Striking circadian neuron diversity and cycling of Drosophila alternative splicing.

Although alternative pre-mRNA splicing (AS) significantly diversifies the neuronal proteome, the extent of AS is still unknown due in part to the large number of diverse cell types in the brain. To address this complexity issue, we used an annotation-free computational method to analyze and compare the AS profiles between small specific groups of Drosophila circadian neurons. The method, the Junction Usage Model (JUM), allows the comprehensive profiling of both known and novel AS events from specific RNA-seq libraries. The results show that many diverse and novel pre-mRNA isoforms are preferentially expressed in one class of clock neuron and also absent from the more standard Drosophila head RNA preparation. These AS events are enriched in potassium channels important for neuronal firing, and there are also cycling isoforms with no detectable underlying transcriptional oscillations. The results suggest massive AS regulation in the brain that is also likely important for circadian regulation

Genome-wide features of neuroendocrine regulation in Drosophila by the basic helix-loop-helix transcription factor DIMMED.

Neuroendocrine (NE) cells use large dense core vesi-cles (LDCVs) to traffic, process, store and secrete neuropeptide hormones through the regulated secre-tory pathway. The dimmed (DIMM) basic helix-loop-helix transcription factor of Drosophila controls the level of regulated secretory activity in NE cells. To pursue its mechanisms, we have performed two in-dependent genome-wide analyses of DIMM’s activi-ties: (i) in vivo chromatin immunoprecipitation (ChIP) to define genomic sites of DIMM occupancy and (ii) deep sequencing of purified DIMM neurons to char-acterize their transcriptional profile. By this com-bined approach, we showed that DIMM binds to con-served E-boxes in enhancers of 212 genes whose expression is enriched in DIMM-expressing NE cells. DIMM binds preferentially to certain E-boxes within first introns of specific gene isoforms. Statistical ma-chine learning revealed that flanking regions of puta-tive DIMM binding sites contribute to its DNA binding specificity. DIMM’s transcriptional repertoire features at least 20 LDCV constituents. In addition, DIMM no-tably targets the pro-secretory transcription factor, creb-A, but significantly, DIMM does not target any neuropeptide genes. DIMM therefore prescribes the scale of secretory activity in NE neurons, by a sys-tematic control of both proximal and distal points in the regulated secretory pathway

Glial wingless/Wnt regulates glutamate receptor clustering and synaptic physiology at the Drosophila neuromuscular junction

Glial cells are emerging as important regulators of synapse formation, maturation, and plasticity through the release of secreted signaling molecules. Here we use chromatin immunoprecipitation along with Drosophila genomic tiling arrays to define potential targets of the glial transcription factor Reversed polarity (Repo). Unexpectedly, we identified wingless (wg), a secreted morphogen that regulates synaptic growth at the Drosophila larval neuromuscular junction (NMJ), as a potential Repo target gene. We demonstrate that Repo regulates wg expression in vivo and that local glial cells secrete Wg at the NMJ to regulate glutamate receptor clustering and synaptic function. This work identifies Wg as a novel in vivo glial-secreted factor that specifically modulates assembly of the postsynaptic signaling machinery at the Drosophila NMJ

Regulation of tubulin functions in Saccharomyces cerevisiae

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2001.Includes bibliographical references.A variety of cellular factors regulate the formation of complex microtubule structures in the cell. Most of these factors modulate microtubule structures by affecting the polymerization of [alpha]/[beta]-tubulin heterodimer subunits to form microtubules. However, there is another class of factors that affect microtubule formation by regulating the steps that precede the polymerization of [alpha]/[beta]-tubulin heterodimer. One of these factors is Rbl2p. Rbl2p was originally isolated in a screen for genes that when overexpressed can rescue the lethality of [beta]-tubulin overexpression. The mammalian homolog of Rbl2p, Cofactor A, was isolated as a non-essential cofactor in an in vitro tubulin folding reaction (Tian et al., 1996). Both Rbl2p and Cofactor A form a heterodimer with monomeric [beta]-tubulin that excludes [beta]-tubulin (Archer et al., 1995; Melki et al., 1996). The assays that identified Rbl2p and its homolog Cofactor A suggest two possible models to explain the ability of Rbl2p/CofactorA to protect cells from free [beta]-tubulin in vivo. First if unfolded [beta]-tubulin is toxic, Rbl2p could protect cells from free [beta]-tubulin by catalyzing the folding of [beta]-tubulin1 as proposed in the in vitro tubulin folding assay. Second, Rbl2p could bind to and sequester free [beta]-tubulin into a Rbl2p/[beta]-tubulin heterodimer. Our data suggest that Rbl2p's in vivo function is more complicated than predicted by either of these two models. Rbl2p binds transiently to a subpopulation of the free [beta]-tubulin and prevents it from interacting with the target of [beta]-tubulin toxicity until it associates with an aggregate of [beta]-tubulin. Cells expressing an allele of [alpha]-tubulin, tubl-729, arrest in the cold as largebudded cells with either short spindles or no microtubules. The spindle defect checkpoint proteins Bublp and Bub3p suppress the cold-sensitivity of tubl-729 cells (Guenette et al., 1995). We tested whether other proteins involved in the mitotic checkpoints can suppress tubl-729 cells. Our results show that an extra genomic copy of MPSl also suppresses the cold-sensitivity of tubl-729 cells, but that genomic copies of BUB2 and MADsl-3 do not. Bub3p rescues the coldsensitivity of tubl-729 cells in which deletion of MAD2 has eliminated the spindle defect checkpoint. This suggests that Bub3p does not act through the spindle defect checkpoint to suppress tubl-729 cells. We present preliminary evidence in support of a model proposing that tubl-729 cells are defective in kinetochoremicrotubule attachment and that Bublp, Bub3p, and Mpslp may suppress the phenotypes of tubl-729 cells by strengthening these attachments.by Katharine Boyer Compton Abruzzi.Ph.D

Biochemical analysis of TREX complex recruitment to intronless and intron-containing yeast genes

The TREX complex is involved in both transcription elongation and mRNA export and is recruited to nascent transcription complexes. We have examined Yra1p, Sub2p and Hpr1p recruitment to nine genes of varying lengths and transcription frequencies. All three proteins increase from the 5′ to the 3′ ends of the four intronless genes examined. A modified chromatin immunoprecipitation assay that includes an RNase step indicates that Sub2p is bound to nascent RNA, Yra1p is associated with both RNA and DNA, and Hpr1p is associated with DNA. Although Hpr1p is recruited similarly to both intronless and intron-containing genes, low Yra1p and Sub2p levels are present on a subset of intron-containing genes. The residual Yra1p and Sub2p recruitment is less RNA-associated, and this correlates with high levels of U1 SnRNP on these genes. These experiments support a model in which TREX is recruited via the transcription machinery and then Yra1p and Sub2p are transferred to the nascent RNA. On some intron-containing genes, retention and/or transfer of Yra1p and Sub2p to nascent RNA are inhibited

Protection from Free β-Tubulin by the β-Tubulin Binding Protein Rbl2p

Free β-tubulin not in heterodimers with α-tubulin can be toxic, disrupting microtubule assembly and function. We are interested in the mechanisms by which cells protect themselves from free β-tubulin. This study focused specifically on the function of Rbl2p, which, like α-tubulin, can rescue cells from free β-tubulin. In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding. Here we show that Rbl2p becomes essential in cells containing a modest excess of β-tubulin relative to α-tubulin. However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay. Rescue of β-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to β-tubulin. The data suggest that Rbl2p binds transiently to free β-tubulin, which then passes into an aggregated form that is not toxic

Dynamic PER repression mechanisms in the Drosophila circadian clock: from on-DNA to off-DNA

Transcriptional feedback loops are central to the generation and maintenance of circadian rhythms. In animal systems as well as Neurospora, transcriptional repression is believed to occur by catalytic post-translational events. We report here in the Drosophila model two different mechanisms by which the circadian repressor PERIOD (PER) inhibits CLOCK/CYCLE (CLK/CYC)-mediated transcription. First, PER is recruited to circadian promoters, which leads to the nighttime decrease of CLK/CYC activity. This decrease is proportional to PER levels on DNA, and PER recruitment probably occurs via CLK. Then CLK is released from DNA and sequestered in a strong, ∼1:1 PER–CLK off-DNA complex. The data indicate that the PER levels bound to CLK change dynamically and are important for repression, first on-DNA and then off-DNA. They also suggest that these mechanisms occur upstream of post-translational events, and that elements of this two-step mechanism likely apply to mammals