Ubiquitin recognition by the Cockayne syndrome group B protein: binding will set you free.

Transcription-coupled nucleotide excision repair (TC-NER) requires the coordinated efforts of many proteins. In this issue of Molecular Cell, Anindya et al. (2010) show that the proteins assemble at the site of DNA damage but cannot begin repair until the Cockayne syndrome group B protein (CSB) binds ubiquitin.

Tethering of the conserved piggyBac transposase fusion protein CSB-PGBD3 to chromosomal AP-1 proteins regulates expression of nearby genes in humans.

The CSB-PGBD3 fusion protein arose more than 43 million years ago when a 2.5-kb piggyBac 3 (PGBD3) transposon inserted into intron 5 of the Cockayne syndrome Group B (CSB) gene in the common ancestor of all higher primates. As a result, full-length CSB is now coexpressed with an abundant CSB-PGBD3 fusion protein by alternative splicing of CSB exons 1-5 to the PGBD3 transposase. An internal deletion of the piggyBac transposase ORF also gave rise to 889 dispersed, 140-bp MER85 elements that were mobilized in trans by PGBD3 transposase. The CSB-PGBD3 fusion protein binds MER85s in vitro and induces a strong interferon-like innate antiviral immune response when expressed in CSB-null UVSS1KO cells. To explore the connection between DNA binding and gene expression changes induced by CSB-PGBD3, we investigated the genome-wide DNA binding profile of the fusion protein. CSB-PGBD3 binds to 363 MER85 elements in vivo, but these sites do not correlate with gene expression changes induced by the fusion protein. Instead, CSB-PGBD3 is enriched at AP-1, TEAD1, and CTCF motifs, presumably through protein-protein interactions with the cognate transcription factors; moreover, recruitment of CSB-PGBD3 to AP-1 and TEAD1 motifs correlates with nearby genes regulated by CSB-PGBD3 expression in UVSS1KO cells and downregulated by CSB rescue of mutant CS1AN cells. Consistent with these data, the N-terminal CSB domain of the CSB-PGBD3 fusion protein interacts with the AP-1 transcription factor c-Jun and with RNA polymerase II, and a chimeric CSB-LacI construct containing only the N-terminus of CSB upregulates many of the genes induced by CSB-PGBD3. We conclude that the CSB-PGBD3 fusion protein substantially reshapes the transcriptome in CS patient CS1AN and that continued expression of the CSB-PGBD3 fusion protein in the absence of functional CSB may affect the clinical presentation of CS patients by directly altering the transcriptional program.

SINEs and LINEs: the art of biting the hand that feeds you.

SINEs and LINEs are short and long interspersed retrotransposable elements, respectively, that invade new genomic sites using RNA intermediates. SINEs and LINEs are found in almost all eukaryotes (although not in Saccharomyces cerevisiae) and together account for at least 34% of the human genome. The noncoding SINEs depend on reverse transcriptase and endonuclease functions encoded by partner LINEs. With the completion of many genome sequences, including our own, the database of SINEs and LINEs has taken a great leap forward. The new data pose new questions that can only be answered by detailed studies of the mechanism of retroposition. Current work ranges from the biochemistry of reverse transcription and integration invitro, target site selection in vivo, nucleocytoplasmic transport of the RNA and ribonucleoprotein intermediates, and mechanisms of genomic turnover. Two particularly exciting new ideas are that SINEs may help cells survive physiological stress, and that the evolution of SINEs and LINEs has been shaped by the forces of RNA interference. Taken together, these studies promise to explain the birth and death of SINEs and LINEs, and the contribution of these repetitive sequence families to the evolution of genomes.

An abundant evolutionarily conserved CSB-PiggyBac fusion protein expressed in Cockayne syndrome.

Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3' terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1-5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein.

On the role of a conserved, potentially helix-breaking residue in the tRNA-binding alpha-helix of archaeal CCA-adding enzymes.

Archaeal class I CCA-adding enzymes use a ribonucleoprotein template to build and repair the universally conserved 3'-terminal CCA sequence of the acceptor stem of all tRNAs. A wealth of structural and biochemical data indicate that the Archaeoglobus fulgidus CCA-adding enzyme binds primarily to the tRNA acceptor stem through a long, highly conserved alpha-helix that lies nearly parallel to the acceptor stem and makes many contacts with its sugar-phosphate backbone. Although the geometry of this alpha-helix is nearly ideal in all available cocrystal structures, the helix contains a highly conserved, potentially helix-breaking proline or glycine near the N terminus. We performed a mutational analysis to dissect the role of this residue in CCA-addition activity. We found that the phylogenetically permissible P295G mutant and the phylogenetically absent P295T had little effect on CCA addition, whereas P295A and P295S progressively interfered with CCA addition (C74>C75>A76 addition). We also examined the effects of these mutations on tRNA binding and the kinetics of CCA addition, and performed a computational analysis using Rosetta Design to better understand the role of P295 in nucleotide transfer. Our data indicate that CCA-adding activity does not correlate with the stability of the pre-addition cocrystal structures visualized by X-ray crystallography. Rather, the data are consistent with a transient conformational change involving P295 of the tRNA-binding alpha-helix during or between one or more steps in CCA addition.

tRNA maturation: RNA polymerization without a nucleic acid template.

The CCA-adding enzyme, which builds and repairs the 3' terminal CCA sequence of tRNA, is the only RNA polymerase that can synthesize a defined nucleotide sequence without using a nucleic acid template. New cocrystal structures tell us how this remarkable enzyme works.

Role of the C-terminal domain of RNA polymerase II in U2 snRNA transcription and 3' processing.

U small nuclear RNAs (snRNAs) and mRNAs are both transcribed by RNA polymerase II (Pol II), but the snRNAs have unusual TATA-less promoters and are neither spliced nor polyadenylated; instead, 3' processing is directed by a highly conserved 3' end formation signal that requires initiation from an snRNA promoter. Here we show that the C-terminal domain (CTD) of Pol II is required for efficient U2 snRNA transcription, as it is for mRNA transcription. However, CTD kinase inhibitors, such as 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), that block mRNA elongation do not affect U2 transcription, although 3' processing of the U2 primary transcript is impaired. We show further that U2 transcription is preferentially inhibited by low doses of UV irradiation or actinomycin D, which induce CTD kinase activity, and that UV inhibition can be rescued by treatment with DRB or H7. We propose that Pol II complexes transcribing snRNAs and mRNAs have distinct CTD phosphorylation patterns. mRNA promoters recruit factors including kinases that hyperphosphorylate the CTD, and the CTD in turn recruits proteins needed for mRNA splicing and polyadenylation. We predict that snRNA promoters recruit factors including a CTD kinase(s) whose snRNA-specific phosphorylation pattern recruits factors required for promoter-coupled 3' end formation.

What role (if any) does the highly conserved CSB-PGBD3 fusion protein play in Cockayne syndrome?

The PGBD3 piggyBac transposon inserted into CSB intron 5 early in the primate lineage. As a result of alternative splicing, the human CSB gene now encodes three proteins: CSB, a CSB-PGBD3 fusion protein that joins the N-terminal CSB domain to the C-terminal PGBD3 transposase domain, and PGBD3 transposase. The fusion protein is as highly conserved as CSB, suggesting that it is advantageous in health; however, expression of the fusion protein in CSB-null cells induces a constitutive interferon (IFN) response. The fusion protein binds in vivo to PGBD3-related MER85 elements, but is also tethered to c-Jun, TEAD1, and CTCF motifs by interactions with the cognate transcription factors. The fusion protein regulates nearby genes from the c-Jun (and to a lesser extent TEAD1 and CTCF) motifs, but not from MER85 elements. We speculate that the fusion protein interferes with CSB-dependent chromatin remodeling, generating double-stranded RNA (dsRNA) that induces an IFN response through endosomal TLR or cytoplasmic RIG-I and/or MDA5 RNA sensors. We suggest that the fusion protein was fixed in primates because an elevated IFN response may help to fight viral infection. We also speculate that an inappropriate IFN response may contribute to the clinical presentation of CS.

PGBD5: a neural-specific intron-containing piggyBac transposase domesticated over 500 million years ago and conserved from cephalochordates to humans.

BACKGROUND: piggyBac domain (PGBD) transposons are found in organisms ranging from fungi to humans. Three domesticated piggyBac elements have been described. In the ciliates Paramecium tetraurelia and Tetrahymena thermophila, homologs known as piggyMacs excise internal eliminated sequences from germline micronuclear DNA during regeneration of the new somatic macronucleus. In primates, a PGBD3 element inserted into the Cockayne syndrome group B (CSB) gene over 43 Mya serves as an alternative 3' terminal exon, enabling the CSB gene to generate both full length CSB and a conserved CSB-PGBD3 fusion protein that joins an N-terminal CSB domain to the C-terminal transposase domain. RESULTS: We describe a fourth domesticated piggyBac element called PGBD5. We show that i) PGBD5 was first domesticated in the common ancestor of the cephalochordate Branchiostoma floridae (aka lancelet or amphioxus) and vertebrates, and is conserved in all vertebrates including lamprey but cannot be found in more basal urochordates, hemichordates, or echinoderms; ii) the lancelet, lamprey, and human PGBD5 genes are syntenic and orthologous; iii) no potentially mobile ancestral PGBD5 elements can be identified in other more deeply rooted organisms; iv) although derived from an IS4-related transposase of the RNase H clan, PGBD5 protein is unlikely to retain enzymatic activity because the catalytic DDD(D) motif is not conserved; v) PGBD5 is preferentially expressed in certain granule cell lineages of the brain and in the central nervous system based on available mouse and human in situ hybridization data, and the tissue-specificity of documented mammalian EST and mRNA clones; vi) the human PGBD5 promoter and gene region is rich in bound regulatory factors including the neuron-restrictive silencer factors NRSF/REST and CoREST, as well as SIN3, KAP1, STAT3, and CTCF; and vii) despite preferential localization within the nucleus, PGBD5 protein is unlikely to bind DNA or chromatin as neither DNase I digestion nor high salt extraction release PGBD5 from fractionated mouse brain nuclei. CONCLUSIONS: We speculate that the neural-specific PGBD5 transposase was domesticated >500 My after cephalochordates and vertebrates split from urochordates, and that PGBD5 may have played a role in the evolution of a primitive deuterostome neural network into a centralized nervous system.

The conserved Cockayne syndrome B-piggyBac fusion protein (CSB-PGBD3) affects DNA repair and induces both interferon-like and innate antiviral responses in CSB-null cells.

Cockayne syndrome is a segmental progeria most often caused by mutations in the CSB gene encoding a SWI/SNF-like ATPase required for transcription-coupled DNA repair (TCR). Over 43Mya before marmosets diverged from humans, a piggyBac3 (PGBD3) transposable element integrated into intron 5 of the CSB gene. As a result, primate CSB genes now generate both CSB protein and a conserved CSB-PGBD3 fusion protein in which the first 5 exons of CSB are alternatively spliced to the PGBD3 transposase. Using a host cell reactivation assay, we show that the fusion protein inhibits TCR of oxidative damage but facilitates TCR of UV damage. We also show by microarray analysis that expression of the fusion protein alone in CSB-null UV-sensitive syndrome (UVSS) cells induces an interferon-like response that resembles both the innate antiviral response and the prolonged interferon response normally maintained by unphosphorylated STAT1 (U-STAT1); moreover, as might be expected based on conservation of the fusion protein, this potentially cytotoxic interferon-like response is largely reversed by coexpression of functional CSB protein. Interestingly, expression of CSB and the CSB-PGBD3 fusion protein together, but neither alone, upregulates the insulin growth factor binding protein IGFBP5 and downregulates IGFBP7, suggesting that the fusion protein may also confer a metabolic advantage, perhaps in the presence of DNA damage. Finally, we show that the fusion protein binds in vitro to members of a dispersed family of 900 internally deleted piggyBac elements known as MER85s, providing a potential mechanism by which the fusion protein could exert widespread effects on gene expression. Our data suggest that the CSB-PGBD3 fusion protein is important in both health and disease, and could play a role in Cockayne syndrome.

Human U2 snRNA genes exhibit a persistently open transcriptional state and promoter disassembly at metaphase.

In mammals, small multigene families generate spliceosomal U snRNAs that are nearly as abundant as rRNA. Using the tandemly repeated human U2 genes as a model, we show by footprinting with DNase I and permanganate that nearly all sequences between the enhancer-like distal sequence element and the initiation site are protected during interphase whereas the upstream half of the U2 snRNA coding region is exposed. We also show by chromatin immunoprecipitation that the SNAPc complex, which binds the TATA-like proximal sequence element, is removed at metaphase but remains bound under conditions that induce locus-specific metaphase fragility of the U2 genes, such as loss of CSB, BRCA1, or BRCA2 function, treatment with actinomycin D, or overexpression of the tetrameric p53 C terminus. We propose that the U2 snRNA promoter establishes a persistently open state to facilitate rapid reinitiation and perhaps also to bypass TFIIH-dependent promoter melting; this open state would then be disassembled to allow metaphase chromatin condensation.

Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases.

CCA-adding enzymes build and repair the 3'-terminal CCA sequence of tRNA. These unusual RNA polymerases use either a ribonucleoprotein template (class I) or pure protein template (class II) to form mock base pairs with the Watson-Crick edges of incoming CTP and ATP. Guided by the class II Bacillus stearothermophilus CCA-adding enzyme structure, we introduced mutations designed to reverse the polarity of hydrogen bonds between the nucleobases and protein template. We were able to transform the CCA-adding enzyme into a (U,G)-adding enzyme that incorporates UTP and GTP instead of CTP and ATP; we transformed the related Aquifex aeolicus CC- and A-adding enzymes into UU- and G-adding enzymes and Escherichia coli poly(A) polymerase into a poly(G) polymerase; and we transformed the B. stearothermophilus CCA-adding enzyme into a poly(C,A) polymerase by mutations in helix J that appear, based on the apoenzyme structure, to sterically limit addition to CCA. We also transformed the B. stearothermophilus CCA-adding enzyme into a dCdCdA-adding enzyme by mutating an arginine that interacts with the incoming ribose 2' hydroxyl. Most importantly, we found that mutations in helix J can affect the specificity of the nucleotide binding site some 20 A away, suggesting that the specificity of both class I and II enzymes may be dictated by an intricate network of hydrogen bonds involving the protein, incoming nucleotide, and 3' end of the tRNA. Collaboration between RNA and protein in the form of a ribonucleoprotein template may help to explain the evolutionary diversity of the nucleotidyltransferase family.

Cockayne syndrome group B protein (CSB) plays a general role in chromatin maintenance and remodeling.

Cockayne syndrome (CS) is an inherited neurodevelopmental disorder with progeroid features. Although the genes responsible for CS have been implicated in a variety of DNA repair- and transcription-related pathways, the nature of the molecular defect in CS remains mysterious. Using expression microarrays and a unique method for comparative expression analysis called L2L, we sought to define this defect in cells lacking a functional CS group B (CSB) protein, the SWI/SNF-like ATPase responsible for most cases of CS. Remarkably, many of the genes regulated by CSB are also affected by inhibitors of histone deacetylase and DNA methylation, as well as by defects in poly(ADP-ribose)-polymerase function and RNA polymerase II elongation. Moreover, consistent with these microarray expression data, CSB-null cells are sensitive to inhibitors of histone deacetylase or poly(ADP-ribose)-polymerase. Our data indicate a general role for CSB protein in maintenance and remodeling of chromatin structure and suggest that CS is a disease of transcriptional deregulation caused by misexpression of growth-suppressive, inflammatory, and proapoptotic pathways.

A model for C74 addition by CCA-adding enzymes: C74 addition, like C75 and A76 addition, does not involve tRNA translocation.

The CCA-adding enzyme adds CCA to the 3'-end of tRNA one nucleotide at a time, using CTP and ATP as substrates. We found previously that tRNA does not rotate or translocate on the enzyme during the addition of C75 and A76. We therefore predicted that the growing 3'-end of tRNA must, upon addition of each nucleotide, refold to reposition the new 3'-hydroxyl equivalently relative to the solitary nucleotidyltransferase motif. Cocrystal structures of the class I archaeal Archaeoglobus fulgidus enzyme, poised for addition of C75 and A76, confirmed this prediction. We have also demonstrated that an evolutionarily flexible beta-turn facilitates progressive refolding of the 3'-terminal C74 and C75 residues during C75 and A76 addition. Although useful cocrystals corresponding to C74 addition have not yet been obtained, we now show experimentally that tRNA does not rotate or translocate during C74 addition. We therefore propose, based on the existing A. fulgidus cocrystal structures, that the same flexible beta-turn functions as a wedge between the discriminator base (N73) and the terminal base pair of the acceptor stem, unstacking and repositioning N73 to attack the incoming CTP. Thus a single flexible beta-turn would orchestrate consecutive addition of all three nucleotides without significant movement of the tRNA on the enzyme surface.

E Pluribus Unum: 3' end formation of polyadenylated mRNAs, histone mRNAs, and U snRNAs.

The 3' ends of almost all eukaryotic RNAs are generated by nucleolytic cleavage. Remarkably, three groups now demonstrate that similar or identical endonucleases of the metallo-beta-lactamase family generate the 3' ends of polyadenylated mRNAs, nonpolyadenylated histone mRNAs, and U snRNAs.

L2L: a simple tool for discovering the hidden significance in microarray expression data.

L2L is a database consisting of lists of differentially expressed genes compiled from published mammalian microarray studies, along with an easy-to-use application for mining the database with the user's own microarray data. As illustrated by re-analysis of a recent study of diabetic nephropathy, L2L identifies novel biological patterns in microarray data, providing insights into the underlying nature of biological processes and disease. L2L is available online at the authors' website [http://depts.washington.edu/l2l/].

Archaeal CCA-adding enzymes: central role of a highly conserved beta-turn motif in RNA polymerization without translocation.

The CCA-adding enzyme (tRNA nucleotidyltransferase) builds and repairs the 3' end of tRNA. A single active site adds both CTP and ATP, but the enzyme has no nucleic acid template, and tRNA does not translocate or rotate during C75 and A76 addition. We modeled the structure of the class I archaeal Sulfolobus shibatae CCA-adding enzyme on eukaryotic poly(A) polymerase and mutated residues in the vicinity of the active site. We found mutations that specifically affected C74, C75, or A76 addition, as well as mutations that progressively impaired addition of CCA. Many of these mutations clustered in an evolutionarily versatile beta-turn located between strands 3 and 4 of the nucleotidyltransferase domain. Our mutational analysis confirms and extends recent crystallographic studies of the highly homologous Archaeoglobus fulgidus enzyme. We suggest that the unusual phenotypes of the beta-turn mutants reflect the consecutive conformations assumed by the beta-turn as it presents the discriminator base N73, then C74, and finally C75 to the active site without translocation or rotation of the tRNA acceptor stem. We also suggest that beta-turn mutants can affect nucleotide selection because the growing 3' end of tRNA must be properly positioned to serve as part of the ribonucleoprotein template that selects the incoming nucleotide.

Closely related CC- and A-adding enzymes collaborate to construct and repair the 3'-terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans.

The 3'-terminal CCA sequence of tRNA is faithfully constructed and repaired by the CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) using CTP and ATP as substrates but no nucleic acid template. Until recently, all CCA-adding enzymes from all three kingdoms appeared to be composed of a single kind of polypeptide with dual specificity for adding both CTP and ATP; however, we recently found that in Aquifex aeolicus, which lies near the deepest root of the eubacterial 16 S rRNA-based phylogenetic tree, CCA addition represents a collaboration between closely related CC-adding and A-adding enzymes (Tomita, K. and Weiner, A. M. (2001) Science 294, 1334-1336). Here we show that in Synechocystis sp. and Deinococcus radiodurans, as in A. aeolicus, CCA is added by homologous CC- and A-adding enzymes. We also find that the eubacterial CCA-, CC-, and A-adding enzymes, as well as the related eubacterial poly(A) polymerases, each fall into phylogenetically distinct groups derived from a common ancestor. Intriguingly, the Thermatoga maritima CCA-adding enzyme groups with the A-adding enzymes, suggesting that these distinct tRNA nucleotidyltransferase activities can intraconvert over evolutionary time.

A single catalytically active subunit in the multimeric Sulfolobus shibatae CCA-adding enzyme can carry out all three steps of CCA addition.

The CCA-adding enzyme ATP(CTP):tRNA nucleotidyltransferase builds and repairs the 3'-terminal CCA sequence of tRNA. Although this unusual RNA polymerase has no nucleic acid template, it can construct the CCA sequence one nucleotide at a time using CTP and ATP as substrates. We found previously that tRNA does not translocate along the enzyme during CCA addition (Yue, D., Weiner, A. M., and Maizels, N. (1998) J. Biol. Chem. 273, 29693-29700) and that a single nucleotidyltransferase motif adds all three nucleotides (Shi, P.-Y., Maizels, N., and Weiner, A. M. (1998) EMBO J. 17, 3197-3206). Intriguingly, the CCA-adding enzyme from the archaeon Sulfolobus shibatae is a homodimer that forms a tetramer upon binding two tRNAs. We therefore asked whether the active form of the S. shibatae enzyme might have two quasi-equivalent active sites, one adding CTP and the other ATP. Using an intersubunit complementation approach, we demonstrate that the dimer is active and that a single catalytically active subunit can carry out all three steps of CCA addition. We also locate one UV light-induced tRNA cross-link on the enzyme structure and provide evidence suggesting the location of another. Our data rule out shuttling models in which the 3'-end of the tRNA shuttles from one quasi-equivalent active site to another, demonstrate that tRNA-induced tetramerization is not required for CCA addition, and support a role for the tail domain of the enzyme in tRNA binding.

Crystal structures of an archaeal class I CCA-adding enzyme and its nucleotide complexes.

CCA-adding enzymes catalyze the addition of CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template and have been divided into two classes based on their amino acid sequences. We have determined the crystal structures of a class I CCA-adding enzyme from Archeoglobus fulgidus (AfCCA) and its complexes with ATP, CTP, or UTP. Although it and the class II bacterial Bacillus stearothermophilus CCA enzyme (BstCCA) have similar dimensions and domain architectures (head, neck, body, and tail), only the polymerase domain is structurally homologous. Moreover, the relative orientation of the head domain with respect to the body and tail domains, which appear likely to bind tRNA, differs significantly between the two enzyme classes. Unlike the class II BstCCA, this enzyme binds nucleotides nonspecifically in the absence of bound tRNA. The shape and electrostatic charge distribution of the AfCCA enzyme suggests a model for tRNA binding that accounts for the phosphates that are protected from chemical modification by tRNA binding to AfCCA. The structures of the AfCCA enzyme and the eukaryotic poly(A) polymerase are very similar, implying a close evolutionary relationship between them.