by
Christine E. Schnitzler;
Kevin Pang;
Meghan L. Powers;
Adam M. Reitzel;
Joseph F. Ryan;
David Simmons;
Takashi Tada;
Morgan Park;
Jyoti Gupta;
Shelise Y. Brooks;
Robert W. Blakesley;
Shozo Yokoyama;
Steven H.D. Haddock;
Mark Q. Martindale;
Andreas D. Baxevanis
Background
Calcium-activated photoproteins are luciferase variants found in photocyte cells of bioluminescent jellyfish (Phylum Cnidaria) and comb jellies (Phylum Ctenophora). The complete genomic sequence from the ctenophore Mnemiopsis leidyi, a representative of the earliest branch of animals that emit light, provided an opportunity to examine the genome of an organism that uses this class of luciferase for bioluminescence and to look for genes involved in light reception. To determine when photoprotein genes first arose, we examined the genomic sequence from other early-branching taxa. We combined our genomic survey with gene trees, developmental expression patterns, and functional protein assays of photoproteins and opsins to provide a comprehensive view of light production and light reception in Mnemiopsis.
Results
The Mnemiopsis genome has 10 full-length photoprotein genes situated within two genomic clusters with high sequence conservation that are maintained due to strong purifying selection and concerted evolution. Photoprotein-like genes were also identified in the genomes of the non-luminescent sponge Amphimedon queenslandica and the non-luminescent cnidarian Nematostella vectensis, and phylogenomic analysis demonstrated that photoprotein genes arose at the base of all animals. Photoprotein gene expression in Mnemiopsis embryos begins during gastrulation in migrating precursors to photocytes and persists throughout development in the canals where photocytes reside. We identified three putative opsin genes in the Mnemiopsis genome and show that they do not group with well-known bilaterian opsin subfamilies. Interestingly, photoprotein transcripts are co-expressed with two of the putative opsins in developing photocytes. Opsin expression is also seen in the apical sensory organ. We present evidence that one opsin functions as a photopigment in vitro, absorbing light at wavelengths that overlap with peak photoprotein light emission, raising the hypothesis that light production and light reception may be functionally connected in ctenophore photocytes. We also present genomic evidence of a complete ciliary phototransduction cascade in Mnemiopsis.
Conclusions
This study elucidates the genomic organization, evolutionary history, and developmental expression of photoprotein and opsin genes in the ctenophore Mnemiopsis leidyi, introduces a novel dual role for ctenophore photocytes in both bioluminescence and phototransduction, and raises the possibility that light production and light reception are linked in this early-branching non-bilaterian animal.
This review will discuss the existing literature that has examined the role of calcineurin (CnA) in the regulation of skeletal muscle mass in conditions associated with hypertrophic growth or atrophy. Muscle mass is determined by the balance between protein synthesis and degradation which is controlled by a number of intracellular signaling pathways, most notably the insulin/IGF/phosphatidylinositol 3-kinase (PI3K)/Akt system. Despite being activated by IGF-1 and having well-described functions in the determination of muscle fiber phenotypes, calcineurin (CnA), a Ca2+-activated serine/threonine phosphatase, and its downstream signaling partners have garnered little attention as a regulator of muscle mass. Compared to other signaling pathways, the relatively few studies that have examined the role of CnA in the regulation of muscle size have produced discordant results. The reasons for these differences is not obvious but may be due to the selective nature of the genetic models studied, fluctuations in the endogenous level of CnA activity in various muscles, and the variable use of CnA inhibitors to inhibit CnA signaling. Despite the inconsistent nature of the outcomes, there is sufficient direct and indirect evidence to conclude that CnA plays a role in the regulation of skeletal muscle mass. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting.
by
M Sherekar;
Sae-Won Han;
Rodolfo Ghirlando;
Simon Messing;
Matthew Drew;
Dana Rabara;
Timothy Waybright;
Puneet Juneja;
Hugh O'Neill;
Christopher B Stanley;
Debsindhu Bhowmik;
Arvind Ramanathan;
Sriram Subramaniam;
Dwight V Nissley;
William Gillette;
Frank McCormick;
Dominic Esposito
Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography–multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.
Natural selection has played an important role in establishing various phenotypes, but the molecular mechanisms of phenotypic adaptation are not well understood. The slow progress is a consequence of mutagenesis experiments in which present-day molecules were used and of the limited scope of statistical methods used to detect adaptive evolution. To fully appreciate phenotypic adaptation, the precise roles of adaptive mutations during phenotypic evolution must be elucidated through the engineering and manipulation of ancestral phenotypes. Experimental and quantum chemical analyses of dim-light vision reveal some surprising results and provide a foundation for a productive study of the adaptive evolution of various phenotypes.
Regulation of transcription elongation is an important mechanism in controlling eukaryotic gene expression. SII is an RNA polymerase II-binding protein that stimulates transcription elongation and also activates nascent transcript cleavage by RNA polymerase II in elongation complexes in vitro (Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). Here we show that SII-dependent in vitro transcription through an arrest site in a human gene is preceded by nascent transcript cleavage. RNA cleavage appeared to be an obligatory step in the SII activation process. Recombinant SII activated cleavage while a truncated derivative lacking polymerase binding activity did not. Cleavage was not restricted to an elongation complex arrested at this particular site, showing that nascent RNA hydrolysis is a general property of RNA polymerase II elongation complexes. These data support a model whereby SII stimulates elongation via a ribonuclease activity of the elongation complex.
Synthesis of accurately initiated transcripts has been reconstituted with RNA polymerase II and a set of five transcription factors purified from rat liver. In addition to three previously identified factors, α, βγ, and δ (Conaway, R.C., and Conaway, J.W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7356-7360), transcription in the reconstituted liver system requires two novel factors designated τ and ε. These five transcription factors comprise two functional classes: (i) promoter recognition factors (τ and ε), which inteact with template DNA to facilitate formation of a stable initial complex that is subsequently recognized and bound by DNA polymerase II, and (ii) RNA chain initiation factors (α, βγ, and δ), which do not participate in formation of the initial complex, but which are essential for transcription initiation.
The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG-bound epoetin β pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo.
Severe hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy.
Background:
The views of people with genetic conditions are crucial to include in public dialogue around developing gene editing technologies. This qualitative study sought to characterize the attitudes of people with inherited retinal conditions (retinitis pigmentosa [RP] and Leber congenital amaurosis [LCA]) toward gene editing.
Methods:
Individuals with RP (N = 9) and LCA (N = 8) participated in semi‐structured qualitative interviews about their experience with and attitudes toward blindness, and their views about gene editing technology for somatic, germline, and enhancement applications.
Results:
Participants saw potential benefits from gene editing in general, but views about its use for retinal conditions varied and were influenced by personal perspectives on blindness. Those who felt more negatively toward blindness, particularly those with later onset blindness, were more supportive of gene editing for retinal conditions. Concerns about both germline and somatic editing included: the importance of informed consent; impacts of gene editing on social attitudes and barriers affecting blind people; and worries about “eliminating” blindness or other traits.
Conclusion:
People with RP and LCA have diverse attitudes toward gene editing technology informed by their own lived experience with disability, and many have concerns about how the ways in which it is discussed and implemented might affect them.
Arif et al. report that phosphorylation of the important metabolism-controlling kinase, S6K1, at two sites near the protein terminus induces its phosphorylation of multiple targets related to lipid metabolism. These insulin-stimulated phosphorylation events in adipocytes (fat cells) might contribute to the known influence of S6K1 on obesity.
Obstacles incurred by RNA polymerase II during primary transcript synthesis have been identified in vivo and in vitro. Transcription past these impediments requires SII, an RNA polymerase II-binding protein. SII also activates a nuclease in arrested elongation complexes and this nascent RNA shortening precedes transcriptional readthrough. Here we show that in the presence of SII and nucleotides, transcript cleavage is detected during SII- dependent elongation but not during SII-independent transcription. Thus, under typical transcription conditions, SII is necessary but insufficient to activate RNA cleavage. RNA cleavage could serve to move RNA polymerase II away from the transcriptional impediment and/or permit RNA polymerase II multiple attempts at RNA elongation. By mapping the positions of the 3'-ends of RNAs and the elongation complex on DNA, we demonstrate that upstream movement of RNA polymerase II is not required for limited RNA shortening (seven to nine nucleotides) and reactivation of an arrested complex. Arrested complexes become elongation competent after removal of no more than nine nucleotides from the nascent RNA's 3'-end. Further cleavage of nascent RNA, however, does result in 'backward' translocation of the enzyme. We also show that one round of RNA cleavage is insufficient for full readthrough at an arrest site, consistent with a previously suggested mechanism of SII action.