A large number of nucleus-encoded messenger RNAs (mRNAs) encoding proteins involved in oxidative phosphorylation have been found to be associated with mitochondria in vivo, indicating organelle-specific mRNA targeting. However, the identification of mitochondrion-bound mRNA (Mtb-RNA) has traditionally relied on cumbersome isolations of polysomes from a large number of input cells and is therefore biased in favour of mRNAs associated through nascent targeting peptides emerging from the polysome during cotranslational import of their encoded proteins, and tends to ignore sequence-directed mRNA targeting. We have, therefore, sought to identify and quantify Mtb-RNAs rapidly in small numbers of cells, independently of their polysomal status. We isolated Mtb-RNAs from tissue-cultured cells under different conditions and assayed them by endpoint or real-time polymerase chain reaction (RT-PCR). We observed that (i) different Mtb-RNAs are differentially affected by cycloheximide-induced polysome arrest, indicating possible artifacts of the use of this translation elongation inhibitor; (ii) several Mtb-RNAs have direct affinity for the mitochondrial surface in vitro, indicating the possibility of targeting through mRNA recognition by surface-bound RNA-binding proteins (RBP); and (iii) mRNA–mitochondrion interactions are stabilized by formaldehyde crosslinking. Our results reveal the importance of sequence-directed targeting of mRNAs to mitochondria.

Eukaryotic cells contain multiple copies of mitochondrial DNA (mtDNA) in discrete organelles or as tubular networks throughout the cytoplasm. The mtDNA copy number is dynamically regulated by mitochondrial biogenesis and mitophagy processes. However, the conditions regulating mtDNA replication, an essential component of biogenesis, are unknown. We observed that short-term (2 h) treatment of rat myoblasts with oligomycin, a specific inhibitor of the mitochondrial F1F0 ATP synthase, resulted in stimulation of mtDNA synthesis from the OH replication origin. This effect was abrogated by Compound C, an antagonist of the AMP-dependent protein kinase (AMPK), a universal intracellular energy sensor, and in AMPK-knockdown cells, indicating that mtDNA replication is regulated by AMPK under oxidative phosphorylation (OXPHOS)-deficient conditions. Using antibody decoration, enzymatically active AMPK, phosphorylated at T172 of the α1 subunit, was found to be located on the mitochondrial surface. Furthermore, oligomycin induced the compartmentalization of several mRNAs encoding OXPHOS components and mtDNA replication factors to mitochondria. Compartmentalization of mRNAs was inhibited by Compound C. We infer that AMPK is locally activated by inhibition of the F1F0 ATP synthase to stimulate association of mtDNA replication factor mRNAs, leading to stimulation of mtDNA synthesis. The findings have implications for the clonal expansion of OXPHOS-deficient mtDNA mutant mitochondria in human patients, with clinical consequences.

Purpose. Zika virus infections have recently been reported in many dengue-endemic areas globally. Both dengue (DENV) and Zika (ZIKV) virus are transmitted by Aedes mosquitoes, raising the possibility of mixed infections in both vector and host. We evaluated DENV and ZIKV prevalence in human and vector samples in Kolkata, a DENV-endemic city. Methodology. Blood samples were collected from 70 patients presenting dengue-like fever symptoms at a hospital in Kolkata during 2015-16. Serum was obtained and tested for DENV infection by DENV NS1-based ELISA. Adult (n= 8) and larval stages (n= 12) of Aedes were also collected. A RT-PCR-based screening of both viruses supplemented by amplicon sequencing was performed. Results. Of the 70 samples, 20 DENV NS1-positive serum samples were used for detailed molecular study for DENV infection. Eighteen of these (90 %) were positive by hemi-nested serotype-specific RT-PCR for DENV1/2/3, with four samples showing evidence of DENV2-3 or DENV1-3 mixed infection. None were ZIKV-positive using NS5 or ENV-based PCR, though weak amplification of a DENV1 NS5 sequence was detected in three serum samples indicating cross-reactivity of the primers. All mosquito samples were ZIKV-negative, whereas 5/8 (63 %) of adult mosquitoes and 11/12 (92 %) of larvae were DENV3-positive. Conclusion. Both host and vector samples showed absence of ZIKV but high prevalence of DENV. The high rate of infection of larvae with DENV is suggestive of trans-ovarial transmission that could contribute to the surge of human infections during each post-monsoon season. It would be important to guard against false positives using the available Zika-reporting primer sets.

RNA binding proteins (RBPs) can regulate the stability and/or translatability of messengerRNAs (mRNAs) through interactions with their 3′-untranslated regions. However, individual mRNAs may be regulated simultaneously or successively by more than one RBP, as well as by Argonaute (AGO)-bound miRNAs; the coordination of these various influences on an individual mRNA is therefore complex and not well studied. In this report we examine the roles of two RBPs that bind to AU-rich elements (ARE) – AUF1 and HuR – in the stability and translation of cyclin D1 (Ccnd1) mRNA in rat myoblasts transiting the G phase of the cell cycle, and their interactions with miRNAs. Knockdown (KD) of AUF1 resulted in (1) transient upregulation of the mRNA level as well as an advancement of translation onset time (TOT) from 6 to 5 h post-serum addition, (2) loss of miRNA loading on AGO1 and AGO2 and (3) reduction in the level of AGO-1 and AGO-2 bound mRNA. In contrast, KD of HuR had no effect on the mRNA level, or on the AGO–mRNA complexes, but delayed TOT by 1 h independent of miRNA let-7. Thus the dynamics of RBP–mRNA binding and –RBP–AGO–miRNA interactions are coordinated to fine tune the expression of Ccnd1 in the G1 phase.

Mammalian Argonaute proteins (AGO1-4), in combination with microRNAs (miRs), bind to target mRNAs to initiate degradation and/or translation repression, but the relationships between these two effects is unclear. Although the AGO isoforms of Drosophila and plants perform different functions, mammalian AGO isoforms are considered to be functionally degenerate in terms of miR loading and downstream silencing effects. However, we found that, in quiescent (G0) rat myoblasts transiting to the G1 phase, cyclin D1 (Ccnd1) mRNA was associated with two functionally distinct AGO-miR complexes. While AGO1-miR-1 down-regulated the mRNA level, AGO2-let-7 delayed the timing of translation. Knockdown (KD) of AGO2, or antisense-mediated depletion of Let-7, caused Ccnd1 translation to occur earlier, but had no significant effect on mRNA abundance. Conversely, down-regulation of either AGO1 or miR-1, resulted in elevated Ccnd1 mRNA levels at early times, but failed to affect the timing of translation. These results show that the two miR-mediated silencing effects, viz. mRNA decay and translation repression, are independent processes induced by individual AGO isoforms in association with specific miRs.

BACKGROUND:There is experimental evidence of many cases of stable macromolecular conformations with charged amino-acids facing lipid, an arrangement thought to be energetically unfavourable.METHODS AND OBJECTIVES:Employing classical electrostatics, we show that, this is not necessarily the case and studied the physical basis of the specific role of proximity of charges to the dielectric interface between two different environments. We illustrate how self and induced energies due to the dielectric medium polarization, on either side of the interface, contribute differentially to the stability of a pair of charges and hence the mutual conformation of the S3b-S4 α-helix pair of the voltage-gated K(+) channel.RESULTS AND CONCLUSION:We show that (1) a pair of opposite charges on either side of lipid-protein interface confers significant stability; (2) hydrophobic media has an important role in holding together two similar repelling charges; (3) dielectric interface has stabilizing effect on a pair of charges, when an ion is closer to its interface than its neighboring charge; (4) in spite of the presence of dielectric interface, there is a nonexistence of any dielectric effect, when an ion is equidistant from its image and neighboring charge. We also demonstrate that, variation in dielectric media of the surrounding environment confers new mutual conformations to S3b-S4 α-helices of voltage sensor domain at zero potential, especially lipid environment on the helix side, which improved stability to the configuration by lowering the potential energy. Our results provide an answer to the long standing question of why charges face hydrophobic lipid membranes in the stable conformation of a protein.

The microenvironment of the injury site can have profound effects on wound healing. Muscle injury results in ischemia leading to short-term local hypoxia, but there are conflicting reports on the role of hypoxia on the myogenic program in vivo and in vitro. In our rat model of mitochondrial restoration (MR), temporary upregulation of mitochondrial activity by a cocktail of organelle-encoded RNAs results in satellite cell proliferation and initiation of myogenesis. We now report that MR leads to a transient hypoxic response in situ. Inhibition of hypoxia by lowering mitochondrial O2 consumption, either by respiratory electron transport inhibitors, or by NO-mediated inhibition of O2 binding to cytochrome c oxidase, resulted in exacerbation of inflammation. Lentivirus-mediated knockdown of hypoxia-inducible factor 1α (HIF1α) or of Notch signaling components had a similar effect, and pharmacologic inhibition of HIF or Notch reduced the number of proliferating Pax7+ cells. In contrast, a prolonged hypoxic response induced either by uncoupling of respiration from oxidative phosphorylation or through HIF stabilization by dimethyloxalylglycine (DMOG) had an immediate anti-inflammatory effect. Although significant satellite cell proliferation occurred in presence of DMOG, expression of differentiation markers was affected. These results emphasize the importance of transient hypoxia as opposed to prolonged hypoxia for myogenesis.

The multi subunit RNA Import Complex (RIC) from the protozoal parasite Leishmania tropica is taken up by mammalian cells and trafficked to mitochondria, with functional RNA delivered to the matrix by an unknown mechanism. By live cell imaging of siRNA mediated knockdown cells we showed that intracellular vesicular transport of the ribonuclear protein complex is dependent on caveolin1, dynamin2 and filaminA but independent of components of the endosomal pathway such as EEA1, Rab5 or Rab7. Moreover, mitochondrial targeting was dependent on TOM20 and TOM22, two components of the outer membrane machinery. By immuno‐selection we could distinguish two types of RNA loaded cytoplasmic vesicles, one containing caveolar components and the other lacking them. In vitro, transfer of RNP from the caveolin negative transport vesicles to mitochondria was dependent on ATP and on TOM22. RIC was transferred to TOM20 deficient mitochondrial membrane but the RNA was not imported into the matrix. These results imply that (1) endosomes are not involved in RIC trafficking and novel interactions between vesicle associated RIC and (2) TOM20 and TOM22 are required for transfer and positioning of RIC on the mitochondrial surface.Supported By: Project Bend of the Council of Scientific and Industrial Research.Ref: Mukherjee, J. (2014). Vesicular transport of a ribonucleoprotein to mitochondria. Biology Open, doi: 10.1242/​bio.20149076.

During myogenesis, satellite stem cells (SCs) are induced to proliferate and differentiate to myogenic precursors. The role of energy sensors such as the AMP-activated protein kinase (AMPK) and the mammalian Target of Rapamycin (mTOR) in SC activation is unclear. We previously observed that upregulation of ATP through RNA-mediated mitochondrial restoration (MR) accelerates SC activation following skeletal muscle injury. We show here that during regeneration, the AMPK-CRTC2-CREB and Raptor-mTORC-4EBP1 pathways were rapidly activated. The phosho-CRTC2-CREB complex was essential for myogenesis and activated transcription of the critical cell cycle regulator cyclin D1 (Ccnd1). Knockdown (KD) of either mTORC or its subunit Raptor delayed SC activation without influencing the differentiation program. KD of 4EBP1 had no effect on SC activation but enhanced myofiber size. mTORC1 positively regulated Ccnd1 translation but destabilized Ccnd1 mRNA. These antithetical effects of mTORC1 were mediated by two microRNAs (miRs) targeted to the 3' untranslated region (UTR) of Ccnd1 mRNA: miR-1 was downregulated in mTORC-KD muscle, and depletion of miR-1 resulted in increased levels of mRNA without any effect on Ccnd1 protein. In contrast, miR-26a was upregulated upon mTORC depletion, while anti-miR-26a oligonucleotide specifically stimulated Ccnd1 protein expression. Thus, mTORC may act as a timer of satellite cell proliferation during myogenesis.

Intracellular trafficking of viruses and proteins commonly occurs via the early endosome in a process involving Rab5. The RNA Import Complex (RIC)-RNA complex is taken up by mammalian cells and targeted to mitochondria. Through RNA interference, it was shown that mito-targeting of the ribonucleoprotein (RNP) was dependent on caveolin 1 (Cav1), dynamin 2, Filamin A and NSF. Although a minor fraction of the RNP was transported to endosomes in a Rab5-dependent manner, mito-targeting was independent of Rab5 or other endosomal proteins, suggesting that endosomal uptake and mito-targeting occur independently. Sequential immunoprecipitation of the cytosolic vesicles showed the sorting of the RNP away from Cav1 in a process that was independent of the endosomal effector EEA1 but sensitive to nocodazole. However, the RNP was in two types of vesicle with or without Cav1, with membrane-bound, asymmetrically orientated RIC and entrapped RNA, but no endosomal components, suggesting vesicular sorting rather than escape of free RNP from endosomes. In vitro, RNP was directly transferred from the Type 2 vesicles to mitochondria. Live-cell imaging captured spherical Cav1(-) RNP vesicles emerging from the fission of large Cav(+) particles. Thus, RNP appears to traffic by a different route than the classical Rab5-dependent pathway of viral transport.

The voltage sensor domain (VSD) of the potassium ion channel KvAP is comprised of four (S1-S4) α-helix proteins, which are encompassed by several charged residues. Apart from these charges, each peptide α-helix having two inherent equal and opposite terminal dipolar charges behave like a macrodipole. The activity of voltage gated ion channel is electrostatic, where all the charges (charged residues and dipolar terminal charges) interact with each other and with the transmembrane potential. There are evidences that the role of the charged residues dominate the stabilization of the conformation and the gating process of the ion channel, but the role of the terminal dipolar charges are never considered in such analysis. Here, using electrostatic theory, we have studied the role of the dipolar terminal charges in aggregation of the S3b-S4 helix pair of KvAP in the absence of any external field (V=0). A system attains stability, when its potential energy reaches minimum values. We have shown that the presence of terminal dipole charges (1) change the total potential energy of the charges on S3b-S4, affecting the stabilization of the α-helix pair within the bilayer lipid membrane and (2) the C- and the N-termini of the α-helices favor a different dielectric medium for enhanced stability. Thus, the dipolar terminal charges play a significant role in the aggregation of the two neighboring α-helices.

Corrigendum to “Role of terminal dipole charges in aggregation of α-helix pair in the voltage gated K channel” [Biochim. Biophys. Acta 1828 (2013) 845–850] Lipika Adhya⁎, Tarunendu Mapder, Samit Adhya a Department of Engineering Physics, B. P. Poddar Institute of Management and Technology, 137, V.I.P. Road, Calcutta-700052, India b Molecular and Human Genetics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Calcutta-700032, India

BACKGROUND:It is well known that α-helices of protein, possessing equal and opposite charged ends, behaves like a macrodipole, but the relative importance of such macrodipoles to the aggregation of a pair of helix in the voltage sensor domain (VSD) of K+ ion channel, has not been assessed. In the VSD, importance has been given primarily to the helically arranged Arginine residues of helix, but the role of the charged residues of S3b is less focused.METHOD AND OBJECTIVE:Applying electrostatic theory, we have studied the interaction between the charges of S3b-S4 α-helix pair of KvAP through virtual mutagenesis.RESULT AND CONCLUSION:We have shown that the terminal charges arising from the inherent dipolar property of α-helices play an important role in affecting the stability of the S3b-S4 pair, and in determining its spatial position at zero transmembrane potential. Moreover, the negatively charged side chain of S3b was found to be the primary stabilizing factor in holding S3b-S4 pair together as a "paddle". Comparison of sequences of S3b helix of K+ channels from different species showed a previously unreported positional conservation of negative residues, highlighting their functional importance. These charges may contribute to the energetic of α-helix movements in an electric field.

Very little is known about how nucleic acids are translocated across membranes. The multi-subunit RNA Import Complex (RIC) from mitochondria of the kinetoplastid protozoon Leishmania tropica induces translocation of tRNAs across artificial or natural membranes, but the nature of the translocation pore remains unknown. We show that subunits RIC6 and RIC9 assemble on the membrane in presence of subunit RIC4A to form complex R3. Atomic Force Microscopy of R3 revealed particles with an asymmetric surface groove of similar to 20 nm rim diameter and similar to 1 nm depth. R3 induced translocation of tRNA into liposomes when the pH of the medium was lowered to similar to 6 in the absence of ATP. R3-mediated tRNA translocation could also be induced at neutral pH by a K+ diffusion potential with an optimum of 60-70 mV. Point mutations in the Cys(2)-His(2) Fe-binding motif of RIC6, which is homologous to the respiratory Complex III Fe-S protein, abrogated import induced by low pH but not by K+ diffusion potential. These results indicate that the R3 complex forms a pore that is gated by a proton-generated membrane potential and that the Fe-S binding region of RIC6 has a role in proton translocation. The tRNA import complex of L. tropica thus contains a novel macromolecular channel distinct from the mitochondrial protein import pore that is apparently involved in tRNA import in some species. (c) 2013 Elsevier Inc. All rights reserved.

Skeletal muscle injury is associated with general down-regulation of mitochondrial function. Postinjury regeneration of skeletal muscle occurs through activation, proliferation, and differentiation of resident stem cells, including satellite cells and endothelial precursor cells. We wanted to determine the role of mitochondrial function in the regeneration process. Using a previously described method for complex-mediated delivery to intracellular mitochondria, a combination of polycistronic RNAs encoding the H strand of the rat mitochondrial genome was administered to injured rat quadriceps muscle, resulting in restoration of mitochondrial mRNA levels, organellar translation, and respiratory capacity. Intramuscular ATP levels were elevated on pcRNA treatment of injured muscle; concomitantly, levels of reactive oxygen species in the injured muscle were reduced. These effects combined to produce a notable increase in the rate of wound resolution, accompanied by reduction of fibrosis and acceleration of myogenesis, vasculogenesis, and resumption of muscle contractile function. There was evidence of proliferation of Pax7+ satellite cells, expression of muscle-specific regulatory factors in a specific time sequence, and formation of new myofibers in the regenerating muscle. RNA-induced wound resolution and satellite cell proliferation were sensitive to mitochondrial inhibitors, indicating the importance of oxidative phosphorylation. These results highlight the activation of endogenous stem cells through mitochondrial restoration as a possible alternative to implantation of cultured stem cells.

Mutations in mitochondrial DNA (mtDNA) generate multi-system disorders due to failure of ATP production. A cybrid containing a 1.9-kb mtDNA deletion from a patient with Kearns Sayre Syndrome is respiration-defective and grows glycolytically. When treated with a ribonucleoprotein (RNP) complex of polycistronic RNA 1 (pcRNA1) containing mtDNA-encoded genes and a multi-subunit carrier complex R8, full-length pcRNA1 was transported to mitochondria. Translation of the pcRNA1-encoded mRNAs was observed in mitochondria from RNP-treated cells. Respiration of the cybrid was rescued to ~90% of normal within hours, switching the cells to aerobic growth. These findings have implications for the development of effective mitochondrial gene therapy.

The association of mitochondrial dysfunction with a variety of human diseases and disabilities has been documented. Mitochondrial gene therapy (MGT) seeks to correct the genetic defect in mitochondrial DNA. For successful MGT, an appreciation of the nature of the dysfunction and of the complexities of mitochondrial disease is necessary. This review summarizes the current status of various MGT protocols described in the literature. Although there are many technical difficulties to be overcome, there are indications that some of them will find clinical applications in the near future.

Genetic dysfunction of mitochondria is pathological, but an effective method of nucleic acid delivery to mitochondria in vivo is lacking. Injection into rodents of tagged polycistronic RNAs (pcRNAs) encoding parts of the organelle genome and bound to a carrier complex, resulted in rapid uptake and concentration of the RNA in many tissues. The delivered RNA was localized to mitochondria. A pan-genomic cocktail of pcRNAs restored mRNA levels, stimulated mitochondrial translation and respiratory capacity of skeletal muscle of middle-aged and old rats. Thus, the carrier-based protocol may be suitable for delivery of functional RNAs to mitochondria in vivo.

Many human diseases are associated with mutations and deletions in mitochondrial DNA (mtDNA). We have generated a cell line, EB delta1, with multiple mtDNA deletions, that is respiration-defective and generates high levels of superoxide, a reactive oxygen species. Treatment of EB delta1 with tagged polycistronic (pc) RNAs, encoding parts of the mitochondrial proteome, bound to a multi-subunit carrier complex, resulted in cellular uptake and transfer of the RNA to mitochondria, restoration of respiration, and suppression of superoxide levels. These findings have implications for correction of mitochondrial defects in age-related disorders due to mtDNA mutations.