced DA and NE neurodegeneration is kinase-dependent and can occur in a cell-autonomous manner. Moreover, these mice provide a substantial advance in animal model development for LRRK2-associated PD and an important platform to investigate molecular mechanisms for how DA neurons degenerate as a result of expression of mutant LRRK2., Competing Interests: The authors declare no conflict of interest.

Polynucleotide ligases comprise a ubiquitous superfamily of nucleic acid repair enzymes that join 3'-OH and 5'-PO 4 DNA or RNA ends. Ligases react with ATP or NAD + and a divalent cation cofactor to form a covalent enzyme-(lysine-Nζ)-adenylate intermediate. Here, we report crystal structures of the founding members of the ATP-dependent RNA ligase family (T4 RNA ligase 1; Rnl1) and the NAD + -dependent DNA ligase family ( Escherichia coli LigA), captured as their respective Michaelis complexes, which illuminate distinctive catalytic mechanisms of the lysine adenylylation reaction. The 2.2-Å Rnl1•ATP•(Mg 2+ ) 2 structure highlights a two-metal mechanism, whereby: a ligase-bound "catalytic" Mg 2+ (H 2 O) 5 coordination complex lowers the p K a of the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; a second octahedral Mg 2+ coordination complex bridges the β and γ phosphates; and protein elements unique to Rnl1 engage the γ phosphate and associated metal complex and orient the pyrophosphate leaving group for in-line catalysis. By contrast, the 1.55-Å LigA•NAD + •Mg 2+ structure reveals a one-metal mechanism in which a ligase-bound Mg 2+ (H 2 O) 5 complex lowers the lysine p K a and engages the NAD + α phosphate, but the β phosphate and the nicotinamide nucleoside of the nicotinamide mononucleotide (NMN) leaving group are oriented solely via atomic interactions with protein elements that are unique to the LigA clade. The two-metal versus one-metal dichotomy demarcates a branchpoint in ligase evolution and favors LigA as an antibacterial drug target.

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy with a unique geographical distribution. The genomic abnormalities leading to NPC pathogenesis remain unclear. In total, 135 NPC tumors were examined to characterize the mutational landscape using whole-exome sequencing and targeted resequencing. An APOBEC cytidine deaminase mutagenesis signature was revealed in the somatic mutations. Noticeably, multiple loss-of-function mutations were identified in several NF-κB signaling negative regulators NFKBIA, CYLD, and TNFAIP3 Functional studies confirmed that inhibition of NFKBIA had a significant impact on NF-κB activity and NPC cell growth. The identified loss-of-function mutations in NFKBIA leading to protein truncation contributed to the altered NF-κB activity, which is critical for NPC tumorigenesis. In addition, somatic mutations were found in several cancer-relevant pathways, including cell cycle-phase transition, cell death, EBV infection, and viral carcinogenesis. These data provide an enhanced road map for understanding the molecular basis underlying NPC., Competing Interests: The authors declare no conflict of interest.

Multiple factors, including host genetics, environmental factors, and Epstein-Barr virus (EBV) infection, contribute to nasopharyngeal carcinoma (NPC) development. To identify genetic susceptibility genes for NPC, a whole-exome sequencing (WES) study was performed in 161 NPC cases and 895 controls of Southern Chinese descent. The gene-based burden test discovered an association between macrophage-stimulating 1 receptor (MST1R) and NPC. We identified 13 independent cases carrying the MST1R pathogenic heterozygous germ-line variants, and 53.8% of these cases were diagnosed with NPC aged at or even younger than 20 y, indicating that MST1R germline variants are relevant to disease early-age onset (EAO) (age of ≤20 y). In total, five MST1R missense variants were found in EAO cases but were rare in controls (EAO vs. control, 17.9% vs. 1.2%, P = 7.94 × 10(-12)). The validation study, including 2,160 cases and 2,433 controls, showed that the MST1R variant c.G917A:p.R306H is highly associated with NPC (odds ratio of 9.0). MST1R is predominantly expressed in the tissue-resident macrophages and is critical for innate immunity that protects organs from tissue damage and inflammation. Importantly, MST1R expression is detected in the ciliated epithelial cells in normal nasopharyngeal mucosa and plays a role in the cilia motility important for host defense. Although no somatic mutation of MST1R was identified in the sporadic NPC tumors, copy number alterations and promoter hypermethylation at MST1R were often observed. Our findings provide new insights into the pathogenesis of NPC by highlighting the involvement of the MST1R-mediated signaling pathways.

ATP-dependent RNA ligases are agents of RNA repair that join 3'-OH and 5'-PO4 RNA ends. Naegleria gruberi RNA ligase (NgrRnl) exemplifies a family of RNA nick-sealing enzymes found in bacteria, viruses, and eukarya. Crystal structures of NgrRnl at three discrete steps along the reaction pathway-covalent ligase-(lysyl-Nζ)-AMP•Mn(2+) intermediate; ligase•ATP•(Mn(2+))2 Michaelis complex; and ligase•Mn(2+) complex-highlight a two-metal mechanism of nucleotidyl transfer, whereby (i) an enzyme-bound "catalytic" metal coordination complex lowers the pKa of the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; and (ii) a second metal coordination complex bridges the β- and γ-phosphates. The NgrRnl N domain is a distinctively embellished oligonucleotide-binding (OB) fold that engages the γ-phosphate and associated metal complex and orients the pyrophosphate leaving group for in-line catalysis with stereochemical inversion at the AMP phosphate. The unique domain architecture of NgrRnl fortifies the theme that RNA ligases have evolved many times, and independently, by fusions of a shared nucleotidyltransferase domain to structurally diverse flanking modules. The mechanistic insights to lysine adenylylation gained from the NgrRnl structures are likely to apply broadly to the covalent nucleotidyltransferase superfamily of RNA ligases, DNA ligases, and RNA capping enzymes.

We examine in Drosophila a group of ∼35 ionotropic receptors (IRs), the IR20a clade, about which remarkably little is known. Of 28 genes analyzed, GAL4 drivers representing 11 showed expression in the larva. Eight drivers labeled neurons of the pharynx, a taste organ, and three labeled neurons of the body wall that may be chemosensory. Expression was not observed in neurons of one taste organ, the terminal organ, although these neurons express many drivers of the Gr (Gustatory receptor) family. For most drivers of the IR20a clade, we observed expression in a single pair of cells in the animal, with limited coexpression, and only a fraction of pharyngeal neurons are labeled. The organization of IR20a clade expression thus appears different from the organization of the Gr family or the Odor receptor (Or) family in the larva. A remarkable feature of the larval pharynx is that some of its organs are incorporated into the adult pharynx, and several drivers of this clade are expressed in the pharynx of both larvae and adults. Different IR drivers show different developmental dynamics across the larval stages, either increasing or decreasing. Among neurons expressing drivers in the pharynx, two projection patterns can be distinguished in the CNS. Neurons exhibiting these two kinds of projection patterns may activate different circuits, possibly signaling the presence of cues with different valence. Taken together, the simplest interpretation of our results is that the IR20a clade encodes a class of larval taste receptors.

The production of purposely made painted or engraved designs on cave walls--a means of recording and transmitting symbolic codes in a durable manner--is recognized as a major cognitive step in human evolution. Considered exclusive to modern humans, this behavior has been used to argue in favor of significant cognitive differences between our direct ancestors and contemporary archaic hominins, including the Neanderthals. Here we present the first known example of an abstract pattern engraved by Neanderthals, from Gorham's Cave in Gibraltar. It consists of a deeply impressed cross-hatching carved into the bedrock of the cave that has remained covered by an undisturbed archaeological level containing Mousterian artifacts made by Neanderthals and is older than 39 cal kyr BP. Geochemical analysis of the epigenetic coating over the engravings and experimental replication show that the engraving was made before accumulation of the archaeological layers, and that most of the lines composing the design were made by repeatedly and carefully passing a pointed lithic tool into the grooves, excluding the possibility of an unintentional or utilitarian origin (e.g., food or fur processing). This discovery demonstrates the capacity of the Neanderthals for abstract thought and expression through the use of geometric forms.

Myeloid cells are key regulators of the tumor microenvironment, governing local immune responses. Here we report that tumor-infiltrating myeloid cells and circulating monocytes in patients with glioblastoma multiforme (GBM) express ligands for activating the Natural killer group 2, member D (NKG2D) receptor, which cause down-regulation of NKG2D on natural killer (NK) cells. Tumor-infiltrating NK cells isolated from GBM patients fail to lyse NKG2D ligand-expressing tumor cells. We demonstrate that lactate dehydrogenase (LDH) isoform 5 secreted by glioblastoma cells induces NKG2D ligands on monocytes isolated from healthy individuals. Furthermore, sera from GBM patients contain elevated amounts of LDH, which correlate with expression of NKG2D ligands on their autologous circulating monocytes. NKG2D ligands also are present on circulating monocytes isolated from patients with breast, prostate, and hepatitis C virus-induced hepatocellular carcinomas. Together, these findings reveal a previously unidentified immune evasion strategy whereby tumors produce soluble factors that induce NKG2D ligands on myeloid cells, subverting antitumor immune responses.

Many biological scenarios generate "dirty" DNA 3'-PO4 ends that cannot be sealed by classic DNA ligases or extended by DNA polymerases. The noncanonical ligase RtcB can "cap" these ends via a unique chemical mechanism entailing transfer of GMP from a covalent RtcB-GMP intermediate to a DNA 3'-PO4 to form DNA3'pp5'G. Here, we show that capping protects DNA 3' ends from resection by Escherichia coli exonucleases I and III and from end-healing by T4 polynucleotide 3' phosphatase. By contrast, the cap is an effective primer for DNA synthesis. E. coli DNA polymerase I and Mycobacterium DinB1 extend the DNAppG primer to form an alkali-labile DNApp(rG)pDNA product. The addition of dNTP depends on pairing of the cap guanine with an opposing cytosine in the template strand. Aprataxin, an enzyme implicated in repair of A5'pp5'DNA ends formed during abortive ligation by classic ligases, is highly effective as a DNA 3' decapping enzyme, converting DNAppG to DNA3'p and GMP. We conclude that the biochemical impact of DNA capping is to prevent resection and healing of a 3'-PO4 end, while permitting DNA synthesis, at the price of embedding a ribonucleotide and a pyrophosphate linkage in the repaired strand. Aprataxin affords a means to counter the impact of DNA capping.

The primary structure and phosphorylation pattern of the tandem Y(1)S(2)P(3)T(4)S(5)P(6)S(7) repeats of the RNA polymerase II carboxyl-terminal domain (CTD) comprise an informational code that coordinates transcription, chromatin modification, and RNA processing. To gauge the contributions of individual CTD coding "letters" to gene expression, we analyzed the poly(A)(+) transcriptomes of fission yeast mutants that lack each of the four inessential CTD phosphoacceptors: Tyr1, Ser2, Thr4, and Ser7. There was a hierarchy of CTD mutational effects with respect to the number of dysregulated protein-coding RNAs, with S2A (n = 227) >> Y1F (n = 71) > S7A (n = 58) >> T4A (n = 7). The majority of the protein-coding RNAs affected in Y1F cells were coordinately affected by S2A, suggesting that Tyr1-Ser2 constitutes a two-letter code "word." Y1F and S2A elicited increased expression of genes encoding proteins involved in iron uptake (Frp1, Fip1, Fio1, Str3, Str1, Sib1), without affecting the expression of the genes that repress the iron regulon, implying that Tyr1-Ser2 transduces a repressive signal. Y1F and S2A cells had increased levels of ferric reductase activity and were hypersensitive to phleomycin, indicative of elevated intracellular iron. The T4A and S7A mutations had opposing effects on the phosphate response pathway. T4A reduced the expression of two genes encoding proteins involved in phosphate acquisition (the Pho1 acid phosphatase and the phosphate transporter SPBC8E4.01c), without affecting the expression of known genes that regulate the phosphate response pathway, whereas S7A increased pho1(+) expression. These results highlight specific cellular gene expression programs that are responsive to distinct CTD cues.

There are many biological contexts in which DNA damage generates "dirty" breaks with 3'-PO4 (or cyclic-PO4) 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'-PO4 to form a "capped" 3' end structure, DNA3'pp5'G. When a suitable DNA 5'-OH end is available, RtcB catalyzes attack of the 5'-OH on DNA3'pp5'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'-PO4 and 5'-OH termini, with implications for DNA repair and DNA rearrangements.

Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. Broadly neutralizing antibodies (BnAbs) are not induced by current vaccines, but are found in plasma in ∼20% of HIV-1-infected individuals after several years of infection. One strategy for induction of unfavored antibody responses is to produce homogeneous immunogens that selectively express BnAb epitopes but minimally express dominant strain-specific epitopes. Here we report that synthetic, homogeneously glycosylated peptides that bind avidly to variable loop 1/2 (V1V2) BnAbs PG9 and CH01 bind minimally to strain-specific neutralizing V2 antibodies that are targeted to the same envelope polypeptide site. Both oligomannose derivatization and conformational stabilization by disulfide-linked dimer formation of synthetic V1V2 peptides were required for strong binding of V1V2 BnAbs. An HIV-1 vaccine should target BnAb unmutated common ancestor (UCA) B-cell receptors of naïve B cells, but to date no HIV-1 envelope constructs have been found that bind to the UCA of V1V2 BnAb PG9. We demonstrate herein that V1V2 glycopeptide dimers bearing Man5GlcNAc2 glycan units bind with apparent nanomolar affinities to UCAs of V1V2 BnAbs PG9 and CH01 and with micromolar affinity to the UCA of a V2 strain-specific antibody. The higher-affinity binding of these V1V2 glycopeptides to BnAbs and their UCAs renders these glycopeptide constructs particularly attractive immunogens for targeting subdominant HIV-1 envelope V1V2-neutralizing antibody-producing B cells.

In animals, each sequence-specific transcription factor typically binds to thousands of genomic regions in vivo. Our previous studies of 20 transcription factors show that most genomic regions bound at high levels in Drosophila blastoderm embryos are known or probable functional targets, but genomic regions occupied only at low levels have characteristics suggesting that most are not involved in the cis-regulation of transcription. Here we use transgenic reporter gene assays to directly test the transcriptional activity of 104 genomic regions bound at different levels by the 20 transcription factors. Fifteen genomic regions were selected based solely on the DNA occupancy level of the transcription factor Kruppel. Five of the six most highly bound regions drive blastoderm patterns of reporter transcription. In contrast, only one of the nine lowly bound regions drives transcription at this stage and four of them are not detectably active at any stage of embryogenesis. A larger set of 89 genomic regions chosen using criteria designed to identify functional cis-regulatory regions supports the same trend: genomic regions occupied at high levels by transcription factors in vivo drive patterned gene expression, whereas those occupied only at lower levels mostly do not. These results support studies that indicate that the high cellular concentrations of sequence-specific transcription factors drive extensive, low-occupancy, nonfunctional interactions within the accessible portions of the genome.

It is now recognized that a number of cognitive, behavioral, and mental health outcomes across the lifespan can be traced to fetal development. Although the direct mediation is unknown, the substantial variance in fetal growth, most commonly indexed by birth weight, may affect lifespan brain development. We investigated effects of normal variance in birth weight on MRI-derived measures of brain development in 628 healthy children, adolescents, and young adults in the large-scale multicenter Pediatric Imaging, Neurocognition, and Genetics study. This heterogeneous sample was recruited through geographically dispersed sites in the United States. The influence of birth weight on cortical thickness, surface area, and striatal and total brain volumes was investigated, controlling for variance in age, sex, household income, and genetic ancestry factors. Birth weight was found to exert robust positive effects on regional cortical surface area in multiple regions as well as total brain and caudate volumes. These effects were continuous across birth weight ranges and ages and were not confined to subsets of the sample. The findings show that (i) aspects of later child and adolescent brain development are influenced at birth and (ii) relatively small differences in birth weight across groups and conditions typically compared in neuropsychiatric research (e.g., Attention Deficit Hyperactivity Disorder, schizophrenia, and personality disorders) may influence group differences observed in brain parameters of interest at a later stage in life. These findings should serve to increase our attention to early influences.

Self-regulation refers to the ability to control behavior, cognition, and emotions, and self-regulation failure is related to a range of neuropsychiatric problems. It is poorly understood how structural maturation of the brain brings about the gradual improvement in self-regulation during childhood. In a large-scale multicenter effort, 735 children (4-21 y) underwent structural MRI for quantification of cortical thickness and surface area and diffusion tensor imaging for quantification of the quality of major fiber connections. Brain development was related to a standardized measure of cognitive control (the flanker task from the National Institutes of Health Toolbox), a critical component of self-regulation. Ability to inhibit responses and impose cognitive control increased rapidly during preteen years. Surface area of the anterior cingulate cortex accounted for a significant proportion of the variance in cognitive performance. This finding is intriguing, because characteristics of the anterior cingulum are shown to be related to impulse, attention, and executive problems in neurodevelopmental disorders, indicating a neural foundation for self-regulation abilities along a continuum from normality to pathology. The relationship was strongest in the younger children. Properties of large-fiber connections added to the picture by explaining additional variance in cognitive control. Although cognitive control was related to surface area of the anterior cingulate independently of basic processes of mental speed, the relationship between white matter quality and cognitive control could be fully accounted for by speed. The results underscore the need for integration of different aspects of brain maturation to understand the foundations of cognitive development.

The primary structure and phosphorylation pattern of the tandem Y(1)S(2)P(3)T(4)S(5)P(6)S(7) repeats of the RNA polymerase II carboxyl-terminal domain (CTD) convey information about the transcription apparatus--a CTD code--to a large ensemble of CTD-binding receptor proteins. Four of the seven coding "letters" of the fission yeast CTD (Tyr1, Pro3, Ser5, Pro6) are essential in vivo, but the grammatical rules of the code are obscure. Here we show that the minimal fission yeast CTD coding unit is a decapeptide Y(1)S(2)P(3)T(4)S(5)P(6)S(7)Y(1)S(2)P(3) and the spacing between coding units is flexible; the coding unit must contain two Tyr1 residues and the spacing between consecutive tyrosines is important; Ser5-PO(4)-Pro6 comprises an essential two-letter code "word" that is read by the mRNA capping apparatus; and a threshold number of Ser5-PO(4)-Pro6 words are needed to comprise a readable "sentence" of CTD information. Bypassing the essentiality of the Ser5 and Pro6 letters by fusion of capping enzymes to the CTD helped reveal how CTD phosphorylation circuits are wired in vivo. We found that the Ser2-PO(4) mark is independent of Ser5, Pro6, Ser7, and Thr4, whereas the Ser5-PO(4) mark is independent of Ser2, Ser7, and Thr4. These results provide unique insights to the reading and writing of the CTD code.

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.

Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from ten different phyla. To gain insight to the mechanism and evolution of this repair system, we determined the crystal structures of the ligase domain of Clostridium thermocellum Pnkp in three functional states along the reaction pathway: apoenzyme, ligase • ATP substrate complex, and covalent ligase-AMP intermediate. The tertiary structure is composed of a classical ligase nucleotidyltransferase module that is embellished by a unique α-helical insert module and a unique C-terminal α-helical module. Structure-guided mutational analysis identified active site residues essential for ligase adenylylation. Pnkp defines a new RNA ligase family with signature structural and functional properties.

RNA 3'-phosphate cyclase (RtcA) synthesizes RNA 2',3' cyclic phosphate ends via three steps: reaction with ATP to form a covalent RtcA-(histidinyl-Nε)-AMP intermediate; transfer of adenylate to an RNA 3'-phosphate to form RNA(3')pp(5')A; and attack of the vicinal O2' on the 3'-phosphorus to form a 2',3' cyclic phosphate and release AMP. Here we report the crystal structures of RtcA•ATP, RtcA•ATP•Mn(2+), and RtcA•ATP•Co(2+) substrate complexes and an RtcA•AMP product complex. Together with the structures of RtcA apoenzyme and the covalent RtcA-AMP intermediate, they illuminate the mechanism of nucleotidyl transfer, especially the stereochemical transitions at the AMP phosphate, the critical role of the metal in orienting the PP(i) leaving group of ATP during step 1, and the protein conformational switches that accompany substrate binding and product release. The octahedral metal complex of RtcA•ATP•Mn(2+) includes nonbridging oxygens from each of the ATP phosphates, two waters, and Glu14 as the sole RtcA component. Whereas the RtcA adenylylation step is metal-catalyzed, the subsequent steps in the cyclization pathway are metal-independent.

The DNA ligase D (LigD) 3'-phosphoesterase (PE) module is a conserved component of the bacterial nonhomologous end-joining (NHEJ) apparatus that performs 3' end-healing reactions at DNA double-strand breaks. Here we report the 1.9 A crystal structure of Pseudomonas aeruginosa PE, which reveals that PE exemplifies a unique class of DNA repair enzyme. PE has a distinctive fold in which an eight stranded beta barrel with a hydrophobic interior supports a crescent-shaped hydrophilic active site on its outer surface. Six essential side chains coordinate manganese and a sulfate mimetic of the scissile phosphate. The PE active site and mechanism are unique vis à vis other end-healing enzymes. We find PE homologs in archaeal and eukaryal proteomes, signifying that PEs comprise a DNA repair superfamily.

Global loss of DNA methylation has been known for decades as an epigenomic aberration associated with carcinogenesis and cancer progression. Loss of DNA methylation affects predominantly repetitive elements, which encompass >50% of the CpG dinucleotides present in the human genome. Because of the lack of an effective approach, no studies have been conducted to reveal such genome-wide methylation changes at a single-base resolution. To precisely determine the CpG sites with methylation loss during progression of pediatric intracranial ependymomas, we exploited a high-throughput bisulfite sequencing approach that simultaneously generates methylation profiles for thousands of Alu elements and their flanking sequences. Comparison of the methylation profiles of normal and tumor tissues revealed that the methylation status of the majority of CpG sites adjacent to or within Alu repeats remain unaltered, while a small set of CpG sites gain or lose methylation in ependymomas. Compared to the CpG sites with stable methylation level between normal control and ependymomas, the differentially methylated CpG sites are enriched in the sequences with low CpG density in the flanking regions of Alu repeats, rather than within the Alu sequences themselves. In addition, the CpG sites that are hypermethylated in ependymomas are proximal to CpG islands, whereas those that are hypomethylated are overrepresented in intergenic regions. Lastly, aberrant methylation of several genomic loci was confirmed to be associated with the aggressive primary tumors and the relapsed ependymomas.

Ubiquitously expressed seven-transmembrane receptors (7TMRs) classically signal through heterotrimeric G proteins and are commonly referred to as G protein-coupled receptors. It is now recognized that 7TMRs also signal through beta-arrestins, which act as versatile adapters controlling receptor signaling, desensitization, and trafficking. Most endogenous receptors appear to signal in a balanced fashion using both beta-arrestin and G protein-mediated pathways. Some 7TMRs are thought to be nonsignaling "decoys" because of their inability to activate typical G protein signaling pathways; it has been proposed that these receptors act to scavenge ligands or function as coreceptors. Here we demonstrate that ligand binding to the decoy receptor CXCR7 does not result in activation of signaling pathways typical of G proteins but does activate MAP kinases through beta-arrestins in transiently transfected cells. Furthermore, we observe that vascular smooth muscle cells that endogenously express CXCR7 migrate to its ligand interferon-inducible T-cell alpha chemoattractant (ITAC), an effect that is significantly attenuated by treatment with either a CXCR7 antagonist or beta-arrestin depletion by siRNA. This example of an endogenous "beta-arrestin-biased" 7TMR that signals through beta-arrestin in the absence of G protein activation demonstrates that some 7TMRs encoded in the genome have evolved to signal through beta-arrestin exclusively and suggests that other receptors that are currently thought to be orphans or decoys may also signal through such nonclassical pathways.

Urodele amphibians and teleost fish regenerate amputated body parts via a process called epimorphic regeneration. A hallmark of this phenomenon is the reactivation of silenced developmental regulatory genes that previously functioned during embryonic patterning. We demonstrate that histone modifications silence promoters of numerous genes involved in zebrafish caudal fin regeneration. Silenced developmental regulatory genes contain bivalent me(3)K4/me(3)K27 H3 histone modifications created by the concerted action of Polycomb (PcG) and Trithorax histone methyltransferases. During regeneration, this silent, bivalent chromatin is converted to an active state by loss of repressive me(3)K27 H3 modifications, occurring at numerous genes that appear to function during regeneration. Loss-of-function studies demonstrate a requirement for a me(3)K27 H3 demethylase during fin regeneration. These results indicate that histone modifications at discreet genomic positions may serve as a crucial regulatory event in the initiation of fin regeneration.

Ligases are essential actors in DNA replication, recombination, and repair by virtue of their ability to seal breaks in the phosphodiester backbone. Ligation proceeds through a nicked DNA-adenylate intermediate (AppDNA), which must be sealed quickly to avoid creating a potentially toxic lesion. Here, we take advantage of ligase-catalyzed AMP-dependent incision of a single supercoiled DNA molecule to observe the step of phosphodiester synthesis in real time. An exponentially distributed number of supercoils was relaxed per successful incision-resealing event, from which we deduce the torque-dependent ligation probability per DNA swivel. Premature dissociation of ligase from nicked DNA-adenylate accounted for approximately 10% of the observed events. The ability of ligase to form a C-shaped protein clamp around DNA is a key determinant of ligation probability per turn and the stability of the ligase-AppDNA intermediate. The estimated rate of phosphodiester synthesis by DNA ligase (400 s(-1)) is similar to the high rates of phosphodiester synthesis by replicative DNA polymerases.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder that results in retinal degeneration, obesity, cognitive impairment, polydactyly, renal abnormalities, and hypogenitalism. Of the 12 known BBS genes, BBS1 is the most commonly mutated, and a single missense mutation (M390R) accounts for approximately 80% of BBS1 cases. To gain insight into the function of BBS1, we generated a Bbs1(M390R/M390R) knockin mouse model. Mice homozygous for the M390R mutation recapitulated aspects of the human phenotype, including retinal degeneration, male infertility, and obesity. The obese mutant mice were hyperphagic and hyperleptinemic and exhibited reduced locomotor activity but no elevation in mean arterial blood pressure. Morphological evaluation of Bbs1 mutant brain neuroanatomy revealed ventriculomegaly of the lateral and third ventricles, thinning of the cerebral cortex, and reduced volume of the corpus striatum and hippocampus. Similar abnormalities were also observed in the brains of Bbs2(-/-), Bbs4(-/-), and Bbs6(-/-) mice, establishing these neuroanatomical defects as a previously undescribed BBS mouse model phenotype. Ultrastructural examination of the ependymal cell cilia that line the enlarged third ventricle of the Bbs1 mutant brains showed that, whereas the 9 + 2 arrangement of axonemal microtubules was intact, elongated cilia and cilia with abnormally swollen distal ends were present. Together with data from transmission electron microscopy analysis of photoreceptor cell connecting cilia, the Bbs1 M390R mutation does not affect axonemal structure, but it may play a role in the regulation of cilia assembly and/or function.

The external layer of the Gram-negative bacterial outer membrane is primarily composed of a protective, selectively permeable LPS. The biosynthesis of LPS relies on UDP-3-O-acyl-glucosamine N-acyltransferase (LpxD), which transfers 3-hydroxy-arachidic acid from acyl carrier protein to the 2' amine of UDP-3-O-myristoyl glucosamine in Chlamydia trachomatis. Our crystallographic study reveals that LpxD is a homotrimer, each subunit of which is constructed from a novel combination of an N-terminal uridine-binding domain, a core lipid-binding domain, and a C-terminal helical extension. Highly conserved residues dominate nucleotide binding. Phe-43 and Tyr-49 form pi-stacking interactions with uracil, and Asn-46 and His-284 form hydrogen bonds with the phosphate groups. These interactions place the glucosamine moiety at the catalytic center formed by two adjacent subunits. Here His-247 and His-284 contribute to a mechanism involving nucleophilic attack by the amine of one substrate on the carbonyl carbon of an acyl carrier protein thioester conjugate. Serendipitously, our study reveals a fatty acid (FA) binding groove near the catalytic center. MS elucidated the presence of a FA mixture binding to LpxD, with palmitic acid the most prevalent. The placement of UDP-N-acetylglucosamine and the FA provides details of N-acyltransferase ligand interactions and allows for a description of structure and reactivity at an early stage of LPS assembly.

The causative agents of malaria have developed a sophisticated machinery for entering multiple cell types in the human and insect hosts. In this machinery, a critical interaction occurs between the unusual myosin motor MyoA and the MyoA-tail Interacting Protein (MTIP). Here we present one crystal structure that shows three different conformations of Plasmodium MTIP, one of these in complex with the MyoA-tail, which reveal major conformational changes in the C-terminal domain of MTIP upon binding the MyoA-tail helix, thereby creating several hydrophobic pockets in MTIP that are the recipients of key hydrophobic side chains of MyoA. Because we also show that the MyoA helix is able to block parasite growth, this provides avenues for designing antimalarials.

DNA ligase D (LigD) is a large polyfunctional protein that participates in a recently discovered pathway of nonhomologous end-joining in bacteria. LigD consists of an ATP-dependent ligase domain fused to a polymerase domain (Pol) and a phosphoesterase module. The Pol activity is remarkable for its dependence on manganese, its ability to perform templated and nontemplated primer extension reactions, and its preference for adding ribonucleotides to blunt DNA ends. Here we report the 1.5-A crystal structure of the Pol domain of Pseudomonas LigD and its complexes with manganese and ATP/dATP substrates, which reveal a minimized polymerase with a two-metal mechanism and a fold similar to that of archaeal DNA primase. Mutational analysis highlights the functionally relevant atomic contacts in the active site. Although distinct nucleoside conformations and contacts for ATP versus dATP are observed in the cocrystals, the functional analysis suggests that the ATP-binding mode is the productive conformation for dNMP and rNMP incorporation. We find that a mutation of Mycobacterium LigD that uniquely ablates the polymerase activity results in increased fidelity of blunt-end double-strand break repair in vivo by virtue of eliminating nucleotide insertions at the recombination junctions. Thus, LigD Pol is a direct catalyst of mutagenic nonhomologous end-joining in vivo. Our studies underscore a previously uncharacterized role for the primase-like polymerase family in DNA repair.

Fcp1 is an essential protein phosphatase that hydrolyzes phosphoserines within the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). Fcp1 plays a major role in the regulation of CTD phosphorylation and, hence, critically influences the function of Pol II throughout the transcription cycle. The basic understanding of Fcp1-CTD interaction has remained ambiguous because two different modes have been proposed: the "dockingsite" model versus the "distributive" mechanism. Here we demonstrate biochemically that Fcp1 recognizes and dephosphorylates the CTD directly, independent of the globular non-CTD part of the Pol II structure. We point out that the recognition of CTD by the phosphatase is based on random access and is not driven by Pol II conformation. Results from three different types of experiments reveal that the overall interaction between Fcp1 and Pol II is not stable but dynamic. In addition, we show that Fcp1 also interacts with a region on the polymerase distinct from the CTD. We emphasize that this non-CTD site is functionally distinct from the docking site invoked previously as essential for the CTD phosphatase activity of Fcp1. We speculate that Fcp1 interaction with the non-CTD site may mediate its stimulatory effect on transcription elongation reported previously.

Epstein-Barr virus (EBV)-associated malignancies display distinct patterns of virus latent gene expression that reflect the complex interplay between the virus and its host cell. In the EBV-associated epithelial tumor nasopharyngeal carcinoma (NPC), the virus-encoded latent membrane protein LMP2A is consistently expressed whereas the oncogenic LMP1 protein appears to be restricted to only a proportion of tumors. In an attempt to understand the contribution of LMP2A to the pathogenesis of NPC, we established carcinoma cell lines stably infected in vitro with either a wild-type recombinant EBV (rEBV) or a mutant rEBV in which LMP2A is deleted (rEBV-2A). An NPC-like pattern of EBV gene expression including LMP2A but not LMP1 was consistently observed in carcinoma cells infected with rEBV. However, carcinoma cells infected with rEBV-2A expressed high levels of LMP1 from the signal transducer and activator of transcription (STAT)-regulated L1-TR promoter. Consistent with this effect, basal STAT activity was reduced in rEBV-infected carcinoma cells, and this repression was relieved in the absence of LMP2A. This modulation of STAT activity correlated with the ability of LMP2A to inhibit the autocrine secretion of IL-6 from carcinoma cell lines. Exogenous IL-6 was able to induce expression of LMP1 by means of STAT3 activation both in rEBV-infected carcinoma cell lines and in the EBV-positive C666-1 NPC cell line. The LMP2A-mediated suppression of IL-6 was a consequence of NF-kappaB inhibition. These data reveal that LMP2A modulates two key transcription factor pathways in carcinoma cells and suggest that this finding may be important in the pathogenesis of EBV-associated tumors.

Transcription and mRNA processing are regulated by phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II, which consists of tandem repeats of a Y(1)S(2)P(3)T(4)S(5)P(6)S(7) heptapeptide. Previous studies showed that members of the plant CTD phosphatase-like (CPL) protein family differentially regulate osmotic stress-responsive and abscisic acid-responsive transcription in Arabidopsis thaliana. Here we report that AtCPL1 and AtCPL2 specifically dephosphorylate Ser-5 of the CTD heptad in Arabidopsis RNA polymerase II, but not Ser-2. An N-terminal catalytic domain of CPL1, which suffices for CTD Ser-5 phosphatase activity in vitro, includes a signature DXDXT acylphosphatase motif, but lacks a breast cancer 1 CTD, which is an essential component of the fungal and metazoan Fcp1 CTD phosphatase enzymes. The CTD of CPL1, which contains two putative double-stranded RNA binding motifs, is essential for the in vivo function of CPL1 and includes a C-terminal 23-aa signal responsible for its nuclear targeting. CPL2 has a similar domain structure but contains only one double-stranded RNA binding motif. Combining mutant alleles of CPL1 and CPL2 causes synthetic lethality of the male but not the female gametes. These results indicate that CPL1 and CPL2 exemplify a unique family of CTD Ser-5-specific phosphatases with an essential role in plant growth and development.

We present the diploid genome sequence of the fungal pathogen Candida albicans. Because C. albicans has no known haploid or homozygous form, sequencing was performed as a whole-genome shotgun of the heterozygous diploid genome in strain SC5314, a clinical isolate that is the parent of strains widely used for molecular analysis. We developed computational methods to assemble a diploid genome sequence in good agreement with available physical mapping data. We provide a whole-genome description of heterozygosity in the organism. Comparative genomic analyses provide important clues about the evolution of the species and its mechanisms of pathogenesis.

Yeast tRNA ligase (Trl1) is an essential enzyme that converts cleaved tRNA half-molecules into spliced tRNAs containing a 2'-PO(4), 3'-5' phosphodiester at the splice junction. Trl1 also catalyzes splicing of HAC1 mRNA during the unfolded protein response. Trl1 performs three reactions: the 2',3'-cyclic phosphate of the proximal RNA fragment is hydrolyzed to a 3'-OH, 2'-PO(4) by a cyclic phosphodiesterase; the 5'-OH of the distal RNA fragment is phosphorylated by a GTP-dependent polynucleotide kinase; and the 3'-OH, 2'-PO(4), and 5'-PO(4) ends are then sealed by an ATP-dependent RNA ligase. The removal of the 2'-PO(4) at the splice junction is catalyzed by the essential enzyme Tpt1, which transfers the RNA 2'-PO(4) to NAD(+) to form ADP-ribose 1"-2"-cyclic phosphate. Here, we show that the bacteriophage T4 enzymes RNA ligase 1 and polynucleotide kinase/phosphatase can fulfill the tRNA and HAC1 mRNA splicing functions of yeast Trl1 in vivo and bypass the requirement for Tpt1. These results attest to the portability of RNA-repair systems, notwithstanding the significant differences in the specificities, mechanisms, and reaction intermediates of the individual yeast and T4 enzymes responsible for the RNA healing and sealing steps. We surmise that Tpt1 and its unique metabolite ADP-ribose 1"-2"-cyclic phosphate do not play essential roles in yeast independent of the tRNA-splicing reaction. Our finding that one-sixth of spliced HAC1 mRNAs in yeast cells containing the T4 RNA-repair system suffered deletion of a single nucleotide at the 3' end of the splice-donor site suggests a model whereby the yeast RNA-repair system evolved a requirement for the 2'-PO(4) for RNA ligation to suppress inappropriate RNA recombination.