🧬 PubMed RNA Editing Daily Digest

RNA binding proteins (RBPs) associate with RNAs in intricate ribonucleoprotein complexes and regulate various aspects of RNA life cycle and, by extension, cell functions. Despite their significance, elucidating the functional contributions of specific RNA-RBP binding events, particularly in long-term phenotypic assays, remains challenging. Here, we harness the specificity of CRISPR/dCas13 to interfere with specific RNA-RBP interactions. We apply this methodology to GA-rich mRNA localization elements which recruit the RNA-binding protein CNBP and serve as platforms for the assembly of mRNA trafficking complexes. We show that dCas13/gRNA binds to target transcripts in a highly specific manner and sterically interferes with CNBP recruitment leading to altered target mRNA localization and cell motility, consistent with the function of the targeted mRNAs. The effectiveness of dCas13/gRNA as a functional interference tool is curtailed by the strength of target mRNA binding as well as by the amount of cytoplasmic gRNA. We describe optimizations and considerations for the stable implementation of this system, to allow the investigation of long-term functional consequences of altered mRNA distributions.

Adenosine deaminase acting on RNA (ADAR)-mediated RNA base editing offers a safer alternative to genome editing for specific clinical applications because of nonpermanent editing of targets. Current guide RNA (gRNA) designs feature a fully complementary specificity domain with an A-C mismatch at the targeted adenosine. However, perfectly matched dsRNA is not the most effective ADAR substrate. Here we introduce MIRROR (mimicking inverted repeats to recruit ADARs using engineered oligoribonucleotides), an approach that implements structural motifs derived from highly edited inverted Alu repeats in human tissues to enable rational gRNA design for ADAR recruitment. We demonstrated that MIRROR is applicable to both short chemically synthesized gRNAs with modifications and long biologically generated gRNAs and surpasses current state-of-the-art approaches in both gRNA forms. It enhances editing efficiency by up to 5.7-fold in multiple human cell types and primary hepatocytes from an alpha-1 antitrypsin deficiency mouse model. Our findings improve programmable RNA editing in vitro and in vivo by rational design through the screening of highly edited natural substrate mimics.

Engineered programmable RNA sensors have been applied in low-cost diagnostics, endogenous RNA detection, and multi-input genetic circuits. However, designing, producing, and screening high-performance RNA sensors remains time-consuming and labor intensive. Here, we present an automated plasmid assembly pipeline using liquid handling robotics to enable high-throughput construction of plasmids with arbitrary sequences. We compare automated and manual assembly methods using the NGS Hamilton Microlab STAR across two plasmid backbones to evaluate efficiency and reliability. As a proof of concept, we use this modular platform to construct a diverse set of programmable RNA regulators, including toehold switch riboregulators targeting viral RNAs, single-nucleotide-specific programmable riboregulators for discrimination of SARS-CoV-2 spike gene mutations, and metal-responsive riboswitches. In total, we construct 174 plasmids and test the designed methods by comparing both manual and automated assembly. We further demonstrate that the assembled toehold switch plasmids are functional in both bacterial and cell-free expression systems.

Transposon-encoded IscB has been established as the evolutionary ancestor of CRISPR-Cas9. This compact RNA-guided endonuclease has since been engineered for genome editing applications. We previously repurposed IscB and Cas9 as efficient RNA editors by removing their double-stranded DNA recognition module, the TAM/PAM-interacting domain. Here, we report four cryo-EM structures of IscB in complex with single-stranded nucleic acid (ssNA) targets to illuminate its mechanistic underpinnings. Structural analysis reveals that IscB initially facilitates formation of a 10-nt seed duplex with ssNA; however, further base-pairing is blocked by an alternatively positioned HNH nuclease that acts as a conformational roadblock. In this intermediate state, neither HNH nor RuvC is competent for target cleavage: the HNH domain is occluded by the roadblock configuration, while the RuvC active site is obstructed by the guide RNA. Only upon full duplex formation do additional base pairs between the guide RNA and ssNA dislodge the HNH roadblock, simultaneously exposing the RuvC nuclease active site. We propose that an analogous conformational checkpoint governs IscB activity during dsDNA target interrogation. Guided by these structural insights, we introduced mutations to either enhance ssNA binding or relieve the conformational checkpoint, both of which significantly improved RNA-targeting efficiency of IscB.

Huntington's disease (HD) is a monogenic autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the