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:The landscape of catalytic RNAs in complex eukaryotes remains poorly charted. Although self-cleaving ribozymes are widespread in microbial and viral genomes, their existence and functional roles in humans remain largely unknown. Here, we introduce a generalizable, genome-wide discovery platform that integrates high-throughput signals from two complementary adapter ligation assays—3P-seq and 5OH-seq—to specifically capture RNA fragments bearing cleavage signatures (2′,3′-cyclic phosphate/3′-phosphate and 5′-hydroxyl termini). By applying a dedicated computational scoring algorithm to human genomic data, we systematically identified four previously unrecognized self-cleaving ribozymes. These ribozymes localize to diverse genomic features: an exon of WDFY1, an intron of PLD5 embedded within a repetitive element, an LTR retrotransposon, and the antisense strand of an SYNJ2BP intron. Truncation analyses and mutational profiling defined minimal functional cores of 21–55 nucleotides, and experiment-assisted secondary structure prediction suggests that they adopt simple structural architectures. Despite their short sequences and simple architectures, we demonstrated that these ribozymes possess robust cleavage activity under multiple biochemical conditions. Most importantly, our experimental results indicate that three of them exhibit significant self-cleavage activity within the cellular environment. Our work establishes a powerful strategy for genome-wide mining of self-cleaving ribozymes, expands the catalogue of human ribozymes from 6 to 10, and reveals new layers of functional complexity in the human RNA world.
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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:Genome-Wide Dual-Selection Unveils Novel Self-Cleaving Ribozymes in the Human Genome
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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:Naturally-occurring catalytic RNA molecules — ribozymes — have attracted a great deal of research interest, yet very few of them have been identified in humans. Here, we developed a genome-wide approach to discover self-cleaving ribozymes and identified one naturally-occurring ribozyme in humans. The secondary structure and biochemical properties of this ribozyme indicate that it belongs to yet un-identified class of small self-cleaving ribozymes. The sequence of the ribozyme exhibits a clear evolutionary path from appearance between ~130 and ~65 million years ago (mya) to gain of self-cleavage activity very recently, ~13–10 mya, in the common ancestor of humans, chimpanzees and gorillas. The ribozyme appears to be functional in vivo and is embedded within an lncRNA belonging to the class of very long intergenic non-coding (vlinc) RNAs. The presence of a catalytic RNA enzyme in lncRNA opens a possibility that these transcripts could function by carrying catalytic RNA domains.
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Project description:Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-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 activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to allow the use of the protein-of-interest 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 protein interactions of the glucocorticoid receptor and SARS-CoV-2 nucleocapsid and NSP7 proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control to match expression levels for proximity labeling proteomics.
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