RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymes
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ABSTRACT:
PROVIDER: PRJEB52899 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR9741130_1.fastq.gz | Fastqsanger.gz | |||
| ERR9741130_2.fastq.gz | Fastqsanger.gz | |||
| ERR9741140_1.fastq.gz | Fastqsanger.gz | |||
| ERR9741140_2.fastq.gz | Fastqsanger.gz | |||
| ERR9741131_1.fastq.gz | Fastqsanger.gz |
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Project description:Self-cleaving ribozymes are RNA molecules that catalyze the cleavage of their own phosphodiester backbones. These ribozymes are found in all domains of life and are also a tool for biotechnical and synthetic biology applications. Self-cleaving ribozymes are also an important model of sequence-to-function relationships for RNA because their small size simplifies synthesis of genetic variants and self-cleaving activity is an accessible readout of the functional consequence of the mutation. Here, we used a high-throughput experimental approach to determine the relative activity for every possible single and double mutant of five self-cleaving ribozymes. From this data, we comprehensively identified non-additive effects between pairs of mutations (epistasis) for all five ribozymes. We analyzed how changes in activity and trends in epistasis map to the ribozyme structures. The variety of structures studied provided opportunities to observe several examples of common structural elements, and the data was collected under identical experimental conditions to enable direct comparison. Heatmap-based visualization of the data revealed patterns indicating structural features of the ribozymes including paired regions, unpaired loops, non-canonical structures, and tertiary structural contacts. The data also revealed signatures of functionally critical nucleotides involved in catalysis. The results demonstrate that the data sets provide structural information similar to chemical or enzymatic probing experiments, but with additional quantitative functional information. The large-scale data sets can be used for models predicting structure and function and for efforts to engineer self-cleaving ribozymes.
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Project description:More than 98% of the mammalian genome is noncoding and approximately half is made up of transposable elements. One of the most abundant is the short interspersed nuclear elements (SINE). Among the million copies of SINEs, B2 accounts for ~350,000 in the mouse genome and have garnered special interest because of emerging roles in gene regulation. Our recent work demonstrated that B2 RNA normally binds stress genes to retard transcription elongation. Though epigenetically silenced, B2s become massively upregulated during thermal stress. Specifically, an interaction between B2 RNA and the Polycomb protein, EZH2, results in cleavage of B2 RNA, release of B2 RNA from RNA Polymerase II, and activation of the stress genes. Although an established RNA-binding protein and histone methyltransferase, EZH2 is not known to be a nuclease. Here, we provide evidence for the surprising conclusion that B2 is a self-cleaving RNA. Contact with EZH2 accelerates cleavage rate by >100-fold, suggesting that EZH2 may assist cleavage as an RNA chaperone. Modification-interference analysis demonstrate that phosphorothioate changes at A and C nucleotides can substitute for EZH2’s function. B2 mutagenesis indicate that nucleotides around positions 45-55 and 100-101 are critical for cleavage reaction. Finally, we demonstrate that another family of SINEs, the ALU elements produce also a self-cleaving RNA. ALUs are intrinsically more auto-reactive than B2s. We propose that the B2/ALU SINEs are a new class of ribozymes whose activity is accelerated by EZH2.
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Project description:During very early embryogenesis of fast cleaving embryos DNA synthesis is short and surveillance mechanisms preserving genome integrity are inefficient implying the possible generation of mutations. To explore this possibility, we have analyzed mutagenesis in Xenopus laevis and Drosophila melanogaster embryos. We report the occurrence of a nucleus-to-cytoplasmic ratio-sensitive high mutation rate in Xenopus. We also show that mutagenesis is dependent upon the master regulator of translesion DNA synthesis (TLS) Rad18 and alleviated by the mismatch repair system. Unexpectedly, using separation of function mutants, we observed a homology-directed repair contribution of Rad18 in reducing the mutation load. While in Drosophila no Rad18 orthologs could be identified, genetic invalidation of TLS in the pre-blastoderm embryo resulted in reduction of both the hatching rate and Single Nucleotide Variations on specific chromosome regions in adult flies. Altogether these findings indicate that in the very early Xenopus and Drosophila embryos TLS strongly contributes to the high mutation rate. This may constitute a previously unforeseen source of genetic diversity contributing to the polymorphisms of each individual with implications for genome evolution and species adaptation.
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Project description:PPIs play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spec-based proteomics overcomes challenges typically associated with other methods, and has quickly become the current state-of-the-art in the field. Nevertheless, tight control of proximity labeling enzymatic acitivty a,d expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleavage mutant to allow the use of the POI in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we build a GOlden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A split-link design, we applied it to identify PPIs of GR, NCAP, and NSP7 by TurboID proximity labeling proteomics. Our results demisntrate that our T2A split-link provides an opportune control that builds upon previously established control requirements in the field.
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