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N 6 -Methyladenosine (m 6 A) is the most abundant post-transcriptional mRNA modification in eukaryotes and exerts many of its effects on gene expression through reader proteins that bind specifically to m 6 A-containing transcripts. Fragile X mental retardation protein (FMRP), an RNA-binding protein, has previously been shown to affect the translation of target mRNAs and trafficking of mRNA granules. Loss of function of FMRP causes fragile X syndrome, the most common form of inherited intellectual disability in humans. Using HEK293T cells, siRNA-mediated gene knockdown, cytoplasmic and nuclear fractions, RNA-Seq, and LC-MS/MS analyses, we demonstrate here that FMRP binds directly to a collection of m 6 A sites on mRNAs. FMRP depletion increased mRNA m 6 A levels in the nucleus. Moreover, the abundance of FMRP targets in the cytoplasm relative to the nucleus was decreased in Fmr1 -KO mice, an effect also observed in highly methylated genes. We conclude that FMRP may affect the nuclear export of m 6 A-modified RNA targets., (© 2019 Hsu et al.)

The amplitude of signaling evoked by stimulation of G protein-coupled receptors may be controlled in part by the GTPase accelerating activity of the regulator of G protein signaling (RGS) proteins. In turn, subcellular targeting, protein-protein interactions, or post-translational modifications such as phosphorylation may shape RGS activity and specificity. We found previously that RGS16 undergoes tyrosine phosphorylation on conserved tyrosine residues in the RGS box. Phosphorylation on Tyr(168) was mediated by the epidermal growth factor receptor (EGFR). We show here that endogenous RGS16 is phosphorylated after epidermal growth factor stimulation of MCF-7 cells. In addition, p60-Src or Lyn kinase phosphorylated recombinant RGS16 in vitro, and RGS16 underwent phosphorylation in the presence of constitutively active Src (Y529F) in EGFR(-) CHO-K1 cells. Blockade of endogenous Src activity by selective inhibitors attenuated RGS16 phosphorylation induced by pervanadate or receptor stimulation. Furthermore, the rate of RGS16 degradation was reduced in cells expressing active Src or treated with pervanadate or a G protein-coupled receptor ligand (CXCL12). Induction of RGS16 tyrosine phosphorylation was associated with increased RGS16 protein levels and enhanced GAP activity in cell membranes. These results suggest that Src mediates RGS16 tyrosine phosphorylation, which may promote RGS16 stability.

Stimulation of beta-adrenergic receptor normally results in signaling by the heterotrimeric G protein G(s), leading to the activation of adenylyl cyclase, production of cAMP, and activation of cAMP-dependent protein kinase (PKA). Here we report that cell death of thymocytes can be induced after stimulation of beta-adrenergic receptor, or by addition of exogenous cAMP. Apoptotic cell death in both cases was observed with the appearance of terminal deoxynucleotidyl transferase-mediated UTP end labeling reactivity and the activation of caspase-3 in S49 T cells. Using thymocytes deficient in either Galpha(s) or PKA, we find that engagement of beta-adrenergic receptors initiated a Galpha(s)-dependent, PKA-independent pathway leading to apoptosis. This alternative pathway involves Src family tyrosine kinase Lck. Furthermore, we show that Lck protein kinase activity can be directly stimulated by purified Galpha(s). Our data reveal a new signaling pathway for Galpha(s), distinct from the classical PKA pathway, that accounts for the apoptotic action of beta-adrenergic receptors.

The 2-methyl branched-chain enoyl-CoA reductase plays a pivotal role in the reversal of beta-oxidation operating in anaerobic mitochondria of the parasitic nematode Ascaris suum. An affinity-purified polyclonal anti-serum against the reductase was used to screen a cDNA library constructed in lambda gt11 with poly(A)+ RNA from adult A. suum muscle. A 1.2-kilobase partial cDNA clone was isolated, subcloned, and sequenced in both directions. Additional sequence at the 5' end of the mRNA was determined by the RACE (rapid amplification of cDNA ends) procedure. Nucleotide sequence analysis of the cDNAs revealed the 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1236 nucleotides and a 3'-untranslated sequence of 109 nucleotides including a short poly(A) tail 14 nucleotides from a polyadenylation signal (AATAAA). The open reading frame encoded a 396-amino acid sequence (M(r) 43,046) including a 16-amino acid leader peptide. Two-dimensional gel electrophoresis of the purified reductase yielded multiple spots with two distinct but overlapping amino-terminal amino acid sequences. Both sequences overlapped with the sequence predicted from the mRNA, and one of the sequences was identical to the predicted sequence. Comparison of the ascarid sequence with that of mammalian acyl-CoA dehydrogenases revealed a high degree of sequence identity, suggesting that these enzymes may have evolved from a common ancestral gene even though the ascarid enzyme functions as a reductase, not as a dehydrogenase. Immunoblotting of A. suum larval stages and adult tissues with antisera that cross-reacted with each of the spots separated on two-dimensional gels suggested that the reductase was only found in adult muscle. Northern blotting using the partial cDNA revealed a hybridization band of about 1.5 kilobases and also suggested that the enzyme was tissue-specific and developmentally regulated in agreement with the results of the immunoblotting.

Electron-transfer flavoprotein:rhodoquinone oxidoreductase (ETF-RO) was purified to homogeneity from anaerobic mitochondria of the parasitic nematode, Ascaris suum. The enzyme has a subunit molecular mass of 64.5 kDa and is similar in many respects to the electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF-UO) characterized in mammalian tissues. EPR spectroscopy of the purified enzyme revealed signals at g = 2.076, 1,936, and 1.883, arising from an iron-sulfur center, as well as signals attributable to a flavin semiquinone. Potentiometric titration on the enzyme with dithionite yielded an oxidation-reduction midpoint potential (Em) for the iron-sulfur center of +25 mV at pH 7.4. The reduction of flavin occurred in two distinct steps, with a flavin semiquinone radical detected as an intermediate. The Em values for the two steps in the complete reduction of flavin were +15 mV and -9 mV, respectively. Physiologically, the ascarid ETF-RO accepts electrons from a low potential quinone, rhodoquinone, and functions in a direction opposite to that of the ETF-UO. Incubations of A. suum submitochondrial particles with NADH, 2-methylcrotonyl-CoA, purified A. suum electron-transfer flavoprotein and 2-methyl branched-chain enoyl-CoA reductase resulted in significant 2-methylbutyryl-CoA formation, which was inhibited by both rotenone and antisera to the purified ETF-RO. Quinone extraction of the submitchondrial particles with dry pentane resulted in almost the complete loss of 2-MBCoA formation by the system. However, the reincorporation of rhodoquinone, but not ubiquinone restored over 50% of the NADH-dependent 2-MBCoA formation.