Authors: Jarred W Rensvold, Shao-En Ong, Athavi Jeevananthan...

Mitochondria are centers of metabolism and signaling whose content and function must adapt to changing cellular environments. The biological signals that initiate mitochondrial restructuring and the cellular processes that drive this adaptive response are largely obscure. To better define these systems, we performed matched quantitative genomic and proteomic analyses of mouse muscle cells as they performed mitochondrial biogenesis. We find that proteins involved in cellular iron homeostasis are highly coordinated with this process and that depletion of cellular iron results in a rapid, dose-dependent decrease of select mitochondrial protein levels and oxidative capacity. We further show that this process is universal across a broad range of cell types and fully reversed when iron is reintroduced. Collectively, our work reveals that cellular iron is a key regulator of mitochondrial biogenesis, and provides quantitative data sets that can be leveraged to explore posttranscriptional and posttranslational processes that are essential for mitochondrial adaptation.

Topics: Animals; Cell Respiration; DNA, Mitochondrial; Gene Expression Regulation; HEK293 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Iron; Iron Chelating Agents; Mice; Mitochondrial Proteins; Mitochondrial Turnover; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Proteomics; RNA, Complementary; RNA, Messenger; Rats; Time Factors; Trans-Activators; Transcription Factors

PubMed: 23318259
DOI: 10.1016/j.celrep.2012.11.029

Authors: Shijian Zhang, Jinlan Wang, Qiang Wang...

Influenza virus transcription is a prototype of primer-dependent initiation. Its replication mechanism is thought to be primer-independent. The internal initiation and realignment model for influenza virus genome replication has been recently proposed (Deng, T., Vreede, F. T., and Brownlee, G. G. (2006) J. Virol. 80, 2337-2348). We obtained new results, which led us to propose a novel model for the initiation of viral RNA (vRNA) replication. In our study, we analyzed the initiation mechanisms of influenza virus vRNA and complementary RNA (cRNA) synthesis in vitro, using purified RNA polymerase (RdRp) and 84-nt model RNA templates. We found that, for vRNA → cRNA →, RdRp initiated replication from the second nucleotide of the 3'-end. Therefore, host RNA-specific ribonucleotidyltransferases are required to add one nucleotide (purine residues are preferred) to the 3'-end of vRNA to make the complete copy of vRNA. This hypothesis was experimentally proven using poly(A) polymerase. For cRNA → vRNA, the dinucleotide primer AG was synthesized from UC (fourth and fifth from the 3'-end) by RdRp pausing at the sixth U of UUU and realigning at the 3'-end of cRNA template; then RdRp was able to read through the entire template RNA. The RdRp initiation complex was not stable until it had read through the UUU of cRNA and the UUUU of vRNA at their respective 3'-ends. This was because primers overlapping with the first U of the clusters did not initiate transcription efficiently, and the initiation product of v84+G (the v84 template with an extra G at its 3'-end), AGC, realigned to the 3'-end.

Topics: Oligoribonucleotides; Orthomyxoviridae; RNA, Complementary; RNA, Viral; RNA-Dependent RNA Polymerase; Viral Proteins; Virus Replication

PubMed: 20858902
DOI: 10.1074/jbc.M110.130062

Authors: Martyna Nowacka, Humberto Fernandes, Agnieszka Kiliszek...

The bacteriophage Mu Com is a small zinc finger protein that binds to its cognate mom mRNA and activates its translation. The Mom protein, in turn, elicits a chemical modification (momification) of the bacteriophage genome, rendering the DNA resistant to cleavage by bacterial restriction endonucleases, and thereby protecting it from defense mechanisms of the host. We examined the basis of specificity in Com-RNA interactions by in vitro selection and probing of RNA structure. We demonstrated that Com recognizes a sequence motif within a hairpin-loop structure of its target RNA. Our data support the model of Com interaction with mom mRNA, in which Com binds to the short hairpin structure proximal to the so-called translation inhibition structure. We also observed that Com binds its target motif weakly if it is within an RNA duplex. These results suggest that the RNA structure, in addition to its sequence, is crucial for Com to recognize its target and that RNA conformational changes may constitute another level of Mom regulation. We determined a crystal structure of a Com binding site variant designed to form an RNA duplex preferentially. Our crystal model forms a 19-mer self-complementary double helix composed of the canonical and non-canonical base pairs. The helical parameters of crystalized RNA indicate why Com may bind it more weakly than a monomeric hairpin form.

Topics: Bacteriophage mu; Base Pairing; Binding Sites; DNA; Genes, Viral; Haemophilus; Nucleic Acid Conformation; Open Reading Frames; Protein Biosynthesis; RNA, Complementary; RNA, Messenger; SELEX Aptamer Technique; Solvents; Transcription, Genetic; Viral Proteins; Zinc Fingers

PubMed: 31022205
DOI: 10.1371/journal.pone.0214481

Authors: Dorte Renneberg, Emilie Bouliong, Ulrich Reber...

Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2'-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10-17 nt in length, show enhanced selectivity and enhanced thermal stability by approximately 1 degrees C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37 degree C. Moreover, tc-DNA-RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human beta-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in beta-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3'-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2'-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2'-O-Me-PS-RNA, tc-DNA shows antisense activity even in the absence of lipofectamine, albeit only at much higher oligonucleotide concentrations.

Topics: Alternative Splicing; Animals; Cattle; DNA; DNA, Antisense; Fetal Blood; Globins; HeLa Cells; Humans; Nucleic Acid Conformation; Nucleic Acid Denaturation; Oligonucleotides; RNA, Complementary; RNA, Messenger; Ribonuclease H; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transfection

PubMed: 12087157
DOI: 10.1093/nar/gkf412

Authors: W Li, Y Li, N Kedersha...

It was reported previously that four baby hamster kidney (BHK) proteins with molecular masses of 108, 60, 50, and 42 kDa bind specifically to the 3'-terminal stem-loop of the West Nile virus minus-stand RNA [WNV 3'(-) SL RNA] (P. Y. Shi, W. Li, and M. A. Brinton, J. Virol. 70:6278-6287, 1996). In this study, p42 was purified using an RNA affinity column and identified as TIAR by peptide sequencing. A 42-kDa UV-cross-linked viral RNA-cell protein complex formed in BHK cytoplasmic extracts incubated with the WNV 3'(-) SL RNA was immunoprecipitated by anti-TIAR antibody. Both TIAR and the closely related protein TIA-1 are members of the RNA recognition motif (RRM) family of RNA binding proteins. TIA-1 also binds to the WNV 3'(-) SL RNA. The specificity of these viral RNA-cell protein interactions was demonstrated using recombinant proteins in competition gel mobility shift assays. The binding site for the WNV 3'(-) SL RNA was mapped to RRM2 on both TIAR and TIA-1. However, the dissociation constant (K(d)) for the interaction between TIAR RRM2 and the WNV 3'(-) SL RNA was 1.5 x 10(-8), while that for TIA-1 RRM2 was 1.12 x 10(-7). WNV growth was less efficient in murine TIAR knockout cell lines than in control cells. This effect was not observed for two other types of RNA viruses or two types of DNA viruses. Reconstitution of the TIAR knockout cells with TIAR increased the efficiency of WNV growth, but neither the level of TIAR nor WNV replication was as high as in control cells. These data suggest a functional role for TIAR and possibly also for TIA-1 during WNV replication.

Topics: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Cell Line; Conserved Sequence; Cricetinae; DNA, Complementary; Evolution, Molecular; Gene Deletion; Kinetics; Membrane Proteins; Mice; Molecular Sequence Data; Nucleic Acid Conformation; Protein Structure, Tertiary; Proteins; RNA, Complementary; RNA, Viral; RNA-Binding Proteins; Sequence Homology, Amino Acid; T-Cell Intracellular Antigen-1; Virus Replication; West Nile virus

PubMed: 12414941
DOI: 10.1128/jvi.76.23.11989-12000.2002

Authors: Sarah E Giron, Sandra K Bordi

BACKGROUND

Nurse anesthesia in California has been instrumental in shaping Certified Registered Nurse Anesthetist (CRNA) practice nationally, but to date, no workforce data has been published on this group of Advanced Practice Registered Nurses.

PURPOSE

The purpose of this workforce study was to explore the demographic information, education, and practice patterns of CRNAs working in California.

METHODS

Survey methodology was conducted to gather workforce data from a sample of licensed CRNAs working in California.

DISCUSSION

California CRNAs provide anesthesia services in very diverse settings among different anesthesia delivery models, including independent practice. CRNAs practice in a majority of California counties and are the sole anesthesia providers for four underserved counties.

CONCLUSION

The CRNA workforce in California is comparatively young, highly educated, and desires more education. CRNAs improve access to care for California patients; however, more ethnically diverse CRNAs and CRNAs with Non-Surgical Pain Management Certification are needed.

Topics: Humans; Nurse Anesthetists; RNA, Complementary; Workforce; Surveys and Questionnaires; California

PubMed: 37827009
DOI: 10.1016/j.outlook.2023.102057

Authors: Carla E Hegeman, Christine P Halter, Thomas G Owens...

The expression of angiosperm chloroplast genes is modified by C-to-U RNA editing. The mechanism for recognition of the approximately 30 C targets of editing is not understood. There is no single consensus sequence surrounding editing sites, though sites can be grouped into small 'clusters' of two to five sites exhibiting some sequence similarity. While complementary RNA that guides nucleotides for alteration has been detected in other RNA modification systems, it is not known whether complementary RNA is involved in chloroplast editing site recognition. We investigated the effect of expressing RNA antisense to the sequences -20 to +6 surrounding the RpoB-2 C target of editing, which is a member of a cluster that includes the PsbL-1 and Rps14-1 sites. Previous experiments had shown that chloroplast rpoB transgene transcripts carrying only these 27 nt were edited in vivo at the proper C. Though transcripts carrying sequences -31 to +60 surrounding the RpoB-2 sites were edited in chloroplast transgenic plants, transcripts carrying the -31 to +62 region followed by the 27 nt complementary region were not edited at all. In contrast, a similar construct, in which the C target as well as the preceding and subsequent nucleotides were mismatched within the 27 nt region, was efficiently edited. The presence of any of the four transgenes carrying RpoB-2 sequences in sense and/or antisense orientation resulted in reduced editing at the PsbL-1 site. Chloroplast transgenic plants expressing the three different antisense RNA constructs exhibited abnormal growth and development, though plants expressing the 92 nt sense transcripts were phenotypically normal.

Topics: Chloroplasts; Cytosine; DNA-Directed RNA Polymerases; Gene Expression Regulation, Plant; Phenotype; Photosynthesis; Plants, Genetically Modified; RNA Editing; RNA, Antisense; RNA, Complementary; RNA, Messenger; Nicotiana; Transgenes

PubMed: 15755747
DOI: 10.1093/nar/gki286

Authors: Peter H Hagedorn, Malene Pontoppidan, Tina S Bisgaard...

Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets. This study design allowed separation of intended- and off-target effects on the transcriptome for each gapmer. Next, we used sequence analysis to identify possible partially complementary binding sites among the potential off-targets, and validated these by measurements of melting temperature and RNase H-cleavage rates. Generally, our observations were as expected in that fewer mismatches or bulges in the gapmer/transcript duplexes resulted in a higher chance of those duplexes being effective substrates for RNase H. Follow-up experiments in mice and cells show, that off-target effects can be mitigated by ensuring that gapmers have minimal sequence complementarity to any RNA besides the intended target, and that they do not have exaggerated binding affinity to the intended target.

Topics: Animals; Apolipoproteins B; Binding Sites; Cells, Cultured; Female; Genetic Therapy; Liver; Mice; Mice, Inbred C57BL; Nucleic Acid Heteroduplexes; Oligonucleotides, Antisense; Proprotein Convertase 9; RNA, Complementary; RNA, Messenger; Ribonuclease H

PubMed: 29790953
DOI: 10.1093/nar/gky397

Authors: L del Río, C Martínez, E Domingo...

Influenza virus-specific RNA has been synthesized in vitro, using cytoplasmic or microsomal fractions of influenza virus-infected MDCK cells. The RNA polymerase activity was stimulated 5-30 times by priming with ApG. About 20-30% of the product was polyadenylated. Most of the in vitro product was of positive polarity, as shown by hybridization to strand specific probes and by T1 fingerprinting of the poly(A)+ and poly(A)- RNA segments encoding haemagglutinin and nucleoprotein. The size of poly(A)- RNA segments, determined on sequencing gels, was indistinguishable from that of virion RNA, whereas poly(A)+ RNA segments contain poly(A) tails approximately 50 nucleotides long. The size of in vitro synthesized RNA segments was also determined by gel electrophoresis of S1-treated double-stranded RNAs, obtained by hybridization of poly(A)+ or poly(A)- RNA fractions with excess of unlabelled virion RNA. The results of these experiments indicate that poly(A)- RNA contains full-length complementary RNA. This conclusion is further substantiated by the presence of additional oligonucleotides in the T1 fingerprints of in vitro synthesized poly(A)- haemagglutinin or nucleoprotein RNA, selected by hybridization to cloned DNA probes corresponding to the 3' termini of the genes.

Topics: Animals; Cells, Cultured; DNA-Directed RNA Polymerases; Dogs; Genes, Viral; Influenza A virus; Poly A; RNA; RNA, Complementary; RNA, Messenger; RNA, Viral; Transcription, Genetic

PubMed: 2410253
DOI: 10.1002/j.1460-2075.1985.tb02342.x

Authors: Genoveva A Nacheva, Alfredo Berzal-Herranz

The transcription patterns of 64 linear double stranded DNA templates obtained with T7 RNA polymerase were investigated. These templates consisted of 17 nucleotide-long sequences under the control of the minimal bacteriophage T7 promoter and represented all possible combinations of nucleotides at positions +8, +10 and +11. Two clearly distinct types of template were identified, which produced the range of transcription patterns observed: (a) those that yielded 17-nucleotide-long RNA as the only detectable run-off product (only 15% of the total), and (b) templates that in addition to the expected full-length RNA, produced other products longer than 17 nucleotides. Self-complementarity analysis of the expected run-off transcripts showed that those obtained from the first type of template were able to form stable intermolecular duplexes with non-base-paired 3'-ends. However, the second type of template yielded RNAs able to generate energetically favorable intermolecular duplexes with 3'-end complementarity, therefore yielding an RNA-primed RNA-template. The gel-purified 17-nucleotide-long RNAs transcribed from the latter yielded longer products when incubated under in vitro transcription conditions in the absence of a DNA template. No extension was observed when assaying the 17-nucleotide RNA products resulting from the first type of template. We observed that just a single nucleotide change within the DNA template could convert the RNA product from an RNA-primed template into a nonextendible dimer thus leading to a drastic switch of the 17-nucleotide product yield from less than 10% to 100%. Further, two type B DNA templates were extended by two nucleotides at the 3'-end, to produce RNA transcripts theoretically unable to form 3'-end base-paired duplexes. The full-length products of these modified DNA templates were found to be nonextendible by T7 RNA polymerase under the standard in vitro transcription conditions.

Topics: Base Pairing; Catalysis; Cell-Free System; DNA; DNA-Directed RNA Polymerases; Gene Library; RNA; RNA, Catalytic; RNA, Complementary; Substrate Specificity; Templates, Genetic; Transcription, Genetic; Viral Proteins

PubMed: 12654001
DOI: 10.1046/j.1432-1033.2003.03510.x