Target binding triggers hierarchical phosphorylation of human Argonaute-2 to promote target release

Argonaute (Ago) proteins play a central role in post-transcriptional gene regulation through RNA interference (RNAi). Agos bind small RNAs (sRNAs) including small interfering RNAs (siRNAs) and microRNAs (miRNAs) to form the functional core of the RNA Induced Silencing Complex (RISC). The sRNA is used as a guide to target mRNAs containing either partially or fully complementary sequences, ultimately leading to down regulation of the corresponding proteins. It was previously shown that the kinase CK1α phosphorylates a cluster of residues in the eukaryotic insertion (EI) of Ago, leading to the alleviation of miRNA-mediated repression through an undetermined mechanism. We show that binding of miRNA-loaded human Ago2 to target RNA with complementarity to the seed and 3′ supplemental regions of the miRNA primes the EI for hierarchical phosphorylation by CK1α. The added negative charges electrostatically promote target release, freeing Ago to seek out additional targets once it is dephosphorylated. The high conservation of potential phosphosites in the EI suggests that such a regulatory strategy may be a shared mechanism for regulating miRNA-mediated repression.

Classification of CRISPR/Cas system and its application in tomato breeding

Remarkable diversity in the domain of genome loci architecture, structure of effector complex, array of protein composition, mechanisms of adaptation along with difference in pre-crRNA processing and interference have led to a vast scope of detailed classification in bacterial and archaeal CRISPR/Cas systems, their intrinsic weapon of adaptive immunity. Two classes: Class 1 and Class 2, several types and subtypes have been identified so far. While the evolution of the effector complexes of Class 2 is assigned solely to mobile genetic elements, the origin of Class 1 effector molecules is still in a haze. Majority of the types target DNA except type VI, which have been found to target RNA exclusively. Cas9, the single effector protein, has been the primary focus of CRISPR-mediated genome editing revolution and is an integral part of Class 2 (type II) system. The present review focuses on the different CRISPR types in depth and the application of CRISPR/Cas9 for epigenome modification, targeted base editing and improving traits such as abiotic and biotic stress tolerance, yield and nutritional aspects of tomato breeding.

Guide RNA acrobatics: positioning consecutive uridines for pseudouridylation by H/ACA pseudouridylation loops with dual guide capacity

Site-specific pseudouridylation of human ribosomal and spliceosomal RNAs is directed by H/ACA guide RNAs composed of two hairpins carrying internal pseudouridylation guide loops. The distal “antisense” sequences of the pseudouridylation loop base-pair with the target RNA to position two unpaired target nucleotides 5′-UN-3′, including the 5′ substrate U, under the base of the distal stem topping the guide loop. Therefore, each pseudouridylation loop is expected to direct synthesis of a single pseudouridine (Ψ) in the target sequence. However, in this study, genetic depletion and restoration and RNA mutational analyses demonstrate that at least four human H/ACA RNAs (SNORA53, SNORA57, SCARNA8, and SCARNA1) carry pseudouridylation loops supporting efficient and specific synthesis of two consecutive pseudouridines (ΨΨ or ΨNΨ) in the 28S (Ψ3747/Ψ3749), 18S (Ψ1045/Ψ1046), and U2 (Ψ43/Ψ44 and Ψ89/Ψ91) RNAs, respectively. In order to position two substrate Us for pseudouridylation, the dual guide loops form alternative base-pairing interactions with their target RNAs. This remarkable structural flexibility of dual pseudouridylation loops provides an unexpected versatility for RNA-directed pseudouridylation without compromising its efficiency and accuracy. Besides supporting synthesis of at least 6% of human ribosomal and spliceosomal Ψs, evidence indicates that dual pseudouridylation loops also participate in pseudouridylation of yeast and archaeal rRNAs.

CRISPR-Based Approaches for the High-Throughput Characterization of Long Non-Coding RNAs

Over the last decade, tens of thousands of new long non-coding RNAs (lncRNAs) have been identified in the human genome. Nevertheless, except for a handful of genes, the genetic characteristics and functions of most of these lncRNAs remain elusive; this is partially due to their relatively low expression, high tissue specificity, and low conservation across species. A major limitation for determining the function of lncRNAs was the lack of methodologies suitable for studying these genes. The recent development of CRISPR/Cas9 technology has opened unprecedented opportunities to uncover the genetic and functional characteristics of the non-coding genome via targeted and high-throughput approaches. Specific CRISPR/Cas9-based approaches were developed to target lncRNA loci. Some of these approaches involve modifying the sequence, but others were developed to study lncRNAs by inducing transcriptional and epigenetic changes. The discovery of other programable Cas proteins broaden our possibilities to target RNA molecules with greater precision and accuracy. These approaches allow for the knock-down and characterization of lncRNAs. Here, we review how various CRISPR-based strategies have been used to characterize lncRNAs with important functions in different biological contexts and how these approaches can be further utilized to improve our understanding of the non-coding genome.

tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

Type VI CRISPR-Cas systems are the only CRISPR variety that cleaves exclusively RNA1,2. In addition to the CRISPR RNA (crRNA)-guided, sequence-specific binding and cleavage of target RNAs, such as phage transcripts, the type VI effector, Cas13, causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from phage spread3,4. We show here that the principal form of collateral RNA degradation elicited by Cas13a protein from Leptotrichia shahii upon target RNA recognition is the cleavage of anticodons of multiple tRNA species, primarily those with anticodons containing uridines. This tRNA cleavage is necessary and sufficient for bacterial dormancy induction by Cas13a. In addition, Cas13a activates the RNases of bacterial toxin-antitoxin modules, thus indirectly causing mRNA and rRNA cleavage, which could provide a back-up defense mechanism. The identified mode of action of Cas13a resembles that of bacterial anticodon nucleases involved in antiphage defense5, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module6,7 encompassing an anticodon nuclease.

Development and Validation of Ten-RNA Binding Protein Signature Predicts Overall Survival in Osteosarcoma

Osteosarcoma is a malignant tumor that originates in the bones with the characteristics of high malignancy, predisposition to metastasis, and poor prognosis. RNA binding proteins (RBPs) are closely related to various tumors, but their relationship with osteosarcoma remains unclear. Based on GTEx and TARGET RNA sequencing data, we applied differential analysis to obtain RBP genes that are differentially expressed in osteosarcoma, and analyzed the functions of these RBPs. After applying univariate and LASSO Cox regression analysis, 10 key prognostic RBPs (TDRD6, TLR8, NXT2, EIF4E3, RPS27L, CPEB3, RBM34, TERT, RPS29, and ZC3HAV1) were screened, and an RBP prognostic risk assessment model for patients with osteosarcoma was established. The independent cohort GSE21257 was used for external verification, and the results showed that the signature has an excellent ability to predict prognosis. In addition, a nomogram that can be used for clinical evaluation was constructed. Finally, the expression levels of 10 prognostic RBPs in osteosarcoma cells and tissues were confirmed through experiments. Our study identified a ten-gene prognostic marker related to RBP, which is of great significance for adjusting the treatment strategy of patients with osteosarcoma and exploring prognostic markers.

Potential Use of CRISPR/Cas13 Machinery in Understanding Virus–Host Interaction

Prokaryotes have evolutionarily acquired an immune system to fend off invading mobile genetic elements, including viral phages and plasmids. Through recognizing specific sequences of the invading nucleic acid, prokaryotes mediate a subsequent degradation process collectively referred to as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR-associated (Cas) (CRISPR–Cas) system. The CRISPR–Cas systems are divided into two main classes depending on the structure of the effector Cas proteins. Class I systems have effector modules consisting of multiple proteins, while class II systems have a single multidomain effector. Additionally, the CRISPR–Cas systems can also be categorized into types depending on the spacer acquisition components and their evolutionary features, namely, types I–VI. Among CRISPR/Cas systems, Cas9 is one of the most common multidomain nucleases that identify, degrade, and modulate DNA. Importantly, variants of Cas proteins have recently been found to target RNA, especially the single-effector Cas13 nucleases. The Cas13 has revolutionized our ability to study and perturb RNAs in endogenous microenvironments. The Cas13 effectors offer an excellent candidate for developing novel research tools in virological and biotechnological fields. Herein, in this review, we aim to provide a comprehensive summary of the recent advances of Cas13s for targeting viral RNA for either RNA-mediated degradation or CRISPR–Cas13-based diagnostics. Additionally, we aim to provide an overview of the proposed applications that could revolutionize our understanding of viral–host interactions using Cas13-mediated approaches.

Antisense Oligonucleotide-Based Therapy of Viral Infections

Nucleic acid-based therapeutics have demonstrated their efficacy in the treatment of various diseases and vaccine development. Antisense oligonucleotide (ASO) technology exploits a single-strand short oligonucleotide to either cause target RNA degradation or sterically block the binding of cellular factors or machineries to the target RNA. Chemical modification or bioconjugation of ASOs can enhance both its pharmacokinetic and pharmacodynamic performance, and it enables customization for a specific clinical purpose. ASO-based therapies have been used for treatment of genetic disorders, cancer and viral infections. In particular, ASOs can be rapidly developed for newly emerging virus and their reemerging variants. This review discusses ASO modifications and delivery options as well as the design of antiviral ASOs. A better understanding of the viral life cycle and virus-host interactions as well as advances in oligonucleotide technology will benefit the development of ASO-based antiviral therapies.

Widespread microRNA degradation elements in target mRNAs can assist the encoded proteins

Binding of microRNAs (miRNAs) to mRNAs normally results in post-transcriptional repression of gene expression. However, extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD). Here, we systematically analyzed Argonaute-CLASH (cross-linking, ligation, and sequencing of miRNA–target RNA hybrids) data and identified numerous candidate TDMD triggers, focusing on their ability to induce nontemplated nucleotide addition at the miRNA 3′ end. When exogenously expressed in various cell lines, eight triggers induce degradation of corresponding miRNAs. Both the TDMD base-pairing and surrounding sequences are essential for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduced miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes a proapoptotic protein, enhances apoptosis. Therefore, we uncovered widespread TDMD triggers in target RNAs and demonstrated an example that could functionally cooperate with the encoded protein.