Molecular mechanisms of RNA-triggered gene silencing machineries

Gene silencing by RNA triggers is an ancient, evolutionarily conserved, and widespread phenomenon. This process, known as RNA interference (RNAi), occurs when double-stranded RNA helices induce cleavage of their complementary mRNAs. Because these RNA molecules can be introduced exogenously as small interfering RNAs (siRNAs), RNAi has become an everyday experimental tool in laboratory research. In addition, the number of RNA-based therapeutics that are currently in clinical trials for a variety of human diseases demonstrate the therapeutic potential of RNAi. In this Account, we focus on our current understanding of the structure and function of various classes of RNAi triggers and how this knowledge has contributed to our understanding of the biogenesis and catalytic functions of siRNA and microRNA in mammalian cells. Mechanistic studies to understand the structure and function of small RNAs that induce RNAi have illuminated broad functions of the ancient RNAi machinery in animals and plants. In addition, such studies have provided insight to identify endogenous physiological gene silencing RNA triggers that engage functional machineries similar to siRNAs. Several endogenous small RNA species have been identified: small noncoding RNAs (microRNAs), piwi-interacting RNAs (piRNAs), and endogenous siRNAs (endo-siRNAs). microRNAs are the most widespread class of small RNAs in mammalian cells. Despite their importance in biology and medicine, the molecular and cellular mechanisms of microRNA biogenesis and function are not fully understood. We provide an overview of the current understanding of how these molecules are synthesized within cells and how they act on gene targets. Interesting questions remain both for understanding the effects of modifications and editing on microRNAs and the interactions between microRNAs and other cellular RNAs such as long noncoding RNAs.     

CHERP Regulates the Alternative Splicing of pre-mRNAs in the Nucleus

Calcium homeostasis endoplasmic reticulum protein (CHERP) is colocalized with the inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum or perinuclear region, and has been involved in intracellular calcium signaling. Structurally, CHERP carries the nuclear localization signal and arginine/serine-dipeptide repeats, like domain, and interacts with the spliceosome. However, the exact function of CHERP in the nucleus remains unknown. Here, we showed that poly(A)⁺ RNAs accumulated in the nucleus of CHERP-depleted U2OS cells. Our global analysis revealed that CHERP regulated alternative mRNA splicing events by interaction with U2 small nuclear ribonucleoproteins (U2 snRNPs) and U2 snRNP-related proteins. Among the five alternative splicing patterns analyzed, intron retention was the most frequently observed event. This was in accordance with the accumulation of poly(A)⁺ RNAs in the nucleus. Furthermore, intron retention and cassette exon choices were influenced by the strength of the 5′ or 3′ splice site, the branch point site, GC content, and intron length. In addition, CHERP depletion induced anomalies in the cell cycle progression into the M phase, and abnormal cell division. These results suggested that CHERP is involved in the regulation of alternative splicing.     

TFIP11, CCNL1 and EWSR1 Protein-protein Interactions,and Their Nuclear Localization

Previous studies using the yeast two-hybrid assay (Y2H) have identified cyclin L1 (CCNL1) and Ewing sarcoma breakpoint region 1 protein (EWSR1) as being interacting partners of tuftelin-interacting protein 11 (TFIP11). All three proteins are functionally related to the spliceosome and involved in pre-mRNA splicing activities. The spliceosome is a dynamic ribonucleoprotein complex responsible for pre-mRNA splicing of intronic regions, and is composed of five small nuclear RNAs (snRNAs) and μ140 proteins. TFIP11 appears to play a role in spliceosome disassembly allowing for the release of the bound lariat-intron. The roles of CCNL1 and EWSR1 in the spliceosome are poorly understood. Using fluorescently-tagged proteins and confocal microscopy we show that TFIP11, CCNL1 and EWSR1 frequently co-localize to speckled nuclear domains. These data would suggest that all three proteins participate in a common cellular activity related to RNA splicing events.     

Canonical and Interior Circular RNAs Function as Competing Endogenous RNAs in Psoriatic Skin

(1) Background: Understanding the function of circular RNAs (circRNAs), a class of noncoding RNA, in psoriatic skin can provide important insights into the complex regulation of genes contributing to the pathogenesis of psoriasis. (2) Methods: A novel method was applied to RNA-seq datasets from 93 skin biopsy samples to comprehensively identify circRNAs of all types, i.e., canonical circRNAs from the intron-exon junctions of mRNAs and interior circRNAs (i-circRNAs) from the interior regions of exons, introns, and intergenic regions. Selected circRNAs were experimentally validated by qRT-PCR and Sanger sequencing. CircRNAs with abundant and differential expression were identified and their putative function as competing endogenous RNAs (ceRNAs) was analyzed by an integrated analysis of circRNAs, microRNAs, and mRNAs. (3) Results: With a comprehensive search using no information of splicing signals, we systematically identified 179 highly abundant circRNAs in psoriatic skin. Many of these were reported for the first time and many were differentially expressed in involved versus normal or uninvolved skin. Validation based on three additional RNA-seq datasets confirmed most of the identified circRNAs in psoriatic skin. Experimental analyses confirmed the expression of the well-known circRNA CDR1as, a canonical circRNA, and a novel i-circRNA in psoriasis. We also identified many circRNAs that may act as ceRNAs to regulate the expression of mRNA genes in psoriasis-related signaling pathways in psoriasis. (4) Conclusions: The result of the study suggested that circRNAs are abundant in psoriatic skin, have distinct characteristics, and contribute to psoriatic pathogenesis.     

Small RNAs Participate in Plant–Virus Interaction and Their Application in Plant Viral Defense

Small RNAs are significant regulators of gene expression, which play multiple roles in plant development, growth, reproductive and stress response. It is generally believed that the regulation of plants’ endogenous genes by small RNAs has evolved from a cellular defense mechanism for RNA viruses and transposons. Most small RNAs have well-established roles in the defense response, such as viral response. During viral infection, plant endogenous small RNAs can direct virus resistance by regulating the gene expression in the host defense pathway, while the small RNAs derived from viruses are the core of the conserved and effective RNAi resistance mechanism. As a counter strategy, viruses evolve suppressors of the RNAi pathway to disrupt host plant silencing against viruses. Currently, several studies have been published elucidating the mechanisms by which small RNAs regulate viral defense in different crops. This paper reviews the distinct pathways of small RNAs biogenesis and the molecular mechanisms of small RNAs mediating antiviral immunity in plants, as well as summarizes the coping strategies used by viruses to override this immune response. Finally, we discuss the current development state of the new applications in virus defense based on small RNA silencing.     

Tecto‐GIRz: Engineered Group I Ribozyme the Catalytic Ability of Which Can Be Controlled by Self‐Dimerization

RNA is a promising biomaterial for self‐assembly of nano‐sized structures with a wide range of applications in nanotechnology and synthetic biology. Several RNA‐based nanostructures have been reported, but most are unrelated to intracellular RNA, which possesses modular structures that are sufficiently large and complex to serve as catalysts to promote sophisticated chemical reactions. In this study, we designed dimeric RNA structures based on the Tetrahymena group I ribozyme. The resulting dimeric RNAs (tecto group I ribozyme; tecto‐GIRz) exhibit catalytic ability that depended on controlled dimerization, by which a pair of ribozymes can be activated to perform cleavage and splicing reactions of two distinct substrates. Modular redesign of complex RNA structures affords large ribozymes for use as modules in RNA nanotechnology and RNA synthetic biology.     

Emerging Functions for snoRNAs and snoRNA-Derived Fragments

The widespread implementation of mass sequencing has revealed a diverse landscape of small RNAs derived from larger precursors. Whilst many of these are likely to be byproducts of degradation, there are nevertheless metabolically stable fragments derived from tRNAs, rRNAs, snoRNAs, and other non-coding RNA, with a number of examples of the production of such fragments being conserved across species. Coupled with specific interactions to RNA-binding proteins and a growing number of experimentally reported examples suggesting function, a case is emerging whereby the biological significance of small non-coding RNAs extends far beyond miRNAs and piRNAs. Related to this, a similarly complex picture is emerging of non-canonical roles for the non-coding precursors, such as for snoRNAs that are also implicated in such areas as the silencing of gene expression and the regulation of alternative splicing. This is in addition to a body of literature describing snoRNAs as an additional source of miRNA-like regulators. This review seeks to highlight emerging roles for such non-coding RNA, focusing specifically on “new” roles for snoRNAs and the small fragments derived from them.     

Bottom‐up Design of Small Molecules that Stimulate Exon 10 Skipping in Mutant MAPT Pre‐mRNA

One challenge in chemical biology is to develop small molecules that control cellular protein content. The amount and identity of proteins are influenced by the RNAs that encode them; thus, protein content in a cell could be affected by targeting mRNA. However, RNA has been traditionally difficult to target with small molecules. In this report, we describe controlling the protein products of the mutated microtubule‐associated protein tau (MAPT) mature mRNA with a small molecule. MAPT mutations in exon 10 are associated with inherited frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP‐17), an incurable disease that is directly caused by increased inclusion of exon 10 in MAPT mRNA. Recent studies have shown that mutations within a hairpin at the MAPT exon 10–intron junction decrease the thermodynamic stability of the RNA, increasing binding to U1 snRNP and thus exon 10 inclusion. Therefore, we designed small molecules that bind and stabilize a mutant MAPT by using Inforna, a computational approach based on information about RNA–small‐molecule interactions. The optimal compound selectively bound the mutant MAPT hairpin and thermodynamically stabilized its folding, facilitating exon 10 exclusion.     

Trans-Splicing Improvement by the Combined Application of Antisense Strategies

Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.