Xia-Jing Tong

Neurexin and neuroligin mediate retrograde synaptic inhibition in C. elegans.

Keep on Neurexin Mutations altering neurexin and neuroligin have been linked to several psychiatric disorders, including autism and schizophrenia. However, it remains uncertain how these mutations alter neural circuit development and function. Working in the nematode Caenorhabditis elegans, Hu et al. (p. 980, published online 2 August) found that neurexin and neuroligin function downstream of a muscle microRNA (miR-1), mediating a retrograde synaptic signal that inhibits neurotransmitter release. This retrograde signal inhibited transmission by adjusting the rate and duration of synaptic vesicle release. Two synaptic adhesion molecules that have been implicated in psychiatric diseases affect the kinetics of synaptic events. The synaptic adhesion molecules neurexin and neuroligin alter the development and function of synapses and are linked to autism in humans. Here, we found that Caenorhabditis elegans neurexin (NRX-1) and neuroligin (NLG-1) mediated a retrograde synaptic signal that inhibited neurotransmitter release at neuromuscular junctions. Retrograde signaling was induced in mutants lacking a muscle microRNA (miR-1) and was blocked in mutants lacking NLG-1 or NRX-1. Release was rapid and abbreviated when the retrograde signal was on, whereas release was slow and prolonged when retrograde signaling was blocked. The retrograde signal adjusted release kinetics by inhibiting exocytosis of synaptic vesicles (SVs) that are distal to the site of calcium entry. Inhibition of release was mediated by increased presynaptic levels of tomosyn, an inhibitor of SV fusion.

Yeast telomerase subunit Est1p has guanine quadruplex–promoting activity that is required for telomere elongation

Telomeres are eukaryotic protein–DNA complexes found at the ends of linear chromosomes that are essential for maintaining genome integrity and are implicated in cellular aging and cancer. The guanine (G)-rich strand of telomeric DNA, usually elongated by the telomerase reverse transcriptase, can form a higher-order structure known as a G-quadruplex in vitro and in vivo. Several factors that promote or resolve G-quadruplexes have been identified, but the functional importance of these structures for telomere maintenance is not well understood. Here we show that the yeast telomerase subunit Est1p, known to be involved in telomerase recruitment to telomeres, can convert single-stranded telomeric G-rich DNA into a G-quadruplex structure in vitro in a Mg2+-dependent manner. Cells carrying Est1p mutants deficient in G-quadruplex formation in vitro showed gradual telomere shortening and cellular senescence, indicating a positive regulatory role for G-quadruplex in the maintenance of telomere length.

UNC-13L, UNC-13S, and Tomosyn form a protein code for fast and slow neurotransmitter release in Caenorhabditis elegans

Synaptic transmission consists of fast and slow components of neurotransmitter release. Here we show that these components are mediated by distinct exocytic proteins. The Caenorhabditis elegans unc-13 gene is required for SV exocytosis, and encodes long and short isoforms (UNC-13L and S). Fast release was mediated by UNC-13L, whereas slow release required both UNC-13 proteins and was inhibited by Tomosyn. The spatial location of each protein correlated with its effect. Proteins adjacent to the dense projection mediated fast release, while those controlling slow release were more distal or diffuse. Two UNC-13L domains accelerated release. C2A, which binds RIM (a protein associated with calcium channels), anchored UNC-13 at active zones and shortened the latency of release. A calmodulin binding site accelerated release but had little effect on UNC-13’s spatial localization. These results suggest that UNC-13L, UNC-13S, and Tomosyn form a molecular code that dictates the timing of neurotransmitter release. DOI: http://dx.doi.org/10.7554/eLife.00967.001

Telomerase-Null Survivor Screening Identifies Novel Telomere Recombination Regulators

Telomeres are protein–DNA structures found at the ends of linear chromosomes and are crucial for genome integrity. Telomeric DNA length is primarily maintained by the enzyme telomerase. Cells lacking telomerase will undergo senescence when telomeres become critically short. In Saccharomyces cerevisiae, a very small percentage of cells lacking telomerase can remain viable by lengthening telomeres via two distinct homologous recombination pathways. These “survivor” cells are classified as either Type I or Type II, with each class of survivor possessing distinct telomeric DNA structures and genetic requirements. To elucidate the regulatory pathways contributing to survivor generation, we knocked out the telomerase RNA gene TLC1 in 280 telomere-length-maintenance (TLM) gene mutants and examined telomere structures in post-senescent survivors. We uncovered new functional roles for 10 genes that affect the emerging ratio of Type I versus Type II survivors and 22 genes that are required for Type II survivor generation. We further verified that Pif1 helicase was required for Type I recombination and that the INO80 chromatin remodeling complex greatly affected the emerging frequency of Type I survivors. Finally, we found the Rad6-mediated ubiquitination pathway and the KEOPS complex were required for Type II recombination. Our data provide an independent line of evidence supporting the idea that these genes play important roles in telomere dynamics.

Sua5p a single-stranded telomeric DNA-binding protein facilitates telomere replication

In budding yeast Saccharomyces cerevisiae, telomere length maintenance involves a complicated network as more than 280 telomere maintenance genes have been identified in the nonessential gene deletion mutant set. As a supplement, we identified additional 29 telomere maintenance genes, which were previously taken as essential genes. In this study, we report a novel function of Sua5p in telomere replication. Epistasis analysis and telomere sequencing show that sua5Δ cells display progressively shortened telomeres at early passages, and Sua5 functions downstream telomerase recruitment. Further, biochemical, structural and genetic studies show that Sua5p specifically binds single‐stranded telomeric (ssTG) DNA in vitro through a distinct DNA‐binding region on its surface, and the DNA‐binding ability is essential for its telomere function. Thus, Sua5p represents a novel ssTG DNA‐binding protein and positively regulates the telomere length in vivo.

A network of autism linked genes stabilizes two pools of synaptic GABAA receptors

Changing receptor abundance at synapses is an important mechanism for regulating synaptic strength. Synapses contain two pools of receptors, immobilized and diffusing receptors, both of which are confined to post-synaptic elements. Here we show that immobile and diffusing GABAA receptors are stabilized by distinct synaptic scaffolds at C. elegans neuromuscular junctions. Immobilized GABAA receptors are stabilized by binding to FRM-3/EPB4.1 and LIN-2A/CASK. Diffusing GABAA receptors are stabilized by the synaptic adhesion molecules Neurexin and Neuroligin. Inhibitory post-synaptic currents are eliminated in double mutants lacking both scaffolds. Neurexin, Neuroligin, and CASK mutations are all linked to Autism Spectrum Disorders (ASD). Our results suggest that these mutations may directly alter inhibitory transmission, which could contribute to the developmental and cognitive deficits observed in ASD. DOI: http://dx.doi.org/10.7554/eLife.09648.001

Est1 Protects Telomeres and Inhibits Subtelomeric Y′-Element Recombination

ABSTRACT In the budding yeast Saccharomyces cerevisiae, the structure and function of telomeres are maintained by binding proteins, such as Cdc13-Stn1-Ten1 (CST), Yku, and the telomerase complex. Like CST and Yku, telomerase also plays a role in telomere protection or capping. Unlike CST and Yku, however, the underlying molecular mechanism of telomerase-mediated telomere protection remains unclear. In this study, we employed both the CDC13-EST1 fusion gene and the separation-of-function allele est1-D514A to elucidate that Est1 provided a telomere protection pathway that was independent of both the CST and Yku pathways. Est1s ability to convert single-stranded telomeric DNA into a G quadruplex was required for telomerase-mediated telomere protection function. Additionally, Est1 maintained the integrity of telomeres by suppressing the recombination of subtelomeric Y′ elements. Our results demonstrate that one major functional role that Est1 brings to the telomerase complex is the capping or protection of telomeres.

Crystal structure of the catalytic core of Saccharomyces cerevesiae histone demethylase Rph1: insights into the substrate specificity and catalytic mechanism

Saccharomyces cerevesiae Rph1 is a histone demethylase orthologous to human JMJD2A (Jumonji-domain-containing protein 2A) that can specifically demethylate tri- and di-methylated Lys³⁶ of histone H3. c-Rph1, the catalytic core of Rph1, is responsible for the demethylase activity, which is essential for the transcription elongation of some actively transcribed genes. In the present work, we report the crystal structures of c-Rph1 in apo form and in complex with Ni²(+) and α-KG [2-oxoglutarate (α-ketoglutarate)]. The structure of c-Rph1 is composed of a JmjN (Jumonji N) domain, a long β-hairpin, a mixed structural motif and a JmjC domain. The α-KG cofactor forms hydrogen-bonding interactions with the side chains of conserved residues, and the Ni²(+) ion at the active site is chelated by conserved residues and the cofactor. Structural comparison of Rph1 with JMJD2A indicates that the substrate-binding cleft of Rph1 is formed with several structural elements of the JmjC domain, the long β-hairpin and the mixed structural motif; and the methylated Lys³⁶ of H3 is recognized by several conserved residues of the JmjC domain. In vitro biochemical results show that mutations of the key residues at the catalytic centre and in the substrate-binding cleft abolish the demethylase activity. In vivo growth phenotype analyses also demonstrate that these residues are essential for its functional roles in transcription elongation. Taken together, our structural and biological data provide insights into the molecular basis of the histone demethylase activity and the substrate specificity of Rph1.

Characterization of the intramolecular G-quadruplex promoting activity of Est1

In the budding yeast Saccharomyces cerevisiae, telomeric DNA includes TG1–3/C1–3A double‐stranded DNA and a protruding G‐rich overhang. Our previous studies revealed that the telomerase regulatory subunit Est1 promotes telomeric single‐stranded DNA to form intermolecular G‐quadruplex in vitro, and this activity is required for telomere replication and protection in vivo. In this study, we further characterized the G‐quadruplex promoting activity of Est1. Here we report that Est1 is able to promote the single‐stranded oligonucleotide of (TGTGTGGG)4, which mimics the natural telomeric DNA, to form intramolecular G‐quadruplex. Therefore, it remains possible that the intramolecular G‐quadruplex promoting activity of Est1 is biologically relevant in telomere replication in vivo.