| 1. |
- Alalam, Hanna, et al.
(författare)
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Global SLAM-Seq for accurate mRNA decay determination and identification of NMD targets.
- 2022
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 28:7
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Tidskriftsartikel (refereegranskat)abstract
- Gene expression analysis requires accurate measurements of global RNA degradation rates, earlier problematic with methods disruptive to cell physiology. Recently, metabolic RNA labeling emerged as an efficient and minimally invasive technique applied in mammalian cells. Here, we have adapted SH-Linked Alkylation for the Metabolic Sequencing of RNA (SLAM-Seq) for a global mRNA stability study in yeast using 4-thiouracil pulse-chase labeling. We assign high-confidence half-life estimates for 67.5 % of expressed ORFs, and measure a median half-life of 9.4 min. For mRNAs where half-life estimates exist in the literature, their ranking order was in good agreement with previous data, indicating that SLAM-Seq efficiently classifies stable and unstable transcripts. We then leveraged our yeast protocol to identify targets of the Nonsense-mediated decay (NMD) pathway by measuring the change in RNA half-lives; instead of steady-state RNA level changes. With SLAM-Seq, we assign 580 transcripts as putative NMD targets, based on their measured half-lives in wild-type and upf3Δ mutants. We find 225 novel targets, and observe a strong agreement with previous reports of NMD targets, 61.2 % of our candidates being identified in previous studies. This indicates that SLAM-Seq is a simpler and more economic method for global quantification of mRNA half-lives. Our adaptation for yeast yielded global quantitative measures of the NMD effect on transcript half-lives, high correlation with RNA half-lives measured previously with more technically challenging protocols, and identification of novel NMD regulated transcripts that escaped prior detection.
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| 2. |
- Banijamali, Elnaz, et al.
(författare)
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RNA:RNA interaction in ternary complexes resolved by chemical probing
- 2022
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Ingår i: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 29:3, s. 317-329
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Tidskriftsartikel (refereegranskat)abstract
- RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.
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| 3. |
- Cavallin, I, et al.
(författare)
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HITS-CLIP analysis of human ALKBH8 reveals interactions with fully processed substrate tRNAs and with specific noncoding RNAs
- 2022
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Ingår i: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 28:12, s. 1568-1581
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Tidskriftsartikel (refereegranskat)abstract
- Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U containing tRNAs obtain 5-methoxycarbonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by Alkylation Repair Homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble-U containing tRNAs. In addition, our both approaches revealed ALKBH8 binding to several other types of non-coding RNAs, in particular C/D box snoRNAs.
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| 4. |
- Destefanis, Eliana, et al.
(författare)
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A mark of disease : How mRNA modifications shape genetic and acquired pathologies
- 2021
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Ingår i: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 27:4, s. 367-389
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Forskningsöversikt (refereegranskat)abstract
- RNA modifications have recently emerged as a widespread and complex facet of gene expression regulation. Counting more than 170 distinct chemical modifications with far-reaching implications for RNA fate, they are collectively referred to as the epitranscriptome. These modifications can occur in all RNA species, including messenger RNAs (mRNAs) and noncoding RNAs (ncRNAs). In mRNAs the deposition, removal, and recognition of chemical marks by writers, erasers and readers influence their structure, localization, stability, and translation. In turn, this modulates key molecular and cellular processes such as RNA metabolism, cell cycle, apoptosis, and others. Unsurprisingly, given their relevance for cellular and organismal functions, alterations of epitranscriptomic marks have been observed in a broad range of human diseases, including cancer, neurological and metabolic disorders. Here, we will review the major types of mRNA modifications and editing processes in conjunction with the enzymes involved in their metabolism and describe their impact on human diseases. We present the current knowledge in an updated catalog. We will also discuss the emerging evidence on the crosstalk of epitranscriptomic marks and what this interplay could imply for the dynamics of mRNA modifications. Understanding how this complex regulatory layer can affect the course of human pathologies will ultimately lead to its exploitation toward novel epitranscriptomic therapeutic strategies.
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| 5. |
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| 6. |
- Fromm, Bastian, et al.
(författare)
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Ancient microRNA profiles of 14,300-yr-old canid samples confirm taxonomic origin and provide glimpses into tissue-specific gene regulation from the Pleistocene
- 2021
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 27:3, s. 324-334
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Tidskriftsartikel (refereegranskat)abstract
- DNA sequencing is the current key technology for historic or ancient biological samples and has led to many exciting discoveries in the field of paleogenomics. However, functional insights into tissue identity, cellular composition, or gene regulation cannot be gained from DNA. Recent analyses have shown that, under favorable conditions, RNA can also be sequenced from ancient samples, enabling studies at the transcriptomic and regulatory level. Analyzing ancient RNA data from a Pleistocene canid, we find hundreds of intact microRNAs that are taxonomically informative, show tissue specificity and have functionally predictive characteristics. With an extraordinary age of 14,300 yr, these microRNA sequences are by far the oldest ever reported. The authenticity of the sequences is further supported by (i) the presence of canid/Caniformia-specific sequences that never evolved outside of this Glade, (ii) tissue-specific expression patterns (cartilage, liver, and muscle) that resemble those of modern dogs, and (iii) RNA damage patterns that are clearly distinct from those of fresh samples. By performing computational microRNA-target enrichment analyses on the ancient sequences, we predict microRNA functions consistent with their tissue pattern of expression. For instance, we find a liver-specific microRNA that regulates carbohydrate metabolism and starvation responses in canids. In summary, we show that straightforward paleotranscriptomic microRNA analyses can give functional glimpses into tissue identity, cellular composition, and gene regulatory activity of ancient samples and biological processes that took place in the Pleistocene, thus holding great promise for deeper insights into gene regulation in extinct animals based on ancient RNA sequencing.
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| 7. |
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| 8. |
- Fromm, Bastian, et al.
(författare)
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The limits of human microRNA annotation have been met
- 2022
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 28:6, s. 781-785
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Tidskriftsartikel (refereegranskat)abstract
- Over the last few years, the number of microRNAs in the human genome has become a controversially debated issue. Several publications reported thousands of putative novel microRNAs not included in the curated microRNA gene database MirGeneDB and the repository miRBase. Recently, by using sequencing of ∼300 human tissues and cell lines, the human RNA atlas, an expanded inventory of human RNA annotations, was published, reporting thousands of putative microRNAs. We, the developers of established microRNA prediction tools and hosts of MirGeneDB, raise concerns about the frequently applied prediction and functional validation strategies, briefly discussing the drawbacks of false positive detections. By means of quantifying well-established biogenesis-derived features, we show that the reported novel microRNAs essentially represent false-positives and argue that the human microRNA complement, at about 550 microRNA genes, is already near complete. Output of available tools must be curated as false predictions will misguide scientists looking for biomarkers or therapeutic targets.
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| 9. |
- Holmqvist, Isak, et al.
(författare)
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FLAME: long-read bioinformatics tool for comprehensive spliceome characterization
- 2021
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Ingår i: Rna. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 27:10, s. 1127-1139
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Tidskriftsartikel (refereegranskat)abstract
- Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this work-flow circumvents issues related to inadequate reference annotations and allows for incorporation of short-read sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long noncoding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing data sets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene.
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| 10. |
- Imbert, Arthur, et al.
(författare)
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FISH-quant v2 : a scalable and modular tool for smFISH image analysis
- 2022
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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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