🧬 PubMed RNA Editing Daily Digest

RNA-binding protein (RBP)-RNA interactions are fundamental for gene regulation and cellular homeostasis. Ataxin-2 is an RBP that has been shown to play an instrumental role in pathophysiological processes by binding to mRNA. Methods such as RNA immunoprecipitation (RIP), cross-linking immunoprecipitation (CLIP), and their variants can be used to study the interactions between Ataxin-2 and its targets, although their high sample requirements and labor-intensive workflows can limit their widespread use. RNA editing-based approaches, such as targets of RBPs identified by editing (TRIBE), provide effective alternatives. TRIBE enables transcriptome-wide identification of RBP targets by inducing site-specific adenosine-to-inosine (A-to-I) editing, which is subsequently detected through high-throughput RNA sequencing in both in vivo and in vitro systems. Compared to in vivo models, cell lines offer a rapid and flexible experimental design.

ADAR RNA editing enzymes deaminate selected adenosines to inosines in dsRNA. In Drosophila, inosine in dsRNA inhibits cleavage by Dcr2 and some ADAR proteins contribute an additional, editing-independent inhibition. The Drosophila AdarG isoform, in particular, has been proposed to inhibit HP1-mediated heterochromatin silencing of repetitive sequences initiated by specific dsRNAs. To address the functions of AdarG, we overexpressed it from new UAS-Adar lines, under the control of a temperature-regulated Act5Cts-GAL4 driver. Overexpression of the adult AdarG isoform or catalytically inactive AdarE374A led to larval lethality with some escaper pupae that show an ecdysone-related, head eversion defect. This indicates an editing-independent effect of high Adar expression. Pupae show aberrantly elevated innate immune and early ecdysone gene transcript expression and no flies eclose. RNAi knockdown of Ecdysone Receptor A (EcRA) or increased expression of the histone H3K9me2,3-associated HP1 protein partially rescue AdarG overexpression defects and normalize gene expression in rescued progeny flies. In other reports, Drosophila mutants with reduced HP1, or egg (SetDB1), Su(var)3-9 double mutants with reduced histone H3K9me2,3 also produce larvae with ecdysone-related and innate immune defects. We show that overexpressed AdarG inhibits histone H3K9me-mediated epigenetic silencing through an editing-independent effect, most likely at the dsRNA/Dcr2/Ago2 initiation stage.

The power of CRISPR/Cas9 mediated genome editing has been harnessed in different facets of entomological research, particularly useful in developing genetic pest management strategies. The edits thus obtained are robust and results in a loss-of-function of the target gene. Recently the development of newer editing approach called Prime editing is yet another addition in the insect editing tool-box. In this regard, the prime editing offers a transformative approach to precise genome manipulation by enabling targeted insertions, deletions, and nucleotide substitutions without double-strand break or donor template. While its application has been explored in mammalian system and plants, its deployment through the delivery of ribonucleoprotein complex (RNP) has been demonstrated for the first-time in the globally significant pest, Spodoptera frugiperda. Using a Cas9 (H840A)-reverse transcriptase fusion protein (PE2) and a customized prime editing guide RNA (pegRNA), we targeted exon 3 of the Tryptophan 2,3-dioxygenase (SfTO) gene to introduce a premature stop codon. Recombinant PE2 protein was expressed in E. coli, purified, and validated functionally through RT-PCR. The Ribonucleoprotein complex was microinjected into G