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Structural determinants of inverted Alu-mediated backsplicing revealed by -MaP and -JuMP

ABSTRACT: Biogenesis of circular RNA usually involves a backsplicing reaction where the downstream donor site is ligated to the upstream acceptor site by the spliceosome. For this reaction to occur, it is hypothesized that these two sites must be in proximity. Inverted repeat sequences, such as Alu elements, in the upstream and downstream introns are predicted to base-pair and represent one mechanism for inducing proximity. Here, we investigate the pre-mRNA structure of the human HIPK3 gene, specifically exon 2, which forms a circular RNA via backsplicing. We leverage multiple chemical probing techniques, including the recently developed SHAPE-JuMP strategy, to characterize both secondary and tertiary interactions in the pre-mRNA that govern backsplicing. Our data confirm that the antisense Alu elements, AluSz(-) and AluSq2(+) in the upstream and downstream introns, form a highly-paired interaction. Circularization requires formation of long-range Alu-mediated base pairs but does not require the full-length AluSq2(+). In addition to confirming long-range base pairs, our SHAPE-JuMP data also identified multiple long-range interactions between non-pairing nucleotides. Genome wide analysis of inverted repeats flanking circular RNAs confirm that their presence favors circularization, but the overall effect is modest. Together these results suggest that secondary structure considerations alone cannot fully explain backsplicing and that additional interactions are key components of the mechanism.

ORGANISM(S): Homo sapiens

PROVIDER: GSE283716 | GEO | 2024/12/13

REPOSITORIES: GEO

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Project description:Transposable elements are viewed as ‘selfish genetic elements’, yet they contribute to gene regulation and genome evolution in diverse ways. More than half of the human genome consists of transposable elements. With over 1 million insertions, Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome. Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability. Alu elements are the main targets of the RNA-editing enzyme ADAR and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC, but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant nuclear RNA helicase, binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to higher levels of circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post-transcriptional regulation of gene expression. This SuperSeries is composed of the SubSeries listed below.

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Structural determinants of inverted Alu-mediated backsplicing revealed by -MaP and -JuMP

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