Retrotransposons are mobile genetic elements that proliferate through an RNA intermediate. Transposons do not encode transcription factors and thus rely on host factors for mRNA expression and survival. Despite information regarding conditions under which elements are upregulated, much remains to be learned about the regulatory mechanisms or factors controlling retrotransposon expression. Here, we report that low oxygen activates the fission yeast Tf2 family of retrotransposons. Sre1, the yeast ortholog of the mammalian membrane-bound transcription factor sterol regulatory element binding protein (SREBP), directly induces the expression and mobilization of Tf2 retrotransposons under low oxygen. Sre1 binds to DNA sequences in the Tf2 long terminal repeat that functions as an oxygen-dependent promoter. We find that Tf2 solo long terminal repeats throughout the genome direct oxygendependent expression of adjacent coding and noncoding sequences, providing a potential mechanism for the generation of oxygen-dependent gene expression.

Animals with germ plasm assemble cytoplasmic RNA granules (germ granules) that segregate with the embryonic germ lineage. How germ granules assemble and recruit RNA is not well understood. Here we characterize the assembly and RNA composition of the germ (P) granules of C. elegans. ~500 maternal mRNAs are recruited into P granules by a sequence independent mechanism that favors mRNAs with low ribosome coverage. Translational activation correlates temporally with P granule exit for two mRNAs that code for germ cell fate regulators. mRNAs are recruited into the granules by MEG-3, an intrinsically disordered protein that condenses with RNA to form nanoscale gels. Our observations reveal parallels between germ granules and stress granules and suggest that cytoplasmic RNA granules are reversible super-assemblies of nanoscale RNA-protein gel condensates.

No abstract

Eukaryotic cells experience fluctuating environmental oxygen concentrations and elicit a physiological response to allow the organism or tissue to adapt. Sre1, the fission yeast Sterol Regulatory Element Binding Protein, is an ER membrane‐bound transcription factor that responds to changes in oxygen‐dependent sterol synthesis as an indirect measure of oxygen availability. Under low oxygen, Sre1 is proteolytically cleaved and the released N‐terminal transcription factor (Sre1N) activates gene expression essential for hypoxic growth. In addition, Sre1N turnover is regulated by oxygen. Ofd1, a prolyl 4‐hydroxylase family member, accelerates Sre1N degradation in the presence of oxygen. However, unlike the prolyl 4‐hydroxylases that regulate mammalian hypoxia inducible factor (HIF), Ofd1 uses multiple domains to regulate Sre1N degradation by oxygen; the Ofd1 N‐terminal dioxygenase domain is required for oxygen sensing and the Ofd1 C‐terminal domain accelerates Sre1N degradation. Proteasomal degradation of Sre1N requires the E2 ubiquitin conjugating enzyme Rhp6 and the N‐end rule E3 ligase Ubr1. Genetic experiments position Ofd1 in this pathway upstream of Ubr1. Thus, oxygen regulates Sre1 activity at two points to mediate adaptation to hypoxic stress: (1) proteolytic cleavage and (2) transcription factor degradation.Research was supported by the NIH‐HL‐077588 and NIH‐AI‐072186.

Wild-type human neuroserpin, a member of the serine protease inhibitor superfamily, is expressed in neurons of the central and peripheral nervous system, as well as in the adult brain. Polymerization of certain mutants of neuroserpin is associated with dementia caused by familial encephalopathy. We have performed hydrogen/deuterium exchange-mass spectrometry in order to monitor the structural stability and flexibility of different regions of the neuroserpin structure. We find that beta-sheet A, a critical region thought to be involved in polymerization, is less stable and more labile in neuroserpin than in other serpins such as alpha-1 antitrypsin and antithrombin. This may explain why wild-type neuroserpin is more susceptible to polymerization than other serpins. Molecular dynamics simulations also indicate that Wild Type neuroserpin shows increases flexibility on the nanosecond timescale as compared with alpha-1 antitrypsin. In the simulations, a novel 2 stranded beta-sheet was formed between the N terminal portion of the reactive center loop and the loop connecting strand 3A to beta-sheet C. This phenomenon occurred repeatedly in multiple independent simulations. If such an interaction in fact occurs in solution, it could contribute to the relatively poor inhibitory efficiency of neuroserpin compared to other serpins by retarding the insertion of the reactive center loop into sheet A after proteolytic cleavage. Simulations of a pathological mutant of neuroserpin showed distortions near the top of the central beta-sheet A, a critical site for polymer formation. This distortion may help explain why the mutant is more prone to polymerize than wild type. 258-Pos Board B58Optimized Quantitation from Proteomic Datasets -Application to Lamin Knockdown and Isoform Modulation During Stem Cell Differentiation Joe Swift, Takamasa Harada, Jae-Won Shin, Hsin-Yao Tang, David W. Speicher, Dennis E. Discher. Label-free mass spectrometry is rapidly emerging as an alternative to antibody-based methods, although there is continued need in either case to maximize measurement accuracy. Here we describe Spectral Ion Fingerprint Recognition (SpIFR) for optimized identification of 'protein fingerprints' within proteomic datasets derived from liquid chromatography tandem mass spectrometry (LC-MS/MS). We focus initially on siRNA knockdown of nuclear lamina proteins that are low in abundance and tightly regulated in stem cells, cancer, and aging. Sets of optimal peptides show that intermediate MS signal -rather than strong signal -is often best for quantitation and produce a high level of accuracy (56%) while also providing global assessments of protein changes. Surprisingly, lamin-A and -C spliceoforms are knocked down to different extents despite siRNA targeting of a shared sequence, and the results also allow the first calculations of ratios for lamin-A/C to lamin-B. From the MS-derived proteomes, SpIFR computes an invariant reference set of 'housekeeping' peptides and enables mapping of species-specific peptides encountered in embr...

Sterol regulatory element binding proteins (SREBPs) are membrane‐bound transcription factors that control lipid homeostasis in mammalian cells. Sterols regulate the activation of SREBP by inhibiting ER‐Golgi transport of SREBP and proteolysis in the Golgi. While much is known about the mechanisms of sterol‐dependent ER retention, less is known about regulation of the nuclear form of SREBP. To identify novel regulators of nuclear SREBP, we designed a genetic selection scheme in Schizosaccharomyces pombe that reports the in vivo activity of nuclear Sre1, the fission yeast ortholog of SREBP. This selection uses a yeast strain containing a ura4+ reporter gene driven by multiple Sre1 DNA binding elements. This scheme allowed us to perform a growth‐dependent, plasmid‐based, high copy suppressor screen for regulators of nuclear Sre1. To date, we have identified both positive and negative candidate regulators of nuclear Sre1, including 2 kinases. This genetic screen should give us a broader view of Sre1 regulation in yeast and identify candidate regulators of SREBP in mammalian cells. Research was funded by a grant from the NIH HL‐077588 and a Burroughs Wellcome Fund Career Award in the Biomedical Sciences (PJE).

Sre1, the fission yeast Sterol Regulatory Element Binding Protein (SREBP), is a principal regulator of low oxygen gene expression whose activation is required for low oxygen growth. Oxygen regulates the activity of Sre1 by two independent mechanisms: Sre1 proteolysis is stimulated by inhibition of oxygen‐dependent sterol synthesis; and oxygen accelerates the degradation of nuclear Sre1. Here, we report the identification of 4‐methyl sterols as a signal for activation of Sre1 proteolysis. These sterols accumulate under low oxygen and activate Sre1 processing when added exogenously. In addition, we describe the identification of an uncharacterized 2‐oxoglutarate, iron‐dependent dioxygenase that is required for the oxygen‐dependent degradation of nuclear Sre1. This bivalent regulation of Sre1 permits the rapid activation of gene expression and adaptation to fluctuations in environmental oxygen. These studies have important implications for our understanding of sterol‐sensing domain proteins and for regulation of SREBP degradation in mammalian cells. Research was funded by a grant from the NIH HL‐077588, a Burroughs Wellcome Fund Career Award in the Biomedical Sciences (PJE), and American Heart Association Predoctoral Fellowships (ALH and BTH).