Many OX PHOS and replication factor mRNAs target mitochondria through specific binding to the organelle surface, independent of cotranslational protein import.

A large number of nucleus-encoded messenger RNAs (mRNAs) encoding proteins involved in oxidative phosphorylation have been found to be associated with mitochondria , 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.

OXPHOS deficiency induces mitochondrial DNA synthesis through non-canonical AMPK-dependent mRNA compartmentalization.

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.

Posttranscriptional regulation of cyclin D1 by ARE-binding proteins AUF1 and HuR in cycling myoblasts.

RNA binding proteins (RBPs) can regulate the stability and/or translatability of messengerRNAs (mRNAs) through interactions with their 30-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.

In-depth molecular analysis of a small cohort of human and Aedes mosquito (adults and larvae) samples from Kolkata revealed absence of Zika but high prevalence of dengue virus.

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.

Non-equivalent Roles of AGO1 and AGO2 in mRNA Turnover and Translation of Cyclin D1 mRNA.

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 ofDrosophilaand 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.

Effect of dielectric interface on charge aggregation in the voltage-gated K(+) ion channel.

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.

Effects of Transient Hypoxia versus Prolonged Hypoxia on Satellite Cell Proliferation and Differentiation In Vivo.

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.

Vesicular transport of a ribonucleoprotein to mitochondria.

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.

Role of the mTORC1 complex in satellite cell activation by RNA-induced mitochondrial restoration: dual control of cyclin D1 through microRNAs.

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.

A voltage-gated pore for translocation of tRNA.

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.

Electrostatic interaction between dipoles and side chains in the voltage sensor domain of K(+) channel.

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.

Role of terminal dipole charges in aggregation of α-helix pair in the voltage gated K(+) channel.

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.

Induction of muscle regeneration by RNA-mediated mitochondrial restoration.

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.

Modulation of mitochondrial respiratory capacity by carrier-mediated transfer of RNA in vivo.

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.

Mitochondrial gene therapy: The tortuous path from bench to bedside.

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.

Suppression of reactive oxygen species in cells with multiple mitochondrial DNA deletions by exogenous protein-coding RNAs.

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.

A mosaic of RNA binding and protein interaction motifs in a bifunctional mitochondrial tRNA import factor from Leishmania tropica.

Proteins that participate in the import of cytosolic tRNAs into mitochondria have been identified in several eukaryotic species, but the details of their interactions with tRNA and other proteins are unknown. In the kinetoplastid protozoon Leishmania tropica, multiple proteins are organized into a functional import complex. RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III).

Proton-guided movements of tRNA within the Leishmania mitochondrial RNA import complex.

The RNA import complex (RIC) from the mitochondrion of the kinetoplastid protozoan Leishmania tropica contains two subunits that directly bind to import signals on two distinct subsets of tRNA and interact with each other allosterically. What happens to the tRNA subsequent to its loading on the complex is unknown. A third subunit-RIC9-has intrinsic affinity for both types of tRNA and is essential for import in vivo.

Targeted mRNA degradation by complex-mediated delivery of antisense RNAs to intracellular human mitochondria.

Mitochondrial dysfunction underlies a large number of acute or progressive diseases, as well as aging. However, proposed therapies for mitochondrial mutations suffer from poor transformation of mitochondria with exogenous DNA, or lack of functionality of the transferred nucleic acid within the organelle. We show that a transfer RNA import complex (RIC) from the parasitic protozoon Leishmania tropica rapidly and efficiently delivered signal-tagged antisense (STAS) RNA or DNA to mitochondria of cultured human cells.

Leishmania mitochondrial tRNA importers.

The RNA Import Complex (RIC) is a multi-subunit protein complex from the mitochondria of the kinetoplastid protozoon Leishmania tropica that induces transport of tRNA across natural and artificial membranes. Leishmania, Trypanosoma and related genera of the order Kinetoplastidae are early diverging, atypical eukaryotes with unique RNA metabolic pathways, including the import of nucleus-encoded tRNAs into the mitochondrion to complement the deletion of all organelle-encoded tRNA genes. Biochemical and genetic studies of RIC are contributing to greater understanding of the mechanism of import.

Necessary and sufficient factors for the import of transfer RNA into the kinetoplast mitochondrion.

The mechanism of active transport of transfer RNA (tRNA) across membranes is largely unknown. Factors mediating the import of tRNA into the kinetoplast mitochondrion of the protozoon Leishmania tropica are organized into a multiprotein RNA import complex (RIC) at the inner membrane. Here, we present the complete characterization of the identities and functions of the subunits of this complex.

The complexity of mitochondrial tRNA import.

Import of nucleus-encoded, cytoplasmic tRNAs into mitochondria to compensate evolutionary loss of the corresponding mitochondrial genes has been documented in a large number of species. Although the phenomenon has been known for more than 25 years, it was only recently that the mechanism of tRNA import started receiving the sustained attention of workers investigating yeast, protozoal and higher plant systems. The purpose of this review is to summarize recent developments that shed new light on the selectivity of the process, the identity of the import apparatus and the nature of the bioenergetic transactions leading to tRNA translocation, and to build a working model of the import complex suggested by these observations.

Functional delivery of a cytosolic tRNA into mutant mitochondria of human cells.

Many maternally inherited and incurable neuromyopathies are caused by mutations in mitochondrial (mt) transfer RNA (tRNA) genes. Kinetoplastid protozoa, including Leishmania, have evolved specialized systems for importing nucleus-encoded tRNAs into mitochondria. We found that the Leishmania RNA import complex (RIC) could enter human cells by a caveolin-1-dependent pathway, where it induced import of endogenous cytosolic tRNAs, including tRNA(Lys), and restored mitochondrial function in a cybrid harboring a mutant mt tRNA(Lys) (MT-TK) gene.

An RNA-binding respiratory component mediates import of type II tRNAs into Leishmania mitochondria.

Transport of tRNAs across the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania requires interactions with specific binding proteins (receptors) in a multi-subunit complex. The allosteric model of import regulation proposes cooperative and antagonistic interactions between two or more receptors with binding specificities for distinct tRNA families (types I and II, respectively). To identify the type II receptor, the gene encoding RIC8A, a subunit of the complex, was cloned.