Project description:We performed a RNA immunoprecipitations experiments using gfp-specific antibodies to precipitate gfp-tagged La proteins from from gfp-La wild type and sumoylation deficient La mutant (K41/200R) cells and found that specific mRNAs are preferentially enriched gfp-La wild type RIPs when compared to sumoylation deficient La mutant (K41/200R) RIPs.
Project description:Caves are populated with a diverse fauna of highly adapted species that tend to exhibit a consistent suite of both regressive and constructive trait modifications. Because molecular studies of cave adaptation have largely concentrated on vertebrate models, our ability to recognize universalities in the genetic trajectories underlying cave adaptation remains limited. We have initiated efforts to elucidate the molecular evolution of the flightless small carrion beetle Ptomaphagus hirtus (Ptomaphagus hirtus), which represents one of the highly endemic signature inhabitants of the Mammoth Cave system of Kentucky. Ptomaphagus hirtus has been considered blind despite the presence of lateral eye rudiments. However, analysis of the Ptomaphagus hirtus adult head transcriptome by deep RNA sequencing reveals the conservation and expression of all essential insect phototransduction genes including a single long wavelength-sensitive opsin. Consistent with the preservation of visual ability, Ptomaphagus hirtus expresses all core members of the clock gene network and exhibits a similar degree of negative phototaxis as does a closely related flight-active species in light-dark choice assays. The structural reduction of the peripheral Ptomaphagus hirtus visual system is reflected by the lack of five eye pigmentation specific genes in the head transcriptome. Taken together our data suggest that wavelength contingent and probably also spatial vision have been lost in Ptomaphagus hirtus, while irradiance vision and contingent behavioral modules have remained preserved. We predict that the adaptive state of Ptomaphagus hirtus is representative for a large number of microphthalmic species adapted to the twilight zone of caves and other subterranean habitats Poly(A)+ transcripts were isolated from a pooled sample of 25 adult Ptomaphagus hirtus heads, reverse transcribed and sequenced on the Illumina GAII
Project description:The Lupus autoantigen (La) is a single-stranded RNA-binding protein that stabilizes RNA polymerase III (pol III) transcripts and supports RNA folding. In addition, La has been implicated in different steps of the mammalian small RNA pathway. Here, we have analyzed effects of La depletion on the Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago protein complexes and our data suggests that La prevents the production and loading of such tRNA fragments. However, one specific isoleucine tRNA escapes this regulation and produces both a functional tRNA as well as a microRNA (miRNA). We demonstrate that fully complementary 5' leader and 3' trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin 5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading. Thus, La functions as gatekeeper to ensure correct tRNA generation and to protect the miRNA pathway from potentially functional tRNA fragments.
Project description:The Lupus autoantigen (La) is a single-stranded RNA-binding protein that stabilizes RNA polymerase III (pol III) transcripts and supports RNA folding. In addition, La has been implicated in different steps of the mammalian small RNA pathway. Here, we have analyzed effects of La depletion on the Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago protein complexes and our data suggests that La prevents the production and loading of such tRNA fragments. However, one specific isoleucine tRNA escapes this regulation and produces both a functional tRNA as well as a microRNA (miRNA). We demonstrate that fully complementary 5' leader and 3' trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin 5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading. Thus, La functions as gatekeeper to ensure correct tRNA generation and to protect the miRNA pathway from potentially functional tRNA fragments.
Project description:We recently identified lysine L-lactylation (KL-la) on histones that can be labelled by L-lactate, the end-product of glycolysis. KL-la has two structural isomers, namely N--(carboxyethyl) lysine (Kce) and lysine D-lactylation (KD-la), which can also be caused by metabolites associated with glycolysis. It is unknown if perturbations of glycolysis can lead to dysregulation of KD-la and Kce, in addition to KL-la. Further, current methods have a difficulty to distinguish among these isomers in cellular contexts. To investigate these questions, we first generated specific antibodies against each one of these three modifications. These reagents enable us to distinguish these three isomers. We demonstrated that KL-la, but not KD-la and Kce, is dynamically regulated by glycolysis. KD-la and Kce occur mainly when the major glycolytic pathway is blocked downstream or when the glyoxalase system is incomplete. This result was also independently confirmed by orthogonal HPLC-mass spectrometry, showing that KL-la is the predominant isomer of lactylation on cellular histones. Finally, we demonstrated that lactyl-CoA, an intermediate between L-lactate and lactylation, is dynamically regulated by glycolysis and is positively correlated with KL-la. Thus, our study clearly shows that KL-la, but not KD-la and Kce, is the major glycolytic- and the Warburg-effect associated responsive modification in cells.
Project description:Spotted hyena (Crocuta crocuta) is the only extant species of the genus Crocuta, which once occupied a much wider range during the Pliocene and Pleistocene. However, its origin and evolutionary history is somewhat contentious due to discordances being found between morphological, nuclear, and mitochondrial data. Due to the limited molecular data from east Asian Crocuta, and the difficulty of extracting ancient DNA from this area, here we present proteomic analysis of cave hyenas from three locations in northern China. This marks the first proteomic data generated from cave hyenas, adding new molecular data to the east Asian populations. Phylogenetic analysis based on these protein sequences reveals two different groups of cave hyenas in east Asia, one of which could not be distinguished from modern spotted hyenas from northern Africa, tentatively the result of previously suggested gene flow between these lineages. With developments of instrumentation and analytical methods, proteomics holds promising potential for the phylogenetic reconstruction of ancient fauna previously thought to be unreachable using ancient DNA.
Project description:Dysregulation of sleep has widespread health consequences and represents an enormous health burden. Short-sleeping individuals are predisposed to the effects of neurodegeneration, suggesting a critical role for sleep in the maintenance of neuronal health. While the effects of sleep on cellular function are not completely understood, growing evidence has identified an association between sleep loss and DNA damage, raising the possibility that sleep facilitates efficient DNA repair. The Mexican tetra fish, Astyanax mexicanus provides a model to investigate the evolutionary basis for changes in sleep and the consequences of sleep loss. Multiple cave-adapted populations of these fish have evolved to sleep for substantially less time compared to surface populations of the same species without identifiable impacts on healthspan or longevity. To investigate whether the evolved sleep loss is associated with DNA damage and aging, we compared the transcriptional response to aging between surface and cave morphs of A. mexicanus