| 51. |
- Imbert, Arthur, et al.
(författare)
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FISH-quant v2 : a scalable and modular tool for smFISH image analysis
- 2022
-
Ingår i: RNA. - : COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. - 1355-8382 .- 1469-9001. ; 28:6, s. 786-795
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Tidskriftsartikel (refereegranskat)abstract
- Regulation of RNA abundance and localization is a key step in gene expression control. Single-molecule RNA fluorescence in situ hybridization (smFISH) is a widely used single-cell-single-molecule imaging technique enabling quantitative studies of gene expression and its regulatory mechanisms. Today, these methods are applicable at a large scale, which in turn come with a need for adequate tools for data analysis and exploration. Here, we present FISH-quant v2, a highly modular tool accessible for both experts and non-experts. Our user-friendly package allows the user to segment nuclei and cells, detect isolated RNAs, decompose dense RNA clusters, quantify RNA localization patterns and visualize these results both at the single-cell level and variations within the cell population. This tool was validated and applied on large-scale smFISH image data sets, revealing diverse subcellular RNA localization patterns and a surprisingly high degree of cell-to-cell heterogeneity.
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| 52. |
- Innocenti, Nicolas, 1986-, et al.
(författare)
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Whole-genome mapping of 5′ RNA ends in bacteria by tagged sequencing: a comprehensive view in Enterococcus faecalis
- 2015
-
Ingår i: RNA. - : RNA Society. - 1355-8382 .- 1469-9001.
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Tidskriftsartikel (refereegranskat)abstract
- Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first snapshot of transcriptional organizations in this bacterium, we used a modified RNA-seq approach enabling to discriminate primary from processed 5' RNA ends. We also validated our approach by confirming known features in Escherichia coli. We mapped 559 transcription start sites (TSSs) and 352 processing sites (PSSs) in E. faecalis. A blind motif search retrieved canonical features of SigA-and SigN-dependent promoters preceding transcription start sites mapped. We discovered 85 novel putative regulatory RNAs, small-and antisense RNAs, and 72 transcriptional antisense organizations. Presented data constitute a significant insight into bacterial RNA landscapes and a step toward the inference of regulatory processes at transcriptional and post-transcriptional levels in a comprehensive manner.
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| 53. |
- Ivanova, Natalia, et al.
(författare)
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Structure probing of tmRNA in distinct stages of trans-translation
- 2007
-
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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| 54. |
- Johansson, Marcus J O, et al.
(författare)
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Dual function of the tRNA(m(5)U54)methyltransferase in tRNA maturation
- 2002
-
Ingår i: RNA. - 1355-8382 .- 1469-9001. ; 8:3, s. 324-335
-
Tidskriftsartikel (refereegranskat)abstract
- A 5-methyluridine (m(5)U) residue at position 54 is a conserved feature of bacterial and eukaryotic tRNAs. The methylation of U54 is catalyzed by the tRNA(m5U54)methyltransferase, which in Saccharomyces cerevisiae is encoded by the nonessential TRM2 gene. In this study, we identified four different strains with mutant forms of tRNA(Ser)CGA. The absence of the TRM2 gene in these strains decreased the stability of tRNA(Ser)CGA and induced lethality. Two alleles of TRM2 encoding catalytically inactive tRNA(m5U54)methyltransferases were able to stabilize tRNA(Ser)CGA in one of the mutants, revealing a role for the Trm2 protein per se in tRNA maturation. Other tRNA modification enzymes interacting with tRNA(Ser)CGA in the maturation process, such as Pus4p, Trm1 p, and Trm3p were essential or important for growth of the tRNA(Ser)CGA mutants. Moreover, Lhp1p, a protein binding RNA polymerase III transcripts, was required to stabilize the mutant tRNAs. Based on our results, we suggest that tRNA modification enzymes might have a role in tRNA maturation not necessarily linked to their known catalytic activity.
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| 55. |
- Kamel, Wael, 1986-, et al.
(författare)
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An Ago2-associated capped transcriptional start site small RNA suppresses adenovirus DNA replication
- 2017
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 23:11, s. 1700-1711
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Tidskriftsartikel (refereegranskat)abstract
- Here we show that the adenovirus major late promoter produces a 31-nucleotide transcriptional start site small RNA (MLP-TSS-sRNA) that retains the 7-methylguanosine (m7G)-cap and is incorporated onto Ago2-containing RNA-induced silencing complexes (RISC) in human adenovirus-37 infected cells. RNA polymerase II CLIP (UV-cross linking immunoprecipitation) experiments suggest that the MLP-TSS-sRNA is produced by promoter proximal stalling/termination of RNA polymerase II transcription at the site of the small RNA 3' end. The MLP-TSS-sRNA is highly stable in cells and functionally active, down-regulating complementary targets in a sequence and dose-dependent manner. The MLP-TSS-sRNA is transcribed from the opposite strand to the adenoviral DNA polymerase and preterminal protein mRNAs, two essential viral replication proteins. We show that the MLP-TSS-sRNA act in trans to reduce DNA polymerase and preterminal protein mRNA expression. As a consequence of this, the MLP-TSS-sRNA has an inhibitory effect on the efficiency of viral DNA replication. Collectively, our results suggest that this novel sRNA may serve a regulatory function controlling viral genome replication during a lytic and/or persistent adenovirus infection in its natural host.
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| 56. |
- Kang, Wenjing, et al.
(författare)
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Survey of 800+data sets from human tissue and body fluid reveals xenomiRs are likely artifacts
- 2017
-
Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 23:4, s. 433-445
-
Tidskriftsartikel (refereegranskat)abstract
- miRNAs are small 22-nucleotide RNAs that can post-transcriptionally regulate gene expression. It has been proposed that dietary plant miRNAs can enter the human bloodstream and regulate host transcripts; however, these findings have been widely disputed. We here conduct the first comprehensive meta-study in the field, surveying the presence and abundances of cross-species miRNAs (xenomiRs) in 824 sequencing data sets from various human tissues and body fluids. We find that xenomiRs are commonly present in tissues (17%) and body fluids (69%); however, the abundances are low, comprising 0.001% of host human miRNA counts. Further, we do not detect a significant enrichment of xenomiRs in sequencing data originating from tissues and body fluids that are exposed to dietary intake (such as liver). Likewise, there is no significant depletion of xenomiRs in tissues and body fluids that are relatively separated from the main bloodstream (such as brain and cerebro-spinal fluids). Interestingly, the majority (81%) of body fluid xenomiRs stem from rodents, which are a rare human dietary contribution but common laboratory animals. Body fluid samples from the same studies tend to group together when clustered by xenomiR compositions, suggesting technical batch effects. Last, we performed carefully designed and controlled animal feeding studies, in which we detected no transfer of plant miRNAs into rat blood, or bovine milk sequences into piglet blood. In summary, our comprehensive computational and experimental results indicate that xenomiRs originate from technical artifacts rather than dietary intake.
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| 57. |
- Karlsson, H, et al.
(författare)
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A robust and versatile method for production and purification of large-scale RNA samples for structural biology
- 2020
-
Ingår i: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 26:8, s. 1023-1037
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Tidskriftsartikel (refereegranskat)abstract
- Recent findings in genome-wide transcriptomics revealed that RNAs are involved in almost every biological process, across all domains of life. The characterization of native RNAs of unknown function and structure is particularly challenging due to their typical low abundance in the cell and the inherent sensitivity toward ubiquitous RNA degrading enzymes. Therefore, robust in vitro synthesis and extensive work-up methods are often needed to obtain samples amenable for biochemical, biophysical, and structural studies. Here, we present a protocol that combines the most recent advances in T7 in vitro transcription methodology with reverse phase ion pairing and ion exchange HPLC purification of RNAs for the production of yield-optimized large-scale samples. The method is easy to follow, robust and suitable for users with little or no experience within the field of biochemistry or chromatography. The complete execution of this method, for example, for production of isotopically labeled NMR samples, can be performed in less than a week.
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| 58. |
- Kirsebom, Leif A.
(författare)
-
Sidney Altman : A brief memoir
- 2022
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Ingår i: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 28:11, s. 1404-1405
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Tidskriftsartikel (populärvet., debatt m.m.)
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| 59. |
- Klaue, Y, et al.
(författare)
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Biochemical analysis and scanning force microscopy reveal productive and non-productive ADAR2 binding to RNA substrates
- 2003
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 9:7, s. 839-846
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Tidskriftsartikel (refereegranskat)abstract
- Scanning force microscopy (SFM) can be used to image biomolecules at high resolution. Here we demonstrate that single-molecule analysis by SFM complements biochemical data on RNA protein binding and can provide information that cannot be obtained by the usual biochemical methods. We have used this method to study the interaction between the RNA editing enzyme ADAR2 and RNA transcripts containing selective and nonselective editing sites. The natural selectively edited R/G site from glutamate receptor subunit B (GluR-B) was inserted into an RNA backbone molecule consisting of a completely double-stranded (ds) central part and incompletely paired ends derived from potato spindle tuber viroid (PSTVd). This molecule was efficiently edited at the R/G site, but promiscuous editing occurred at nonselective sites in the completely double-stranded region. The construct was also used to analyze binding of ADAR2 to wild-type and modified R/G editing sites in relation to binding at other nonselectively edited sites. Editing analysis together with SFM allow us to differentiate between binding and enzymatic activity. ADAR2 has been reported to have a general affinity to dsRNA. However, we show that there is a prominent bias for stable binding at sites selectively edited over other edited sites. On the other hand, promiscuous editing at nonselective sites apparently results from transient binding of the enzyme to the substrate. Furthermore, we find distinct sites with nonproductive binding of the enzyme.
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| 60. |
- Krzywkowski, Tomasz, et al.
(författare)
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Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection
- 2019
-
Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 25:1, s. 82-89
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Tidskriftsartikel (refereegranskat)abstract
- Many approaches exist to detect RNA using complementary oligonucleotides. DNA ligation-based techniques can improve discrimination of subtle sequence variations, but they have been difficult to implement for direct RNA analysis due to the infidelity and inefficiency of most DNA ligases on RNA. In this report, we have systematically studied if ribonucleotide substitutions in padlock probes can provide higher catalytic efficiencies for Chlorella virus DNA ligase (PBCV-1DNA ligase) and T4 RNA ligase 2 (T4Rnl2) on RNA. We provide broad characterization of end-joining fidelity for both enzymes in RNA-templated 3'-OH RNA/5'-pDNA chimeric probe ligation. Both ligases showed increased ligation efficiency toward chimeric substrates on RNA. However, end-joining fidelity of PBCV-1 DNA ligase remained poor, while T4Rnl2 showed a somewhat better end-joining fidelity compared to PBCV-1 DNA ligase. The recently presented invader padlock (iLock) probes overcome the poor end-joining fidelity of PBCV-1 DNA ligase by the requirement of target-dependent 5' flap removal prior to ligation. Here we show that two particular ribonucleotide substitutions greatly improve the activation and ligation rate of chimeric iLock probes on RNA. We characterized the end-joining efficiency and fidelity of PBCV-1 DNA ligase and T4Rnl2 with chimeric iLock probes on RNA and found that both enzymes exhibit high ligation fidelities for single nucleotide poly-morphisms on RNA. Finally, we applied the chimeric probe concept to directly differentiate between human and mouse ACTB mRNA in situ, demonstrating chimeric padlock and iLock probes as superior to their DNA equivalents.
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