Distinct nuclear and cytoplasmic machineries cooperatively promote the inheritance of RNAi in Caenorhabditis elegans

Epigenetic information can be inherited over multiple generations, which is termed as transgenerational epigenetic inheritance (TEI). Although the mechanism(s) of TEI remains poorly understood, noncoding RNAs have been demonstrated to play important roles in TEI. In many eukaryotes, double‐stranded RNA (dsRNA) triggers the silencing of cellular nucleic acids that exhibit sequence homology to the dsRNA via a process termed RNA interference (RNAi). In Caenorhabditis elegans, dsRNA‐directed gene silencing is heritable and can persist for a number of generations after its initial induction. During the process, small RNAs and the RNAi machinery mediate the initiation, transmission and re‐establishment of the gene silencing state. In this review, we summarise our current understanding of the underlying mechanism(s) of transgenerational inheritance of RNAi in C. elegans and propose that multiple RNAi machineries may act cooperatively to promote TEI.     

Identification and characterization of small RNAs involved in RNA silencing

Double-stranded RNA (dsRNA) is a potent trigger of sequence-specific gene silencing mechanisms known as RNA silencing or RNA interference. The recognition of the target sequences is mediated by ribonucleoprotein complexes that contain 21- to 28-nucleotide (nt) guide RNAs derived from processing of the trigger dsRNA. Here, we review the experimental and bioinformatic approaches that were used to identify and characterize these small RNAs isolated from cells and tissues. The identification and characterization of small RNAs and their expression patterns is important for elucidating gene regulatory networks.     

How does precursor RNA structure influence RNA processing and gene expression?

RNA is a fundamental biomolecule that has many purposes within cells. Due to its single-stranded and flexible nature, RNA naturally folds into complex and dynamic structures. Recent technological and computational advances have produced an explosion of RNA structural data. Many RNA structures have regulatory and functional properties. Studying the structure of nascent RNAs is particularly challenging due to their low abundance and long length, but their structures are important because they can influence RNA processing. Precursor RNA processing is a nexus of pathways that determines mature isoform composition and that controls gene expression. In this review, we examine what is known about human nascent RNA structure and the influence of RNA structure on processing of precursor RNAs. These known structures provide examples of how other nascent RNAs may be structured and show how novel RNA structures may influence RNA processing including splicing and polyadenylation. RNA structures can be targeted therapeutically to treat disease.     

Recent insights into noncanonical 5′ capping and decapping of RNA

The 5′ N⁷-methylguanosine cap is a critical modification for mRNAs and many other RNAs in eukaryotic cells. Recent studies have uncovered an RNA 5′ capping quality surveillance mechanism, with DXO/Rai1 decapping enzymes removing incomplete caps and enabling the degradation of the RNAs, in a process we also refer to as “no-cap decay.” It has also been discovered recently that RNAs in eukaryotes, bacteria, and archaea can have noncanonical caps (NCCs), which are mostly derived from metabolites and cofactors such as NAD, FAD, dephospho-CoA, UDP-glucose, UDP-N-acetylglucosamine, and dinucleotide polyphosphates. These NCCs can affect RNA stability, mitochondrial functions, and possibly mRNA translation. The DXO/Rai1 enzymes and selected Nudix (nucleotide diphosphate linked to X) hydrolases have been shown to remove NCCs from RNAs through their deNADding, deFADding, deCoAping, and related activities, permitting the degradation of the RNAs. In this review, we summarize the recent discoveries made in this exciting new area of RNA biology.     

RNA干扰在疾病治疗方面的应用研究

RNA干扰是由双链RNA引起的序列特异的基因沉默现象。由于RNA干扰能在细胞组织及动物模型中沉默疾病相关基因,因此,RNA干扰也是各种疾病治疗的有效手段。在哺乳动物细胞内诱导RNA干扰可以通过导入小干扰RNA（siRNA）,或是以质粒、病毒为载体表达短的发夹RNA（shRNA）而实现。本文介绍了RNA干扰在疾病治疗方面的应用,并就其面临的挑战进行讨论。     

A novel method for constructing pathogen-regulated small RNA cDNA library

Pathogen-responsive endogenous small non-coding RNAs regulate gene expression in relation to plant immune responses by serving as RNA silencing machinery. Decay caused by the bacterium, Erwinia carotovora subsp. carotovora (Ecc), often leads to soft rot disease in the plant Brassica campestris L. ssp. pekinensis (Bcp). To discover endogenous small RNA species in Bcp in response to Ecc infection, we developed a highly efficient approach for cloning pathogen-regulated small RNAs. A group of degenerate stem-loop reverse primers was designed to synthesize first single-stranded cDNA (sscDNA) and the sscDNA was then tailed with a poly(C) at its 3′ end to create a forward priming site. A novel cDNA/RNA subtractive hybridization was performed to capture Ecc-regulated small RNAs and this subsequently allowed construction of small RNA cDNA libraries for sequencing.     

Tau‐induced nuclear envelope invagination causes a toxic accumulation of mRNA in Drosophila

The nucleus is a spherical dual‐membrane bound organelle that encapsulates genomic DNA. In eukaryotes, messenger RNAs (mRNA) are transcribed in the nucleus and transported through nuclear pores into the cytoplasm for translation into protein. In certain cell types and pathological conditions, nuclei harbor tubular invaginations of the nuclear envelope known as the “nucleoplasmic reticulum.” Nucleoplasmic reticulum expansion has recently been established as a mediator of neurodegeneration in tauopathies, including Alzheimer's disease. While the presence of pore‐lined, cytoplasm‐filled, nuclear envelope invaginations has been proposed to facilitate the rapid export of RNAs from the nucleus to the cytoplasm, the functional significance of nuclear envelope invaginations in regard to RNA export in any disorder is currently unknown . Here, we report that polyadenylated RNAs accumulate within and adjacent to tau‐induced nuclear envelope invaginations in a Drosophila model of tauopathy. Genetic or pharmacologic inhibition of RNA export machinery reduces accumulation of polyadenylated RNA within and adjacent to nuclear envelope invaginations and reduces tau‐induced neuronal death. These data are the first to point toward a possible role for RNA export through nuclear envelope invaginations in the pathogenesis of a neurodegenerative disorder and suggest that nucleocytoplasmic transport machinery may serve as a possible novel class of therapeutic targets for the treatment of tauopathies.     

Unveiling the druggable RNA targets and small molecule therapeutics

The increasing appreciation for the crucial roles of RNAs in infectious and non-infectious human diseases makes them attractive therapeutic targets. Coding and non-coding RNAs frequently fold into complex conformations which, if effectively targeted, offer opportunities to therapeutically modulate numerous cellular processes, including those linked to undruggable protein targets. Despite the considerable skepticism as to whether RNAs can be targeted with small molecule therapeutics, overwhelming evidence suggests the challenges we are currently facing are not outside the realm of possibility. In this review, we highlight the most recent advances in molecular techniques that have sparked a revolution in understanding the RNA structure-to-function relationship. We bring attention to the application of these modern techniques to identify druggable RNA targets and to assess small molecule binding specificity. Finally, we discuss novel screening methodologies that support RNA drug discovery and present examples of therapeutically valuable RNA targets.     

RNA-binding proteins in RNA interference

Short RNAs (21–27 nt) silence genes that contain homologous nucleotide sequences; this is known as RNA silencing. This review considers the generation of short RNAs from their precursors: double-stranded RNAs, capable of inducing RNA interference, and hairpin RNAs, whose processing yields microRNAs, as well as the properties of RNA-binding domains that were initially identified in proteins operating in RNA interference. The interactions between these domains and known RNA-binding modules within proteins involved in RNA interference and microRNA generation are described.