| 1. |
- Baranowska, Izabella, et al.
(författare)
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Sensory ataxic neuropathy in golden retriever dogs is caused by a deletion in the mitochondrial tRNATyr gene
- 2009
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Ingår i: PLoS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 5:5, s. e1000499-
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Tidskriftsartikel (refereegranskat)abstract
- Sensory ataxic neuropathy (SAN) is a recently identified neurological disorder in golden retrievers. Pedigree analysis revealed that all affected dogs belong to one maternal lineage, and a statistical analysis showed that the disorder has a mitochondrial origin. A one base pair deletion in the mitochondrial tRNA(Tyr) gene was identified at position 5304 in affected dogs after re-sequencing the complete mitochondrial genome of seven individuals. The deletion was not found among dogs representing 18 different breeds or in six wolves, ruling out this as a common polymorphism. The mutation could be traced back to a common ancestor of all affected dogs that lived in the 1970s. We used a quantitative oligonucleotide ligation assay to establish the degree of heteroplasmy in blood and tissue samples from affected dogs and controls. Affected dogs and their first to fourth degree relatives had 0-11% wild-type (wt) sequence, while more distant relatives ranged between 5% and 60% wt sequence and all unrelated golden retrievers had 100% wt sequence. Northern blot analysis showed that tRNA(Tyr) had a 10-fold lower steady-state level in affected dogs compared with controls. Four out of five affected dogs showed decreases in mitochondrial ATP production rates and respiratory chain enzyme activities together with morphological alterations in muscle tissue, resembling the changes reported in human mitochondrial pathology. Altogether, these results provide conclusive evidence that the deletion in the mitochondrial tRNA(Tyr) gene is the causative mutation for SAN.
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| 2. |
- Darfeuille, Fabien, et al.
(författare)
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An antisense RNA inhibits translation by competing with standby ribosomes
- 2007
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 26:3, s. 381-392
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Tidskriftsartikel (refereegranskat)abstract
- Most antisense RNAs in bacteria inhibit translation by competing with ribosomes for translation initiation regions (TIRs) on nascent mRNA. We propose a mechanism by which an antisense RNA inhibits translation without binding directly to a TIR. The tisAB locus encodes an SOS-induced toxin, and IstR-1 is the antisense RNA that counteracts toxicity. We show that full-length tisAB mRNA (+1) is translationally inactive and endonucleolytic processing produces an active mRNA (+42). IstR-1 binding inhibits translation of this mRNA, and subsequent RNase III cleavage generates a truncated, inactive mRNA (+106). In vitro translation, toeprinting, and structure mapping suggest that active, but not inactive, tisAB mRNAs contain an upstream ribosome loading or “standby” site. Standby binding is required for initiation at the highly structured tisB TIR. This may involve ribosome sliding to a transiently open tisB TIR. IstR-1 competes with ribosomes by base pairing to the standby site located 100 nucleotides upstream.
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| 3. |
- Djupedal, Ingela, et al.
(författare)
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Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA
- 2009
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 28:24, s. 3832-3844
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Tidskriftsartikel (refereegranskat)abstract
- The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 50-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1D cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity. The EMBO Journal (2009) 28, 3832-3844. doi: 10.1038/emboj.2009.351; Published online 26 November 2009
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| 4. |
- Ivanova, Natalia, et al.
(författare)
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Structure probing of tmRNA in distinct stages of trans-translation
- 2007
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 13:5, s. 713-722
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Tidskriftsartikel (refereegranskat)abstract
- Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helperprotein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work weused lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery atdifferent steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reactedwith Ala-tmRNA EF-Tu GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was d dprobed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we alsoanalyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. Weobserved some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNAconformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA isin complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.
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| 5. |
- Lindell, Magnus, et al.
(författare)
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Lead(II) cleavage analysis of RNase P RNA in vivo.
- 2005
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Ingår i: RNA. - 1355-8382. ; 11:9, s. 1348-54
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Tidskriftsartikel (refereegranskat)abstract
- The overall conformation of M1 RNA, the catalytic RNA subunit of RNase P in Escherichia coli, was analyzed in vivo and, in the presence of the C5 protein subunit, in vitro by lead(II) acetate probing. The partial cleavage patterns obtained are congruent with previous structure mapping performed in vitro. Most of the known major and minor cleavages in M1 RNA were supported and could be mapped onto a secondary structure model. The data obtained indicate that C5 has only minor effects on the overall structure of the RNA subunit. The similar cleavage patterns obtained in vitro and in vivo furthermore suggest that the intracellular environment does not greatly alter the overall conformation of M1 RNA within the holoenzyme complex. Moreover, our data indicate that M1 RNA in vivo is present in at least two states-the major fraction is bound to tRNA substrates and a minor fraction is substrate free. Finally, both in this and previous work we found that lead(II) probing data from in vivo experiments conducted on longer RNAs (tmRNA and M1 RNA) generally gives superior resolution compared to parallel in vitro experiments. This may reflect the absence of alternative conformers present in vitro and the more natural state of these RNAs in the cell due to proper, co-transcriptional folding pathways and possibly the presence of RNA chaperones.
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| 6. |
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| 7. |
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| 8. |
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| 9. |
- Udekwu, Klas I, et al.
(författare)
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Hfq-dependent regulation of OmpA synthesis is mediated by an an-tisense RNA.
- 2005
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Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 19:19, s. 2355-2366
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Tidskriftsartikel (refereegranskat)abstract
- This paper shows that the small RNA MicA (previously SraD) is an antisense regulator of ompA in Escherichia coli. MicA accumulates upon entry into stationary phase and down-regulates the level of ompA mRNA. Regulation of ompA (outer membrane protein A), previously attributed to Hfq/mRNA binding, is lost upon deletion of the micA gene, whereas overexpression of MicA inhibits the synthesis of OmpA. In vitro, MicA binds to the ompA mRNA leader. Enzymatic and chemical probing was used to map the structures of MicA, the ompA mRNA leader, and the complex formed upon binding. MicA binding generates a footprint across the ompA Shine-Dalgarno sequence, consistent with a 12 + 4 base-pair interaction, which is additionally supported by the effect of mutations in vivo and by bioinformatics analysis of enterobacterial micA/ompA homolog sequences. MicA is conserved in many enterobacteria, as is its ompA target site. In vitro toeprinting confirmed that binding of MicA specifically interferes with ribosome binding. We propose that MicA, when present at high levels, blocks ribosome binding at the ompA translation start site, which—in line with previous work—secondarily facilitates RNase E cleavage and subsequent mRNA decay. MicA requires the presence of the Hfq protein, although the mechanistic basis for this remains unclear.
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| 10. |
- Udekwu, Klas I., et al.
(författare)
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Sigma E controls biogenesis of the antisense RNA MicA
- 2007
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 35:4, s. 1279-1288
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Tidskriftsartikel (refereegranskat)abstract
- Adaptation stress responses in the Gram-negative bacterium Escherichia coli and its relatives involve a growing list of small regulatory RNAs (sRNAs). Previous work by us and others showed that the antisense RNA MicA downregulates the synthesis of the outer membrane protein OmpA upon entry into stationary phase. This regulation is Hfq-dependent and occurs by MicA-dependent translational inhibition which facilitates mRNA decay. In this article, we investigate the transcriptional regulation of the micA gene. Induction of MicA is dependent on the alarmone ppGpp, suggestive of alternative σ factor involvement, yet MicA accumulates in the absence of the general stress/stationary phase σS. We identified stress conditions that induce high MicA levels even during exponential growth - a phase in which MicA levels are low (ethanol, hyperosmolarity and heat shock). Such treatments are sensed as envelope stress, upon which the extracytoplasmic sigma factor σE is activated. The strict dependence of micA transcription on σE is supported by three observations. Induced overexpression of σE increases micA transcription, an ΔrpoE mutant displays undetectable MicA levels and the micA promoter has the consensus σE signature. Thus, MicA is part of the σE regulon and downregulates its target gene, ompA, probably to alleviate membrane stress.
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| 11. |
- Unoson, Cecilia, et al.
(författare)
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A small SOS-induced toxin is targeted against the inner membrane in Escherichia coli
- 2008
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 70:1, s. 258-70
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Tidskriftsartikel (refereegranskat)abstract
- We previously reported on an SOS-induced toxin, TisB, in Escherichia coli and its regulation by the RNA antitoxin IstR-1. Here, we addressed the mode of action of TisB. By placing the tisB reading frame downstream of a controllable promoter on a plasmid, toxicity could be analysed in the absence of the global SOS response. Upon induction of TisB, cell growth was inhibited and plating efficiency decreased rapidly. The onset of toxicity correlated with a drastic decrease in transcription, translation and replication rates. Cellular RNA was degraded, but in vitro experiments showed that TisB did not affect translation or transcription directly. Thus, these effects are downstream consequences of membrane damage: TisB is predicted to be hydrophobic and membrane spanning, and Western analyses demonstrated that this peptide was strictly localized to the cytoplasmic membrane fraction. Membrane damage and cell killing under tisB multicopy expression are also seen by live/death staining and the formation of ghost cells. This is reminiscent of another toxin, Hok of plasmid R1, which also targets the membrane. The biological significance of the istR/tisB locus is still elusive; deletion of the entire locus gave no fitness phenotype in competition experiments.
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| 12. |
- Unoson, Cecilia, et al.
(författare)
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Dealing with stable structures at ribosome binding sites : bacterial translation and ribosome standby.
- 2007
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Ingår i: RNA Biology. - 1547-6286. ; 4:3, s. 113-117
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial ribosomes have great difficulties to initiate translation on stable structures within mRNAs. Translational coupling and induced structure changes are strategies to open up inhibitory RNA structures encompassing ribosome binding sites (RBS). There are, however, mRNAs in which stable structures are not unfolded, but that are nevertheless efficiently initiated at high rates. de Smit and van Duin(1) proposed a "ribosome standby" model to theoretically solve this paradox: the 30S ribosome binds nonspecifically to an accessible site on the mRNA (standby site), waiting for a transient opening of a stable RBS hairpin. Upon unfolding, the 30S subunit relocates to form a productive initiation complex. Recent reports have provided experimental support for this model. This review will describe and compare two different flavors of standby sites, their properties, and their likely implications. We also discuss the possibility that ribosome standby may be a more general strategy to obtain high translation rates.
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| 13. |
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| 14. |
- Vogel, Jörg, et al.
(författare)
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Target identification of small noncoding RNAs in bacteria
- 2007
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Ingår i: Current Opinion in Microbiology. - : Elsevier BV. - 1369-5274 .- 1879-0364. ; 10:3, s. 262-270
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Forskningsöversikt (refereegranskat)abstract
- Small noncoding RNAs have been discovered at a staggering rate in Escherichia coli and many other bacteria. Most of the sRNAs of known function regulate gene expression by binding to specific mRNAs or proteins. Given the scores of sRNAs of unknown function, the identification of their cellular targets has become urgent. Here, we review the diverse strategies that have been used to identify and validate bacterial sRNA targets. These include the pulse-expression of sRNAs followed by global transcriptome analysis (microarrays), new biocomputational prediction algorithms, and novel gfp reporter gene fusions to validate candidate target gene regulation.
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| 15. |
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| 16. |
- Wagner, Gerhart E. H.
(författare)
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Kill the messenger : bacterial antisense RNA promotes mRNA decay.
- 2009
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Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 16:8, s. 804-806
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial antisense RNAs target translation initiation regions (TIR s) to compete with ribosome binding, thus repressing translation and—secondarily—causing degradation of the naked mRNA. A new study reports on an antisense RNA that directly accelerates mRNA decay by targeting a sequence deep within the coding region, far downstream of the TIR.
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| 17. |
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| 18. |
- Wagner, Gerhart E. H., et al.
(författare)
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Small regulatory RNAs in bacteria
- 2006
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Ingår i: Small RNAs. - Berlin, Heidelberg : Springer Verlag Berlin Heidelberg. - 9783540281306 ; , s. 1-29
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- In recent years, small regulatory RNAs have been discovered at a staggering rate both in prokaryotes and eukaryotes. By now it is clear that post-transcriptional regulation of gene expression mediated by such RNAs is the rule rather than—as previously believed—the exception. In this chapter, we focus on small RNAs (sRNAs) encoded by bacterial chromosomes. The strategies for their discovery, their biological roles, and their mechanisms of action are discussed. Even though the number of well-characterized sRNAs in, for example, the best studied model enterobacterium Escherichia coli, is still small, the emerging pattern suggests that antisense mechanisms predominate. In terms of their roles in bacterial physiology, most of these RNAs appear to be involved in stress response regulation. Some other examples indicate functions in regulation of virulence. Two aspects of sRNA-mediated control arising from recent observations are addressed as well. Firstly, some sRNAs need proteins (notably Hfq) as helpers in their antisense activities—at this point the reason for this requirement is not understood. Secondly, only limited sequence complementarity is generally observed in antisense–target RNA pairs. This raises the fundamental question of how specific recognition is accomplished, and what the structure/sequence determinants for rapid and productive interaction are.
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