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1.  Corrigendum 
RNA Biology  2015;12(9):1070.
doi:10.1080/15476286.2015.1082401
PMCID: PMC4615175  PMID: 26383778
2.  Pseudouridine: Still mysterious, but never a fake (uridine)! 
RNA Biology  2015;11(12):1540-1554.
Pseudouridine (Ψ) is the most abundant of >150 nucleoside modifications in RNA. Although Ψ was discovered as the first modified nucleoside more than half a century ago, neither the enzymatic mechanism of its formation, nor the function of this modification are fully elucidated. We present the consistent picture of Ψ synthases, their substrates and their substrate positions in model organisms of all domains of life as it has emerged to date and point out the challenges that remain concerning higher eukaryotes and the elucidation of the enzymatic mechanism.
doi:10.4161/15476286.2014.992278
PMCID: PMC4615568  PMID: 25616362
enzymatic mechanism; modified nucleoside; pseudouridine; regulation; rRNA; RNA Modification; tRNA; snRNA
3.  The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry 
RNA Biology  2015;11(12):1568-1585.
The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems.
doi:10.4161/15476286.2014.992280
PMCID: PMC4615682  PMID: 25616408
biological ribonucleic acids; LC-MS/MS; MALDI-MS/MS; modified RNA; modified bases; RNA sequencing; rRNA; tRNA; tandem mass spectrometry
4.  Distribution and frequencies of post-transcriptional modifications in tRNAs 
RNA Biology  2015;11(12):1619-1629.
Functional tRNA molecules always contain a wide variety of post-transcriptionally modified nucleosides. These modifications stabilize tRNA structure, allow for proper interaction with other macromolecules and fine-tune the decoding of mRNAs during translation. Their presence in functionally important regions of tRNA is conserved in all domains of life. However, the identities of many of these modified residues depend much on the phylogeny of organisms the tRNAs are found in, attesting for domain-specific strategies of tRNA maturation. In this work we present a new tool, tRNAmodviz web server (http://genesilico.pl/trnamodviz) for easy comparative analysis and visualization of modification patterns in individual tRNAs, as well as in groups of selected tRNA sequences. We also present results of comparative analysis of tRNA sequences derived from 7 phylogenetically distinct groups of organisms: Gram-negative bacteria, Gram-positive bacteria, cytosol of eukaryotic single cell organisms, Fungi and Metazoa, cytosol of Viridiplantae, mitochondria, plastids and Euryarchaeota. These data update the study conducted 20 y ago with the tRNA sequences available at that time.
doi:10.4161/15476286.2014.992273
PMCID: PMC4615829  PMID: 25611331
comparative analysis; evolution; modified nucleotides; post-transcriptional modification; RNA maturation; tRNA; tRNA modifications; tRNA sequence; web server
5.  Elongator, a conserved complex required for wobble uridine modifications in Eukaryotes 
RNA Biology  2015;11(12):1519-1528.
Elongator is a 6 subunit protein complex highly conserved in eukaryotes. The role of this complex has been controversial as the pleiotropic phenotypes of Elongator mutants have implicated the complex in several cellular processes. However, in yeast there is convincing evidence that the primary and probably only role of this complex is in formation of the 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) side chains on uridines at wobble position in tRNA. In this review we summarize the cellular processes that have been linked to the Elongator complex and discuss its role in tRNA modification and regulation of translation. We also describe additional gene products essential for formation of ncm5 and mcm5 side chains at U34 and their influence on Elongator activity.
doi:10.4161/15476286.2014.992276
PMCID: PMC4615193  PMID: 25607684
elongator complex; KTI genes; SIT4; SAP genes; tRNA wobble uridine modifications,; translation; Kluveromyces lactis γ-toxin
6.  Post-transcriptional modifications are very important after all 
RNA Biology  2015;11(12):1481-1482.
doi:10.1080/15476286.2014.1005439
PMCID: PMC4615288  PMID: 25607350
7.  Modification of the wobble uridine in bacterial and mitochondrial tRNAs reading NNA/NNG triplets of 2-codon boxes 
RNA Biology  2015;11(12):1495-1507.
Posttranscriptional modification of the uridine located at the wobble position (U34) of tRNAs is crucial for optimization of translation. Defects in the U34 modification of mitochondrial-tRNAs are associated with a group of rare diseases collectively characterized by the impairment of the oxidative phosphorylation system. Retrograde signaling pathways from mitochondria to nucleus are involved in the pathophysiology of these diseases. These pathways may be triggered by not only the disturbance of the mitochondrial (mt) translation caused by hypomodification of tRNAs, but also as a result of nonconventional roles of mt-tRNAs and mt-tRNA-modifying enzymes. The evolutionary conservation of these enzymes supports their importance for cell and organismal functions. Interestingly, bacterial and eukaryotic cells respond to stress by altering the expression or activity of these tRNA-modifying enzymes, which leads to changes in the modification status of tRNAs. This review summarizes recent findings about these enzymes and sets them within the previous data context.
doi:10.4161/15476286.2014.992269
PMCID: PMC4615368  PMID: 25607529
GTPBP3; mitochondrial diseases; MELAS; MnmA; MnmE; MnmG; MnmC; MTO1; TrmL; TRMU/MTU1; tRNA modification
8.  RNAa in action: From the exception to the norm 
RNA Biology  2015;11(10):1221-1225.
Small RNA programmed Argonautes are sophisticated cellular effector platforms known to be involved in a diverse array of functions ranging from mRNA cleavage, translational inhibition, DNA elimination, epigenetic silencing, alternative splicing and even gene activation. First observed in human cells, small RNA-induced gene activation, also known as RNAa, involves the targeted recruitment of Argonaute proteins to specific promoter sequences followed by induction of stable epigenetic changes which promote transcription. The existence of RNAa remains contentious due to its elusive mechanism. A string of recent studies in C. elegans provides unequivocal evidence for RNAa's fundamental role in sculpting the epigenetic landscape and maintaining active transcription of endogenous genes and supports the presence of a functionally sophisticated network of small RNA-Argonaute pathways consisting of opposite yet complementary “yin and yang” regulatory elements. In this review, we summarize key findings from recent studies of endogenous RNAa in C. elegans, with an emphasis on the Argonaute protein CSR-1.
doi:10.4161/15476286.2014.972853
PMCID: PMC4615537  PMID: 25602906
Argonaute; epigenetic memory; gene expression; RNAa; RNAe
9.  Identity elements for the aminoacylation of metazoan mitochondrial tRNAArg have been widely conserved throughout evolution and ensure the fidelity of the AGR codon reassignment 
RNA Biology  2015;11(10):1313-1323.
Eumetazoan mitochondrial tRNAs possess structures (identity elements) that require the specific recognition by their cognate nuclear-encoded aminoacyl-tRNA synthetases. The AGA (arginine) codon of the standard genetic code has been reassigned to serine/glycine/termination in eumetazoan organelles and is translated in some organisms by a mitochondrially encoded tRNASerUCU. One mechanism to prevent mistranslation of the AGA codon as arginine would require a set of tRNA identity elements distinct from those possessed by the cytoplasmic tRNAArg in which the major identity elements permit the arginylation of all 5 encoded isoacceptors. We have performed comparative in vitro aminoacylation using an insect mitochondrial arginyl-tRNA synthetase and tRNAArgUCG structural variants. The established identity elements are sufficient to maintain the fidelity of tRNASerUCU reassignment. tRNAs having a UCU anticodon cannot be arginylated but can be converted to arginine acceptance by identity element transplantation. We have examined the evolutionary distribution and functionality of these tRNA elements within metazoan taxa. We conclude that the identity elements that have evolved for the recognition of mitochondrial tRNAArgUCG by the nuclear encoded mitochondrial arginyl-tRNA synthetases of eumetazoans have been extensively, but not universally conserved, throughout this clade. They ensure that the AGR codon reassignment in eumetazoan mitochondria is not compromised by misaminoacylation. In contrast, in other metazoans, such as Porifera, whose mitochondrial translation is dictated by the universal genetic code, recognition of the 2 encoded tRNAArgUCG/UCU isoacceptors is achieved through structural features that resemble those employed by the yeast cytoplasmic system.
doi:10.1080/15476286.2014.996094
PMCID: PMC4615739  PMID: 25603118
arginyl-tRNA synthetase; codon reassignment; identity elements; metazoan; mitochondrial tRNA
10.  TDP-43 regulates β-adducin (Add2) transcript stability 
RNA Biology  2015;11(10):1280-1290.
TDP-43 is an RNA-binding protein involved in several steps of mRNA metabolism including transcription, splicing and stability. It is also involved in ALS and FTD, neurodegenerative diseases characterized by TDP-43 nuclear depletion. We previously identified TDP-43 as a binder of the downstream element (DSE) of the β-Adducin (Add2) brain-specific polyadenylation site (A4 PAS), suggesting its involvement in pre-mRNA 3′ end processing. Here, by using chimeric minigenes, we showed that TDP-43 depletion in HeLa and HEK293 cells resulted in down-regulation of both the chimeric and endogenous Add2 transcripts. Despite having confirmed TDP-43-DSE in vitro interaction, we demonstrated that the in vivo effect was not mediated by the TDP-43-DSE interaction. In fact, substitution of the Add2 DSE with viral E-SV40 and L-SV40 DSEs, which are not TDP-43 targets, still resulted in decreased Add2 mRNA levels after TDP-43 downregulation. In addition, we failed to show interaction between TDP-43 and key polyadenylation factors, such as CstF-64 and CPSF160 and excluded TDP-43 involvement in pre-mRNA cleavage and regulation of polyA tail length. These evidences allowed us to exclude the pre-hypothesized role of TDP43 in modulating 3′ end processing of Add2 pre-mRNA. Finally, we showed that TDP-43 regulates Add2 gene expression levels by increasing Add2 mRNA stability. Considering that Add2 in brain participates in synapse assembly, synaptic plasticity and their stability, and its genetic inactivation in mice leads to LTP, LTD, learning and motor-coordination deficits, we hypothesize that a possible loss of Add2 function by TDP-43 depletion may contribute to ALS and FTD disease states.
doi:10.1080/15476286.2014.996081
PMCID: PMC4615836  PMID: 25602706
Add2; DSE; mRNA stability; TDP-43; 3′ end processing; 3′UTR
11.  RNA-guided isomerization of uridine to pseudouridine—pseudouridylation 
RNA Biology  2015;11(12):1483-1494.
Box H/ACA ribonucleoproteins (RNPs), each consisting of one unique guide RNA and 4 common core proteins, constitute a family of complex enzymes that catalyze, in an RNA-guided manner, the isomerization of uridines to pseudouridines (Ψs) in RNAs, a reaction known as pseudouridylation. Over the years, box H/ACA RNPs have been extensively studied revealing many important aspects of these RNA modifying machines. In this review, we focus on the composition, structure, and biogenesis of H/ACA RNPs. We explain the mechanism of how this enzyme family recognizes and specifies its target uridine in a substrate RNA. We discuss the substrates of box H/ACA RNPs, focusing on rRNA (rRNA) and spliceosomal small nuclear RNA (snRNA). We describe the modification product Ψ and its contribution to RNA function. Finally, we consider possible mechanisms of the bone marrow failure syndrome dyskeratosis congenita and of prostate and other cancers linked to mutations in H/ACA RNPs.
doi:10.4161/15476286.2014.972855
PMCID: PMC4615163  PMID: 25590339
dyskeratosis congenita; H/ACA; prostate cancer; pseudouridine; ribonucleoproteins; RNA-guided; RNA modification; rRNA; spliceosomal small nuclear RNA; snoRNA
12.  Epigenome-wide analysis of piRNAs in gene-specific DNA methylation 
RNA Biology  2015;11(10):1301-1312.
PIWI-interacting RNAs (piRNAs) have long been associated with the silencing of transposable elements (TEs). However, over 20,000 unique species of piRNAs mapped to the human genome are more than the relatively few presumably required to regulate the known human transposon classes. Here, we present the results of the first genome-wide effort to study the effects of piRNAs on gene specific DNA methylation. We found that exon-derived piRNAs consist almost universally of species with 10 or fewer genomic copies, whereas piRNAs existing in high copies originate predominately from intronic and intergenic regions. Genome-wide methylation profiling following transfection of human somatic cells with piRNA mimics revealed methylation changes at numerous genic loci in single copy piRNA-transfected cells. Moreover, genomic regions directly adjacent to differentially methylated CpG sites were enriched for sequence matches to the transfected piRNAs. These findings suggest that a subset of single copy piRNAs may be able to induce DNA methylation at non-TE genic loci, a process that may be mediated in part by direct binding to either genomic DNA or nascent mRNA near target CpG sites.
doi:10.1080/15476286.2014.996091
PMCID: PMC4615395  PMID: 25590657
DNA methylation; gene regulation; genomic distribution; human somatic cells; piRNA
13.  DExD-box RNA-helicases in Listeria monocytogenes are important for growth, ribosomal maturation, rRNA processing and virulence factor expression 
RNA Biology  2015;11(11):1457-1466.
RNA-helicases are proteins required for the unwinding of occluding secondary RNA structures, especially at low temperatures. In this work, we have deleted all 4 DExD-box RNA helicases in various combinations in the Gram-positive pathogen Listeria monocytogenes. Our results show that 3 out of 4 RNA-helicases were important for growth at low temperatures, whereas the effect was less prominent at 37°C. Over-expression of one RNA-helicase, Lmo1450, was able to overcome the reduced growth of the quadruple mutant strain at temperatures above 26°C, but not at lower temperatures. The maturation of ribosomes was affected in different degrees in the various strains at 20°C, whereas the effect was marginal at 37°C. This was accompanied by an increased level of immature 23S rRNA precursors in some of the RNA-helicase mutants at low temperatures. Although the expression of the PrfA regulated virulence factors ActA and LLO decreased in the quadruple mutant strain, this strain showed a slightly increased infection ability. Interestingly, even though the level of the virulence factor LLO was decreased in the quadruple mutant strain as compared with the wild-type strain, the hly-transcript (encoding LLO) was increased. Hence, our results could suggest a role for the RNA-helicases during translation. In this work, we show that DExD-box RNA-helicases are involved in bacterial virulence gene-expression and infection of eukaryotic cells.
doi:10.1080/15476286.2014.996099
PMCID: PMC4615572  PMID: 25590644
bacterial infection; DExD-box RNA-helicases; hly; Listeria monocytogenes; LLO; translation
14.  Let there be light: Regulation of gene expression in plants 
RNA Biology  2015;11(10):1215-1220.
Gene expression regulation relies on a variety of molecular mechanisms affecting different steps of a messenger RNA (mRNA) life: transcription, processing, splicing, alternative splicing, transport, translation, storage and decay. Light induces massive reprogramming of gene expression in plants. Differences in alternative splicing patterns in response to environmental stimuli suggest that alternative splicing plays an important role in plant adaptation to changing life conditions. In a recent publication, our laboratories showed that light regulates alternative splicing of a subset of Arabidopsis genes encoding proteins involved in RNA processing by chloroplast retrograde signals. The light effect on alternative splicing is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. These results point at alternative splicing regulation by retrograde signals as an important mechanism for plant adaptation to their environment.
doi:10.4161/15476286.2014.972852
PMCID: PMC4615654  PMID: 25590224
alternative splicing; chloroplast; light; photoreceptors; retrograde signaling; RNA
15.  Small indels induced by CRISPR/Cas9 in the 5′ region of microRNA lead to its depletion and Drosha processing retardance 
RNA Biology  2015;11(10):1243-1249.
MicroRNA knockout by genome editing technologies is promising. In order to extend the application of the technology and to investigate the function of a specific miRNA, we used CRISPR/Cas9 to deplete human miR-93 from a cluster by targeting its 5’ region in HeLa cells. Various small indels were induced in the targeted region containing the Drosha processing site and seed sequences. Interestingly, we found that even a single nucleotide deletion led to complete knockout of the target miRNA with high specificity. Functional knockout was confirmed by phenotype analysis. Furthermore, de novo microRNAs were not found by RNA-seq. Nevertheless, expression of the pri-microRNAs was increased. When combined with structural analysis, the data indicated that biogenesis was impaired. Altogether, we showed that small indels in the 5’ region of a microRNA result in sequence depletion as well as Drosha processing retard.
doi:10.1080/15476286.2014.996067
PMCID: PMC4615719  PMID: 25590615
CRISPR/Cas9; microRNA depletion; microRNA biogenesis; miR-93; RNA-guided DNA cleavage
16.  Guardian small RNAs and sex determination 
RNA Biology  2015;11(10):1238-1242.
The W chromosome of the silkworm Bombyx mori has been known to determine femaleness for more than 80 years. However, the feminizing gene has not been molecularly identified, because the B. mori W chromosome is almost fully occupied by a large number of transposable elements. The W chromosome-derived feminizing factor of B. mori was recently shown to be a female-specific PIWI-interacting RNA (piRNA). piRNAs are small RNAs that potentially repress invading “non-self” elements (e.g., transposons and virus-like elements) by associating with PIWI proteins. Our results revealed that female-specific piRNA precursors, which we named Fem, are transcribed from the sex-determining region of the W chromosome at the early embryonic stage and are processed into a single mature piRNA (Fem piRNA). Fem piRNA forms a complex with Siwi (silkworm Piwi), which cleaves a protein-coding mRNA transcribed from the Z chromosome. RNA interference of this Z-linked gene, which we named Masc, revealed that this gene encodes a protein required for masculinization and dosage compensation. Fem and Masc both participate in the ping-pong cycle of the piRNA amplification loop by associating with the 2 B. mori PIWI proteins Siwi and BmAgo3 (silkworm Ago3), respectively, indicating that the piRNA-mediated interaction between the 2 sex chromosomes is the primary signal for the B. mori sex determination cascade. Fem is a non-transposable repetitive sequence on the W chromosome, whereas Masc is a single-copy protein-coding gene. It is of great interest how the piRNA system recognizes “self ”Masc mRNA as “non-self” RNA.
doi:10.1080/15476286.2014.996060
PMCID: PMC4615651  PMID: 25588029
amplification cycles; ping-pong; piRNA; PIWI proteins; silkworm; sex determination
17.  Re-editing the paradigm of Cytidine (C) to Uridine (U) RNA editing 
RNA Biology  2015;11(10):1233-1237.
Cytidine (C) to Uridine (U) RNA editing is a post-trancriptional modification that until recently was known to only affect Apolipoprotein b (Apob) RNA and minimally require 2 components of the C to U editosome, the deaminase APOBEC1 and the RNA-binding protein A1CF. Our latest work has identified a novel RNA-binding protein, RBM47, as a core component of the editosome, which can substitute A1CF for the editing of ApoB mRNA. In addition, new RNA species that are subjected to C to U editing have been identified. Here, we highlight these recent discoveries and discuss how they change our view of the composition of the C to U editing machinery and expand our knowledge of the functional attributes of C to U RNA editing.
doi:10.1080/15476286.2014.996054
PMCID: PMC4615559  PMID: 25585043
APOBEC1; Apolipoprotein b; A1CF; Intestine; RBM47; RNA editing
18.  Functions of double-stranded RNA-binding domains in nucleocytoplasmic transport 
RNA Biology  2015;11(10):1226-1232.
The double-stranded RNA-binding domain (dsRBD) is a small protein domain found in eukaryotic, prokaryotic and viral proteins, whose central property is to bind to double-stranded RNA (dsRNA). Aside from this major function, recent examples of dsRBDs involved in the regulation of the sub-cellular localization of proteins, suggest that the participation of dsRBDs in nucleocytoplasmic trafficking is likely to represent a widespread auxiliary function of this type of RNA-binding domain. Overall, dsRBDs from proteins involved in many different biological processes have been reported to be implicated in nuclear import and export, as well as cytoplasmic, nuclear and nucleolar retention. Interestingly, the function of dsRBDs in nucleocytoplasmic trafficking is often regulated by their dsRNA-binding capacity, which can either enhance or impair the transport from one compartment to another. Here, we present and discuss the emerging function of dsRBDs in nucleocytoplasmic transport.
doi:10.4161/15476286.2014.972856
PMCID: PMC4615638  PMID: 25584639
ADAR1; cytoplasmic retention; double-stranded RNA-binding motif; dsRBM; dicer; nucleocytoplasmic shuttling; nuclear localization signal; nuclear retention; RNA-binding protein; RNA editing
19.  Identification of miRNAs that modulate glucocerebrosidase activity in Gaucher disease cells 
RNA Biology  2015;11(10):1291-1300.
Gaucher disease is an autosomal recessive disorder caused by deficiency of the enzyme glucocerebrosidase. Although it is a monogenic disease, there is vast phenotypic heterogeneity, even among patients with the same genotype. MicroRNAs (miRNAs) are small non-coding RNAs involved in many biological processes and diseases. To determine whether miRNAs can affect glucocerebrosidase activity, we performed a screen of 875 different miRNA mimics. The screen was performed using Gaucher fibroblasts, and glucocerebrosidase activity was used as the initial outcome parameter. We found several miRNAs that either up- or down-regulated glucocerebrosidase activity. In follow-up assays, we confirmed that one specific miRNA (miR-127–5p) down-regulated both glucocerebrosidase activity and protein levels by down-regulation of LIMP-2, the receptor involved in proper trafficking of glucocerebrosidase from the endoplasmic reticulum to the lysosome. A conditioned media assay demonstrated that cells treated with this miRNA secreted glucocerebrosidase into the extracellular environment, supporting impaired LIMP-2 function. Two other miRNAs, miR-16–5p and miR-195–5p, were found to up-regulate glucocerebrosidase activity by greater than 40% and to enhance expression and protein levels of the enzyme. In conclusion, we show that miRNAs can alter glucocerebrosidase activity in patient cells, indicating that miRNAs can potentially act as modifiers in Gaucher disease.
doi:10.1080/15476286.2014.996085
PMCID: PMC4615671  PMID: 25584808
Gaucher Disease; GBA; GCase; glucocerebrosidase; mimics; miRNA; screen
20.  The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets 
RNA Biology  2015;11(10):1250-1261.
Human antigen R (HuR) is a 32 kDa protein with 3 RNA Recognition Motifs (RRMs), which bind to Adenylate and uridylate Rich Elements (AREs) of mRNAs. Whereas the N-terminal and central domains (RRM1 and RRM2) are essential for AREs recognition, little is known on the C-terminal RRM3 beyond its implication in HuR oligomerization and apoptotic signaling. We have developed a detergent-based strategy to produce soluble RRM3 for structural studies. We have found that it adopts the typical RRM fold, does not interact with the RRM1 and RRM2 modules, and forms dimers in solution. Our NMR measurements, combined with Molecular Dynamics simulations and Analytical Ultracentrifugation experiments, show that the protein dimerizes through a helical region that contains the conserved W261 residue. We found that HuR RRM3 binds to 5′-mer U-rich RNA stretches through the solvent exposed side of its β-sheet, located opposite to the dimerization site. Upon mimicking phosphorylation by the S318D replacement, RRM3 mutant shows less ability to recognize RNA due to an electrostatic repulsion effect with the phosphate groups. Our study brings new insights of HuR RRM3 as a domain involved in protein oligomerization and RNA interaction, both functions regulated by 2 surfaces on opposite sides of the RRM domain.
doi:10.1080/15476286.2014.996069
PMCID: PMC4615805  PMID: 25584704
dimerization; Human antigen R (HuR); Nuclear Magnetic Resonance (NMR); RNA binding protein (RBP); RNA binding; RNA recognition motif (RRM); Serine Phosphorylation
21.  Genome wide chromatin occupancy of mrhl RNA and its role in gene regulation in mouse spermatogonial cells 
RNA Biology  2015;11(10):1262-1279.
Mrhl RNA is a nuclear lncRNA encoded in the mouse genome and negatively regulates Wnt signaling in spermatogonial cells through p68/Ddx5 RNA helicase. Mrhl RNA is present in the chromatin fraction of mouse spermatogonial Gc1-Spg cells and genome wide chromatin occupancy of mrhl RNA by ChOP (Chromatin oligo affinity precipitation) technique identified 1370 statistically significant genomic loci. Among these, genes at 37 genomic loci also showed altered expression pattern upon mrhl RNA down regulation which are referred to as GRPAM (Genes Regulated by Physical Association of Mrhl RNA). p68 interacted with mrhl RNA in chromatin at these GRPAM loci. p68 silencing drastically reduced mrhl RNA occupancy at 27 GRPAM loci and also perturbed the expression of GRPAM suggesting a role for p68 mediated mrhl RNA occupancy in regulating GRPAM expression. Wnt3a ligand treatment of Gc1-Spg cells down regulated mrhl RNA expression and also perturbed expression of these 27 GRPAM genes that included genes regulating Wnt signaling pathway and spermatogenesis, one of them being Sox8, a developmentally important transcription factor. We also identified interacting proteins of mrhl RNA associated chromatin fraction which included Pc4, a chromatin organizer protein and hnRNP A/B and hnRNP A2/B1 which have been shown to be associated with lincRNA-Cox2 function in gene regulation. Our findings in the Gc1-Spg cell line also correlate with the results from analysis of mouse testicular tissue which further highlights the in vivo physiological significance of mrhl RNA in the context of gene regulation during mammalian spermatogenesis.
doi:10.1080/15476286.2014.996070
PMCID: PMC4615903  PMID: 25584904
Chromatin occupancy; gene regulation; GRPAM; mrhl RNA; p68
22.  mRNP granules 
RNA Biology  2014;11(8):1019-1030.
Messenger ribonucleoprotein (mRNP) granules are dynamic, self-assembling structures that harbor non-translating mRNAs bound by various proteins that regulate mRNA translation, localization, and turnover. Their importance in gene expression regulation is far reaching, ranging from precise spatial-temporal control of mRNAs that drive developmental programs in oocytes and embryos, to similarly exquisite control of mRNAs in neurons that underpin synaptic plasticity, and thus, memory formation. Analysis of mRNP granules in their various contexts has revealed common themes of assembly, disassembly, and modes of mRNA regulation, yet new studies continue to reveal unexpected and important findings, such as links between aberrant mRNP granule assembly and neurodegenerative disease. Continued study of these enigmatic structures thus promises fascinating new insights into cellular function, and may also suggest novel therapeutic strategies in various disease states.
doi:10.4161/15476286.2014.972208
PMCID: PMC4615263  PMID: 25531407
stress granules; P-bodies; neuronal transport granules; germ granules; mRNA; translation; repression; decay; neurodegenerative disease
23.  Proteins involved in the degradation of cytoplasmic mRNA in the major eukaryotic model systems 
RNA Biology  2014;11(9):1122-1136.
The process of mRNA decay and surveillance is considered to be one of the main posttranscriptional gene expression regulation platforms in eukaryotes. The degradation of stable, protein-coding transcripts is normally initiated by removal of the poly(A) tail followed by 5’-cap hydrolysis and degradation of the remaining mRNA body by Xrn1. Alternatively, the exosome complex degrades mRNA in the 3’>5’direction. The newly discovered uridinylation-dependent pathway, which is present in many different organisms, also seems to play a role in bulk mRNA degradation. Simultaneously, to avoid the synthesis of incorrect proteins, special cellular machinery is responsible for the removal of faulty transcripts via nonsense-mediated, no-go, non-stop or non-functional 18S rRNA decay. This review is focused on the major eukaryotic cytoplasmic mRNA degradation pathways showing many similarities and pointing out main differences between the main model-species: yeast, Drosophila, plants and mammals.
doi:10.4161/rna.34406
PMCID: PMC4615280  PMID: 25483043
Arabidopsis thaliana; Drosophila melanogaster; human; mRNA decay; mRNA surveillance; Saccharomyces cerevisiae; Schizosaccharomyces pombe
24.  RNA imaging in living cells – methods and applications 
RNA Biology  2014;11(8):1083-1095.
Numerous types of transcripts perform multiple functions in cells, and these functions are mainly facilitated by the interactions of the RNA with various proteins and other RNAs. Insight into the dynamics of RNA biosynthesis, processing and cellular activities is highly desirable because this knowledge will deepen our understanding of cell physiology and help explain the mechanisms of RNA-mediated pathologies. In this review, we discuss the live RNA imaging systems that have been developed to date. We highlight information on the design of these systems, briefly discuss their advantages and limitations and provide examples of their numerous applications in various organisms and cell types. We present a detailed examination of one application of RNA imaging systems: this application aims to explain the role of mutant transcripts in human disease pathogenesis caused by triplet repeat expansions. Thus, this review introduces live RNA imaging systems and provides a glimpse into their various applications.
doi:10.4161/rna.35506
PMCID: PMC4615301  PMID: 25483044
live-cell imaging systems; RNA nuclear foci; RNA localization; RNA fluorescence imaging; triplet repeat diseases
25.  Requirement for CRIF1 in RNA interference and Dicer-2 stability 
RNA Biology  2014;11(9):1171-1179.
RNA interference (RNAi) is a eukaryotic gene-silencing system. Although the biochemistry of RNAi is relatively well defined, how this pathway is regulated remains incompletely understood. To identify genes involved in regulating the RNAi pathway, we screened for genetic mutations in Drosophila that alter the efficiency of RNAi. We identified the Drosophila homolog of the mammalian CR6-interacting factor 1 (CRIF1), also known as growth arrest and DNA-damage-inducible 45-gamma interacting protein (Gadd45GIP1), as a potential new regulator of the RNAi pathway. Loss-of-function mutants of Drosophila CRIF1 (dCRIF) are deficient in RNAi-mediated target gene knock-down, in the biogenesis of small interfering RNA (siRNA) molecules, and in antiviral immunity. Moreover, we show that dCRIF may function by interacting with, and stabilizing, the RNase III enzyme Dicer-2. Our results suggest that dCRIF may play an important role in regulating the RNAi pathway.
doi:10.4161/rna.34381
PMCID: PMC4615304  PMID: 25483042
Drosophila; RNAi; Dcr-2; Gadd45GIP

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