FUS is sequestered in nuclear aggregates in ALS patient fibroblasts.

Mutations in the RNA-binding protein FUS have been shown to cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We investigate whether mutant FUS protein in ALS patient-derived fibroblasts affects normal FUS functions in the nucleus. We investigated fibroblasts from two ALS patients possessing different FUS mutations and a normal control. Fibroblasts from these patients have their nuclear FUS protein trapped in SDS-resistant aggregates. Genome-wide analysis reveals an inappropriate accumulation of Ser-2 phosphorylation on RNA polymerase II (RNA Pol II) near the transcription start sites of 625 genes for ALS patient cells and after small interfering RNA (siRNA) knockdown of FUS in normal fibroblasts. Furthermore, both the presence of mutant FUS protein and siRNA knockdown of wild-type FUS correlate with altered distribution of RNA Pol II within fibroblast nuclei. A loss of FUS function in orchestrating Ser-2 phosphorylation of the CTD of RNA Pol II is detectable in ALS patient-derived fibroblasts expressing mutant FUS protein, even when the FUS protein remains largely nuclear. A likely explanation for this loss of function is the aggregation of FUS protein in nuclei. Thus our results suggest a specific mechanism by which mutant FUS can have biological consequences other than by the formation of cytoplasmic aggregates.

RNA seeds higher-order assembly of FUS protein.

The abundant nuclear RNA binding protein FUS binds the C-terminal domain (CTD) of RNA polymerase II in an RNA-dependent manner, affecting Ser2 phosphorylation and transcription. Here, we examine the mechanism of this process and find that RNA binding nucleates the formation of higher-order FUS ribonucleoprotein assemblies that bind the CTD. Both the low-complexity domain and the arginine-glycine rich domain of FUS contribute to assembly. The assemblies appear fibrous by electron microscopy and have characteristics of ß zipper structures. These results support the emerging view that the pathologic protein aggregation seen in neurodegenerative diseases such as amyotrophic lateral sclerosis may occur via the exaggeration of functionally important assemblies of RNA binding proteins.

FUS binds the CTD of RNA polymerase II and regulates its phosphorylation at Ser2.

Mutations in the RNA-binding protein FUS (fused in sarcoma)/TLS have been shown to cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS), but the normal role of FUS is incompletely understood. We found that FUS binds the C-terminal domain (CTD) of RNA polymerase II (RNAP2) and prevents inappropriate hyperphosphorylation of Ser2 in the RNAP2 CTD at thousands of human genes. The loss of FUS leads to RNAP2 accumulation at the transcription start site and a shift in mRNA isoform expression toward early polyadenylation sites. Thus, in addition to its role in alternative RNA splicing, FUS has a general function in orchestrating CTD phosphorylation during RNAP2 transcription.

Multiple POT1-TPP1 proteins coat and compact long telomeric single-stranded DNA.

Telomeres are nucleoprotein complexes that cap and protect the ends of linear chromosomes. In humans, telomeres end in 50-300 nt of G-rich single-stranded DNA (ssDNA) overhangs. Protection of telomeres 1 (POT1) binds with nanomolar affinity to the ssDNA overhangs and forms a dimer with another telomere-end binding protein called TPP1. Whereas most previous studies utilized telomeric oligonucleotides comprising single POT1-TPP1 binding sites, here, we examined 72- to 144-nt tracts of telomeric DNA containing 6-12 POT1-TPP1 binding sites. Using electrophoretic mobility gel shift assays, size-exclusion chromatography, and electron microscopy, we analyzed telomeric nucleoprotein complexes containing POT1 alone, POT1-TPP1, and a truncated version of POT1 (POT1-N) that maintains its DNA-binding domain. The results revealed that POT1-N and POT1-TPP1 can completely coat long telomeric ssDNA substrates. Furthermore, we show that ssDNA coated with human POT1-TPP1 heterodimers forms compact, potentially ordered structures.

Functional interaction between telomere protein TPP1 and telomerase.

Human chromosome end-capping and telomerase regulation require POT1 (Protection of Telomeres 1) and TPP1 proteins, which bind to the 3' ssDNA extension of human telomeres. POT1-TPP1 binding to telomeric DNA activates telomerase repeat addition processivity. We now provide evidence that this POT1-TPP1 activation requires specific interactions with telomerase, rather than it being a DNA substrate-specific effect. First, telomerase from the fish medaka, which extends the same telomeric DNA primer as human telomerase, was not activated by human POT1-TPP1. Second, mutation of a conserved glycine, Gly100 in the TEN (telomerase essential N-terminal) domain of TERT, abolished the enhancement of telomerase processivity by POT1-TPP1, in contrast to other single amino acid mutations. Chimeric human-fish telomerases that contained the human TEN domain were active but not stimulated by POT1-TPP1, showing that additional determinants of processivity lie outside the TEN domain. Finally, primers bound to mouse POT1A and human TPP1 were activated for extension by human telomerase, whereas mPOT1A-mTPP1 was most active with mouse telomerase, indicating that these mammalian telomerases have specificity for their respective TPP1 proteins. We suggest that a sequence-specific interaction between TPP1 in the TPP1-POT1-telomeric DNA complex and the G100 region of the TEN domain of TERT is necessary for high-processivity telomerase action.

How telomeric protein POT1 avoids RNA to achieve specificity for single-stranded DNA.

The POT1-TPP1 heterodimer, the major telomere-specific single-stranded DNA-binding protein in mammalian cells, protects chromosome ends and contributes to the regulation of telomerase. The recent discovery of telomeric RNA raises the question of how POT1 faithfully binds telomeric ssDNA and avoids illicit RNA binding that could result in its depletion from telomeres. Here we show through binding studies that a single deoxythymidine in a telomeric repeat dictates the DNA versus RNA discrimination by human POT1 and mouse POT1A. We solve the crystal structure of hPOT1 bound to DNA with a ribouridine in lieu of the critical deoxythymidine and show that this substitution results in burying the 2(')-hydroxyl group in a hydrophobic region (Phe62) of POT1 in addition to eliminating favorable hydrogen-bonding interactions at the POT1-nucleic acid interface. At amino acid 62, Phe discriminates against RNA binding and Tyr allows RNA binding. We further show that TPP1 greatly augments POT1's discrimination against RNA.

Mutation in TERT separates processivity from anchor-site function.

Telomerase shows repeat-addition processivity (RAP): synthesis of multiple telomeric DNA repeats without primer dissociation. Leu14 mutants in the telomerase essential N-terminal domain of Tetrahymena thermophila telomerase reverse transcriptase retain full activity and anchor-site function but lose RAP, suggesting models for how this domain facilitates DNA translocation.

Multiple yeast genes, including Paf1 complex genes, affect telomere length via telomerase RNA abundance.

Twofold reductions in telomerase RNA levels cause telomere shortening in both humans and the yeast Saccharomyces cerevisiae. To test whether multiple genes that affect telomere length act by modulating telomerase RNA abundance, we used real-time reverse transcription-PCR to screen S. cerevisiae deletion strains reported to maintain shorter or longer telomeres to determine the levels of their telomerase RNA (TLC1) abundance. Of 290 strains screened, 5 had increased TLC1 levels; 4 of these maintained longer telomeres. Twenty strains had decreased TLC1 levels; 18 of these are known to maintain shorter telomeres. Four strains with decreased TLC1 RNA levels contained deletions of subunits of Paf1C (polymerase II-associated factor complex). While Paf1C had been implicated in the transcription of both polyadenylated and nonpolyadenylated RNAs, Paf1C had not been associated previously with the noncoding telomerase RNA. In Paf1C mutant strains, TLC1 overexpression partially rescues telomere length and cell growth defects, suggesting that telomerase RNA is a critical direct or indirect Paf1C target. Other factors newly identified as affecting TLC1 RNA levels include cyclin-dependent kinase, the mediator complex, protein phosphatase 2A, and ribosomal proteins L13B and S16A. This report establishes that a subset of telomere length genes act by modulating telomerase RNA abundance.

The POT1-TPP1 telomere complex is a telomerase processivity factor.

Telomeres were originally defined as chromosome caps that prevent the natural ends of linear chromosomes from undergoing deleterious degradation and fusion events. POT1 (protection of telomeres) protein binds the single-stranded G-rich DNA overhangs at human chromosome ends and suppresses unwanted DNA repair activities. TPP1 is a previously identified binding partner of POT1 that has been proposed to form part of a six-protein shelterin complex at telomeres. Here, the crystal structure of a domain of human TPP1 reveals an oligonucleotide/oligosaccharide-binding fold that is structurally similar to the beta-subunit of the telomere end-binding protein of a ciliated protozoan, suggesting that TPP1 is the missing beta-subunit of human POT1 protein. Telomeric DNA end-binding proteins have generally been found to inhibit rather than stimulate the action of the chromosome end-replicating enzyme, telomerase. In contrast, we find that TPP1 and POT1 form a complex with telomeric DNA that increases the activity and processivity of the human telomerase core enzyme. We propose that POT1-TPP1 switches from inhibiting telomerase access to the telomere, as a component of shelterin, to serving as a processivity factor for telomerase during telomere extension.

A new model for Schizosaccharomyces pombe telomere recognition: the telomeric single-stranded DNA-binding activity of Pot11-389.

The protection of telomeres 1 (Pot1) proteins specifically recognize the single-stranded 3' end of the telomere, an activity essential for sustained cellular viability and proliferation. The current model for the telomeric single-stranded DNA (ssDNA) binding activity of Schizosaccharomyces pombe Pot1 is based on a 20 kDa fragment, Pot1pN. Recent biochemical studies suggest that SpPot1 contains a larger ssDNA-binding domain and we have identified a novel ssDNA-binding domain similar in size to the human Pot1 domain. This domain, Pot1(1-389), binds extremely tightly to an oligonucleotide consisting of two conserved hexameric S. pombe telomere repeats, d(GGTTACGGTTAC), with an affinity approximately 4000-fold tighter than Pot1pN binds its cognate ssDNA. The Pot1(1-389)/ssDNA complex exhibits a half-life of 53 min, consistent with that estimated for full-length SpPot1 and significantly longer than that of Pot1pN. Single nucleotide substitutions reveal that, in contrast to Pot1pN, tandem trinucleotide repeats (GTT) within d(GGTTACGGTTAC) are specifically recognized by Pot1(1-389). Interestingly, certain single nucleotide substitutions that impacted Pot1pN binding exhibited no effect on binding affinity by Pot1(1-389). However, these substitutions reduced binding affinity when simultaneously substituted in each hexameric repeat. The non-additive nature of these substitutions suggests that certain nucleotides are coupled through the ability of the flexible ssDNA oligonucleotide to adopt alternate, thermodynamically equivalent conformations. The biochemical behavior of Pot1(1-389) is more similar to that of the full-length SpPot1 protein than to that of Pot1pN, making Pot1(1-389) a valuable domain for the future study of how full-length SpPot1 interacts with telomeric ssDNA.

Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase.

Telomerase, a ribonucleoprotein enzyme, adds telomeric DNA repeats to the ends of linear chromosomes. Here we report the first high-resolution structure of any portion of the telomerase reverse transcriptase, the telomerase essential N-terminal (TEN) domain from Tetrahymena thermophila. The structure, which seems to represent a novel protein fold, shows phylogenetically conserved amino acid residues in a groove on its surface. These residues are crucial for telomerase catalytic activity, and several of them are required for sequence-specific binding of a single-stranded telomeric DNA primer. The positively charged C terminus, which becomes ordered upon interaction with other macromolecules, is involved in binding RNA in a non-sequence-specific manner. The TEN domain's ability to bind both RNA and telomeric DNA, coupled with the notably strong effects on activity upon mutagenesis of single surface residues, suggest how this domain contributes to telomerase catalysis.

Human POT1 disrupts telomeric G-quadruplexes allowing telomerase extension in vitro.

The POT1 (protection of telomeres 1) protein binds the ssDNA overhangs at the ends of chromosomes in diverse eukaryotes. POT1 is essential for chromosome end-protection, as best demonstrated in fission yeast. In human cells, hPOT1 is also involved in telomere-length regulation. We now show that telomeric oligonucleotides, such as d[GGG(TTAGGG)(3)], which form intramolecular G-quadruplexes through Hoogsteen base-pairing, serve as only marginal primers for extension by recombinant human telomerase; telomerase stalls after every nucleotide addition. Addition of hPOT1 to the reaction restores the normal processive elongation pattern seen with primers that cannot form G-quadruplexes. hPOT1 does not act catalytically but, instead, forms a stoichiometric complex with the DNA, freeing its 3' tail. An antisense oligonucleotide, which base-pairs near the 5' end of the telomeric sequence, leaving a telomerase-extendable 3' tail, duplicates the effect of hPOT1 on activation of G-quadruplex primers. Thus, hPOT1 may function simply by trapping the unfolded forms of these telomeric primers in an equilibrium population. We propose an additional role for hPOT1 in telomere maintenance: disrupting G-quadruplex structures in telomeric DNA, thereby allowing proper elongation by telomerase.

Soluble domains of telomerase reverse transcriptase identified by high-throughput screening.

Telomerase is a ribonucleoprotein complex responsible for extending the ends of eukaryotic chromosomes. Structural and biophysical studies of this enzyme have been limited by the inability to produce large amounts of recombinant protein. Here we perform a high-throughput screen to map regions of the Tetrahymena thermophila TERT (Telomerase Reverse Transcriptase) protein that are overexpressed in a soluble form in Escherichia coli using a GFP-fusion system. Many of the soluble protein domains identified do not coincide with domains inferred from multiple sequence alignment, so screening for fluorescent colonies provided information not otherwise readily obtained. The method revealed an essential, independently folded N-terminal domain that was expressed and purified with high yield and found to be suitable for structural analysis. These results provide a tool for future structural and biophysical studies of TERT.

POT1 stimulates RecQ helicases WRN and BLM to unwind telomeric DNA substrates.

Defects in human RecQ helicases WRN and BLM are responsible for the cancer-prone disorders Werner syndrome and Bloom syndrome. Cellular phenotypes of Werner syndrome and Bloom syndrome, including genomic instability and premature senescence, are consistent with telomere dysfunction. RecQ helicases are proposed to function in dissociating alternative DNA structures during recombination and/or replication at telomeric ends. Here we report that the telomeric single-strand DNA-binding protein, POT1, strongly stimulates WRN and BLM to unwind long telomeric forked duplexes and D-loop structures that are otherwise poor substrates for these helicases. This stimulation is dependent on the presence of telomeric sequence in the duplex regions of the substrates. In contrast, POT1 failed to stimulate a bacterial 3'-5'-helicase. We find that purified POT1 binds to WRN and BLM in vitro and that full-length POT1 (splice variant 1) precipitates a higher amount of endogenous WRN protein, compared with BLM, from the HeLa nuclear extract. We propose roles for the cooperation of POT1 with RecQ helicases WRN and BLM in resolving DNA structures at telomeric ends, in a manner that protects the telomeric 3' tail as it is exposed during unwinding.

Switching human telomerase on and off with hPOT1 protein in vitro.

POT1 (protection of telomeres 1) protein binds the G-rich single-stranded telomeric DNA at the ends of chromosomes. In human cells hPOT1 is involved in telomere length regulation, but the mechanism of this regulation remains unknown. Examination of the high-resolution crystal structure of the hPOT1-TTAGGGTTAG complex suggested that it would not be extended by telomerase, a hypothesis that we confirm by in vitro assays with recombinant telomerase. On the other hand, when hPOT1 is bound at a position one telomeric repeat before the 3'-end, leaving an 8-nucleotide 3'-tail, the complex is extended with improved activity and processivity. Thus, depending on its location relative to the DNA 3'-end, hPOT1 can either inhibit telomerase action or form a preferred substrate for telomerase. We propose that another factor catalyzes the interconversion of these states in vivo.

Structure of human POT1 bound to telomeric single-stranded DNA provides a model for chromosome end-protection.

The POT1 (protection of telomeres 1) protein binds the single-stranded overhang at the ends of chromosomes in diverse eukaryotes. It is essential for chromosome end-protection in the fission yeast Schizosaccharomyces pombe, and it is involved in regulation of telomere length in human cells. Here, we report the crystal structure at a resolution of 1.73 A of the N-terminal half of human POT1 (hPOT1) protein bound to a telomeric single-stranded DNA (ssDNA) decamer, TTAGGGTTAG, the minimum tight-binding sequence indicated by in vitro binding assays. The structure reveals that hPOT1 contains two oligonucleotide/ oligosaccharide-binding (OB) folds; the N-terminal OB fold binds the first six nucleotides, resembling the structure of the S. pombe Pot1pN-ssDNA complex, whereas the second OB fold binds and protects the 3' end of the ssDNA. These results provide an atomic-resolution model for chromosome end-capping.

DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA.

Telomeres, specialized protein-DNA complexes that cap the ends of linear chromosomes, are essential for protecting chromosomes from degradation and end-to-end fusions. The Pot1 (protection of telomeres 1) protein is a widely distributed eukaryotic end-capping protein, having been identified in fission yeast, microsporidia, plants and animals. Schizosaccharomyces pombe Pot1p is essential for telomere maintenance, and human POT1 has been implicated in telomerase regulation. Pot1 binds telomeric single-stranded DNA (ssDNA) with exceptionally high sequence specificity, the molecular basis of which has been unknown. Here we describe the 1.9-A-resolution crystal structure of the amino-terminal DNA-binding domain of S. pombe Pot1p complexed with ssDNA. The protein adopts an oligonucleotide/oligosaccharide-binding (OB) fold with two loops that protrude to form a clamp for ssDNA binding. The structure explains the sequence specificity of binding: in the context of the Pot1 protein, DNA self-recognition involving base-stacking and unusual G-T base pairs compacts the DNA. Any sequence change disrupts the ability of the DNA to form this structure, preventing it from contacting the array of protein hydrogen-bonding groups. The structure also explains how Pot1p avoids binding the vast excess of RNA in the nucleus.